The Source of Self - Log in to Wiley Online Library

NATURAL GENETIC ENGINEERING AND NATURAL GENOME EDITING

The Source of Self Genetic Parasites and the Origin of Adaptive Immunity Luis P. Villarreal Center for Virus Research, Department of Molecular Biology and Biochemistry, University of California, Irvine, California, USA Stable colonization of the host by viruses (genetic parasites) can alter the systems of host identity and provide immunity against related viruses. To attain the needed stability, some viruses of prokaryotes (P1 phage) use a strategy called an addiction module. The linked protective and destructive gene functions of an addiction module insures both virus persistence but will also destroy cells that interrupt this module and thereby prevent infection by competitors. Previously, I have generalized this concept to also include persistent and lytic states of virus infection, which can be considered as a virus addiction module.1 Such states often involve defective viruses. In this report, I examine the origin of the adaptive immune system from the perspective of a virus addiction module. The likely role of both endogenous and exogenous retroviruses, DNA viruses, and their defective elements is considered in the origin of all the basal components of adaptive immunity (T-cell receptor, RAG-mediated gene rearrangement, clonal lymphocyte proliferation, antigen surface presentation, apoptosis, and education of immune cells). It is concluded that colonization by viruses and their defectives provides a more coherent explanation for the origin of adaptive immunity. Key words: virus; evolution; immunity; adaptive immunity; endogenous virus

Introduction A core purpose of biological communication is to differentiate self from nonself at the organismal level. The recognition of kin, mates, competitors, prey, and predators all depend on such communication. However, the ability to recognize self and communicate this is also a characteristic of individual cells that make up multicellular organisms. Such recognition is also fundamental for all immune systems. In multicellular organisms, it can also be important to both support and destroy self cells, as seen with apoptosis during development. Thus all cells have systems that communicate self-

Address for correspondence: Luis P. Villarreal, Center for Virus Research, Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, USA. Voice: 949-824-6074; fax: 949-824-9437. [email protected]

recognition and can react in both supportive and destructive modes and use similar mechanisms for pathogen detection and destruction. Typically, such systems are defined from the context of immune systems directed at pathogens. However, a more basic definition is that these systems constitute systems of identity that can also respond in a destructive way. The origin of identity systems thus provides a more fundamental way to think of the origin of immunity systems. In a previous monograph, I presented arguments and evidence that persisting viruses and related (defective) genetic parasites can generate and alter the cellular mechanisms used to determine self-identity.1 A core example is found in bacteria colonized with persisting (temperate) viruses. Such viruses frequently employ immune and addiction strategies that allow stable maintenance of the virus. However,

Natural Genetic Engineering and Natural Genome Editing: Ann. N.Y. Acad. Sci. 1178: 194–232 (2009). c 2009 New York Academy of Sciences. doi: 10.1111/j.1749-6632.2009.05020.x

194

Villarreal: Viral Origin of Adaptive Immunity

these same strategies also limit the replication of related viruses to allow stable persistence (providing immunity). The existence of addiction modules was first established in the episomal persisting P1 phage of Escherichia coli.2,3 Addiction modules involve sets of complementary functions or genes that are beneficial or protective matched with (counteracting) more stable functions that are harmful or destructive.4 If the parasite genome should be lost, the more stable harmful component of the addiction module is no longer inhibited and self-destruction is induced.5 Violation of addiction modules can occur following sexual reproduction that leads to loss of the virus mediated protection, or invasion by a second parasite or pathogen that can also disrupt the module. These addiction modules can also be thought of as toxin/antitoxin (T/A) gene sets. Addiction modules thus inherently provide identity systems. Because they can result from persistent virus colonization, they offer a different perspective to understand the external origin and evolution of systems of immunity and self-destruction (such as apoptosis). However, a more generalized version of addiction modules can also be created by the direct action of acute and persisting viruses.1 In this application, a potentially lethal virus can be held at bay by the immunity function of the same virus that has persistently colonized its host. Furthermore, even defective viral elements can directly contribute to establishing such persisting states. Because defective viral elements have also been considered as selfish DNA, the presence of such elements suggest past events of persistent viral colonization. Thus, a host cell that is persistently colonized by a virus that is also prevalent and potentially lethal is protected from the same and similar viruses. This colonized host has acquired a new self-identity and is immune to related viruses. However, because a virus is transmissible (communicating) it can act on other individuals in the same population, thus it affects groups. Because of this, viruses have the capacity to affect the survival of host groups, and promote states of group addiction or group identity. For ex-

195

ample, a bacterial colony that is colonized by a specific temperate phage is able to kill an otherwise identical adjacent (communicating) bacterial colony that is not colonized or otherwise immune. In this way, groups of organisms (including multicellular organisms) may be able to acquire viral mediated mechanisms that define group identity and promote symbiosis and multicellularity.6,7 Given these viral consequences, the emergence of adaptive immunity in jawed vertebrates, such as fish, is now examined. Fish represent a major evolutionary transition.8,9 Fish make up 50% of all vertebrate species on Earth and greatly outnumber their related ancestral tunicate (lamprey) species. The adaptive immune system first emerged in fish and is usually portrayed as a highly sophisticated immunity system able to protect against yet unseen (anticipated) pathogens. It has invented lymphocytes that can now recognize and kill virus infected “self-cells.” Despite this view as a highly sophisticated immune system, it is ironic that the adaptive immune system itself appears to host numerous viruses. Indeed, the renewing white blood cells, specific to the adaptive immune system, are themselves able to support a surprisingly diverse set of viruses not known to infect many of the ancestral animal species. These include negative stranded RNA viruses (vesicular stomatitis virus, parainfluenza virus, measles virus, respiratory syncytial virus), retroviruses (murine leukemia virus [MLV], endogeneous type C viruses, FeLV, HIV, HTLV), single strand DNA viruses (porcine parvovirus, minute virus of mice), and double stranded DNA viruses (papovaviruses, group C adenoviruses, 8 distinct lineages of human herpesviruses, and leporipoxviruses).10 It is also interesting that viral mediated mass die offs have been often described for natural populations of vertebrate fish.11–15 Thus, such observations seem at odds with the idea that this highly sophisticated adaptive immunity is more capable of preventing viral pathogens. However, if the evolution of adaptive immunity is evaluated as the acquisition of a more sophisticated

196

system of cellular identity (now able to recognize virus infected self-cells), its origin as an antiviral system may become clear. If we think of adaptive immunity as a system based on addiction (identity) modules, in which self-identity is acquired during development (immune system education), we can also consider the possibility that colonizing viruses might be able to explain the origin of the various novel components of this adaptive system. These components were absent from what are though to be the immediate ancestors of the jawed vertebrates, the tunicates and hagfish.16,17 Unlike their ancestors, in jawed vertebrates, self-identity is acquired during immune development and subsequent violation of this self-identity (such as virus infection) induces self-destruction (immunity). Adapting a Virus-first Perspective Let us start by considering how white blood cells, able to recognize nonself, proliferate, and kill virus infected self-cells, might have evolved. Ancestral jawless vertebrate animals all have similar types of lymphocytes,18 but these cells appear to lack antiviral activity (and also support many fewer viral parasites). Indeed, no viruses specific to their immune cells have yet been described. Yet other invertebrate oceanic animals (such as mollusk and shrimp) do support many types of viruses (although retroviruses are rare)19 yet still lack blood cells that can kill virus infected cells.20 However, as discussed below, some viruses can kill these blood cells by inducing apoptosis. Genomic viruses (ERVs) differ considerably between invertebrates and vertebrates. The genomes of tunicates and hagfish are much less colonized by retroviral elements and their derivatives (reverse transcriptase [RT] mediates long interspersed retrotransposable elements [LINES] and short interspersed retrotransposable elements [SINES]). These genomes also lack all the core elements needed for the adaptive immune system; TCR genes (T-cell receptors,

Annals of the New York Academy of Sciences

members of the Ig superfamily), the RAG1/2 mediated system of Ig gene rearrangement, the resulting T-cell specific rearrangement and clonal selection, and the use of the major histocompatibility complex (MHC) (Ig superfamily) genes to recognize nonself, proliferate, and kill nonself cells.9,16,21 Yet invertebrate animals are not defenseless against viruses. Indeed the simplest animal model, such as Caenorhabditis elegans and sea urchin, have highly effective antiviral systems based on RNA interference (RNAi) that appear to effectively control essentially all known viral infections.22–27 Given that the immune cells of vertebrates are themselves prone to infection by numerous viruses as mentioned above, what then was the selective pressure for the emergence of these adaptive immune cells? Let us now examine the likely role of viral colonization in the development of white blood cells of adaptive immunity. Starting Material, Invertebrate White Blood Cells, and Virus A main interest is to understand how the blood cells of ancestral oceanic animals acquired the ability to kill virus infected self-cells. Bony fish clearly have cytotoxic T cells that respond to MHC mediated antigen presentation. Phagocytic blood cells were present in the blood of the earliest examples of animals, such as sponges, snails, and hydra.28 Such cells were maintained during the evolution of all invertebrate marine animals, but in no case did they evolve into pathogen specific cytotoxic T lymphocyte (CTL)-like cells. Mollusks, arthropods, echinoderms, and tunicates all show essentially similar phagocytic blood cells and how these cells respond to pathogens is also similar.20,29,30 The main reaction of these nonproliferative amoebocyte cells is to wall off invaders, using calcium (bone-like) deposits. Apoptosis does not appear to be a normal part of the developmental program of these cells. Some invertebrate blood cells can also be cytotoxic


by expressing lytic granules, but this reaction is not antigen specific. Thus, these cells resemble vertebrate natural killer (NK) cells (not CTLs) in this less specific activity. However, complex surface recognition of foreign cells (identity systems) did develop in some jawless vertebrates (see below). But such recognition capacity is not a characteristic of invertebrate blood cells nor are the components used by such systems related to the components of the adaptive immune system. Thus, in contrast to jawed vertebrate blood cells, invertebrate blood cells are mainly phagocytic and do not undergo complex antiviral induced proliferation and progressive differentiation. Nor do invertebrates have bone morrow or lymphoid organs, which support lymphocyte differentiation. Instead, as mentioned, these lower animals (including shrimp), have efficient innate immune systems (such as RNAi), that appears to be most important for the antiviral response.31 However, some prevalent viruses of invertebrates (e.g., white spot syndrome virus [WSSV] below) do induce considerable and nonproductive damage via apoptosis in invertebrate blood cells. Viruses and Mollusks Yet invertebrate oceanic animals are highly successful species relative to vertebrates. For example, mussels are the biomass dominant species of the deep sea and they represent an impressive 110,000 species of only Mollusca.32 But the oceans have a vast quantity of viruses, especially large dsDNA viruses, that these filter feeders must encounter. And it is clear that viruses are a major factor in the habitat and survival of mussels and other oceanic invertebrates,11,19,33,34 such as the viral mediated mass die offs seen with farmed oysters.35,36 In particular, it appears herpes-related viruses are common to these animals and may induce apoptosis in diseased tissue.37 There is overwhelming evidence that mollusks have many other intimate but poorly understood relationships with

197

viruses of various types. In deep sea species, for example, gut tissues can often be seen (by electron microscopy) to harbor virus-like inclusions, and some of these agents appear to be associated with pathogenesis of big shelled individuals.32 At least some of these viruses have asymptomatic relationships with their mollusk host, yet these same viruses may also be capable of pathogenic infection. An example of this relationship is found with the mollusk Herpesvirus family. In particular, OsHV-1 (Oyster herpes virus in adult Pacific Crassostrea) is a prevalent infection found in natural populations.36,38 Outbreaks by this virus were observed to account for a high mortality in larvae and juvenile oysters, especially in farmed shellfish.39 Yet surveys of natural adult populations frequently show that an asymptomatic OsHV can be found in gonad tissue of 90% of the individuals. Thus, both disease-free persistence and disease-associated acute infections are seen with the same virus in the same host. Connective tissue and ganglions were the main sites of virus replication.40 It is worth noting that the gonad tissue (site of persistence) is also the main tissue that shows ongoing differentiation in bivalves, in contrast to vertebrates that also have differentiating lymphocytes. It is thought that persistence in gonads may allow vertical (persistent) virus transmission. Thus, it seems that most adults in natural populations of oysters are survivors of herpesvirus colonization. It is interesting that crosses between such infected males and uninfected females results in decreased survival of offspring whereas crosses with infected females had higher survival rates.41 This suggests that viral persistence can affect group survival. Field studies of commercial oyster farms, such as Tomales Bay, California,38,42 also support the close association between virus and host population. Most adult oysters here also had OsHV but were colonized by a version of the virus that appears to be specific to the host population. The OsHV virion is clearly similar to mammalian HSV-1 and CMV (i.e., capsid T = 16, a characteristic of herpes).36 Thus, OsHV seems to be clearly related to vertebrate

198

animal herpesviruses. Also, OsHV has many usual proteins in addition to some unusual HSV like-ones, such as ion channel membrane proteins. It is interesting to note ion channel proteins are crucial signal transducers of the adaptive immune system. In oceanic DNA viruses, however, the most diverse source of novel gated ion channel proteins are found in the viruses of green algae, such as PBCV-1.43–45 In these algal viruses, ion channels appear to be used early in infection and may preclude other viruses from an infected host.46,47 Because many of such host-like viral proteins are structurally simpler and appear to be phylogenetically basal to their host protein homologues, and are often more diverse then their host counterparts, it appears clear that these channel proteins originate from viral, not host sources. There are other less characterized herpesviruses of bivalve species that are also known to show high mortality in farmed populations.48–50 For example, major losses were caused by a herpes-like virus causing ganglioneuritis of farmed abalone species in four separate farms of the Victorian coast of Australia. Here, the virus had an affinity for nervous tissue, a tissue developmentally related to lymphocytes. It is interesting to note that similar viruses have been reported to induce tumors in abalone nervous tissue.51 Besides herpesviruses, a lot of aquatic birnaviruses are also known to infect shellfish (and fish). Clearly, marine viruses have major consequences to bivalves, although their viral evolutionary ecology remains poorly studied. However, it is clear that despite being filter feeders that are able to support various types of virus infection, and despite lacking all the elements of the adaptive immune system, bivalves are highly successful species and can also be extremely long lived. This longevity is of relevance to the origin of the adaptive immune system. It has often been rationalized that because most invertebrates and insects have short life spans (reproducing every season), they do not need adaptive immune systems that will resist reexposure to seasonal infectious agents.


Yet some invertebrates can be very long lived. For example, some large individuals of Quahog clam (a.k.a. hard or chowder clam) have been estimated to live as long as 250 years. Similar, large clams found in deep sea hydrothermal vents also live over 200 years. In invertebrate insects, individual queen termites and queen bees are known to live 10–25 years, in hives that are clearly also prone to virus colonization. This life span is well beyond the average life expectancy for most vertebrates. Because few, if any, vertebrates have attained the highly extended life spans of giant clams, the selective pressures for the adaptive immune system of vertebrates cannot be explained solely by longevity. Thus, the selective pressures for and advantages of the adaptive immune system are not clear. Transition to Vertebrate Genome: ERV Invasion and Displacement Comparative genomics between extant jawless vertebrates with those of jawed fish should provide clues regarding the genetic alterations associated with the origin of the adaptive immune system. Bony fish show a remarkable pattern of colonization and expansion by several families of endogenous retroviruses (gypsylike ERVs, see chromoviruses below). They also show a considerable expansion of overall DNA content. Yet similar ERV elements were present but in much smaller numbers in extant representatives of the ancestral genomes.52 Relative to tunicates and jawed vertebrates, most invertebrate genomes are compact and relatively overrepresented by the presence of DNA transposon elements. With the notable exception of Tc1 transposons,53 the large majority of DNA transposons appear to have been lost with the emergence of vertebrates.54 What selective pressures might have led to such overall displacement of these genetic parasites? Shrimp genomes, in contrast to bony fish, resemble those of simple animals, such as C. elegans.


Like C. elegans, shrimp have maintained relatively low but conserved copies of gypsy-like ERVS and long terminal repeat (LTR) elements55–57 and also a significant presence of DNA elements (i.e., high Tc1 elements). There is, however, one clear and general distinction between shrimp and C. elegans DNA. Shrimp genomes have large quantities of satellite DNA sequences (similar to what found in annelids). Shrimp satellite DNA can be highly abundant (up to 100,000 copies/genome) and are often species specific.58,59 Generally, satellites reside within the heterochromatic component of shrimp DNA so their expression should be silenced. Yet they can be highly transcribed during shrimp development for unknown reasons. Thus, although shrimp genomes have been highly altered by their own peculiar genetic parasites, vertebrate-like ERV or LINE elements are not overrepresented. Rather, non-LTR, DNA-types of transposons seem to have been more active in shrimps (similar to those found in Dictyostelium repeat sequence, DIRS). Curiously, the mitochondrial genomes of some shrimp species do show unusually large number of pseudogenes60 suggesting the action of retroposon activity during shrimp evolution.59 Thus shrimp genomes have been much affected by genetic parasites, but the makeup of these genetic parasites are distinct from those seen in vertebrate fish. And although shrimp have effective antiviral systems, they resemble those of lower animals (i.e., RNAi, interferon), not those of vertebrates.20,61 Tunicate Genomes: Before the Great ERV Colonization Tunicates have maintained the compact genomes characteristic of most invertebrates.62–64 Indeed, the tunicate genome are the smallest of all animal genomes so far characterized with 65–75 MBP and with only about 15,000 genes. Tunicate Oikopleura dioica has the most compact genome of any animal yet examined. Its DNA has about 20 times the gene

199

density of human DNA, and it also has very little heterochromatin. Some have proposed that tunicates have undergone a seemingly massive elimination of retro-elements in comparison to jawed vertebrate genomes as most known families of retrotransposons are absent. However, given the history and discussion presented above, it seems much more likely vertebrates underwent a significant colonization and expansion of LTR and LINE elements relative to tunicates. The tunicate genome has six clades of non-LTR retrotransposons,65 and compared to jawed vertebrates, its genome is LTR underrepresented. Yet, most notable in this regard, it has an astonishing level of diversity for the Gypsy/Ty3 elements (a Chromovirus ERV related to a new family called Odin).52,66,67 Furthermore, most of these LTR elements are not corrupted. Some specific examples are the Tor3 and Tor 4b elements, both of which appear to have even maintained an intact env ORF, which is normally deleted in most ERV elements. Oddly, however, this env resembles the env of paramyxovirus, not other retroviruses. In summary, although tunicate genomes maintain a highly compact nature prior to the great ERV expansion, they have been colonized by a low number of specific but diverse chromoviral (gypsy ERV-like) elements. But in contrast to their jawed vertebrate counterparts, these elements did not undergo large-scale genomic expansion. And also in contrast to other invertebrates, no Ty1/copia or Bel-like elements were maintained. The jawless vertebrates do show some variation in genomic changes regarding their DNA transposons. Both the diversity and percentage of such elements is considerably higher in the invertebrates relative to jawed vertebrates.68 In general, lower vertebrates have many more DNA type transposons than are seen in mammals (about 4% of their genome) and these appear to have been acquired from external sources.53 In tunicates, although low in diversity, these DNA elements can be very abundant (albeit mostly inactive). Ciona, for example, has 15,000 copies of Tc1 alone. Because Tc1 bears

200


some similarity to RAG1/2 recombination of the adaptive immune system, this is of some interest, yet Ciona lacks RAG homologues,69 as found in other invertebrates. The more limited diversity of DNA transposons in tunicate genomes also suggests that the elimination of some DNA transposon elements may have occurred during their evolution. Yet, curiously, DIRS1 elements, also characteristic of invertebrate animal genomes, are maintained in the tunicate genomes but completely absent from the genomes of jawed vertebrates. It thus seems clear that a significant transition in the composition of genetic parasites occurred within the jawless vertebrates in the transition to jawed vertebrates. For example, in amphioxus, we don’t see a massive duplication of genome seen in jawed vertebrates, nor has the genome undergone major rearrangement as seen in Ciona. Comparing these compact tunicate genomes to that of the compact pufferfish genome, this fish has a low percentage of repetitive DNA, but a much higher diversity of autonomous RT elements.

Self-Identity in Jawless Vertebrates Tunicates lack most genes of adaptive immunity, although they have some elements used for innate immunity as found in jawed vertebrates.70,71 For example, several complement components (C3, C4, C5), are present and expressed on tunicate blood cells but do not appear to be linked to adaptive immunity (MHC).16,72–74 Interesting that in the solitary ascidian, Halocynthis, it has a C3 along with a regulator of complement activation (RCA) that prevents self-destruction. This clearly resembles some type of addiction/identity module. In mammals/tetrapods, the C3 component underwent an expansion to 11 copies/cell for unknown reasons. Colonial tunicates (e.g., Botryllus), do differentiate self from nonself colony members because mixed tissues are re-

jected,75,76 This capacity is associated with an MHC-like polymorphic gene locus known as Fu/HC.77,78 However, these genes have no similarity to the vertebrate MHC locus. When cells with different Fu/HC loci are mixed, they will reject all tissue not sharing at least one allele. Rejection is via the induction of massive phagocytosis and an oxidative burst, thus this blood cell response more resembles that of NK cells79 and is not similar to antigen specific T cell rejection.80,81 Nor does this Fu/HC system appear to represent a pathogen response system because it is mainly active against other tunicate colonies. It thus appears to provide a self or group identity system.82 However, some minimal domains related to adaptive immune function can be found in tunicates. These include the presence of 2 Ig-like domain proteins and also an MHC-like peptide binding domain. But these domains have no known role in immune recognition or tissue rejection and the adjoining genes are very distinct from those of vertebrates. Ciona does have a relative to JAM/CTX (junctional adhesion molecule), discussed below. As will be presented, these are “virus” membrane receptors that resemble an Ig antigen receptor.70 The absence of RAG in the sea squirt is especially interesting, because it was found as a DNA transposase (parasites) in the echinoderm genome.83,84 Yet no invertebrate uses RAG genes as a component of an identity/immunity systems, let alone as an antiviral system. Thus this RAG-related element seems to have independently colonized the echinoderm and vertebrate genome, but not their respective ancestors. This clearly supports the concept that the basic elements of the adaptive immune system were acquired via complex genetic colonization from genetic parasites. Thus the Ciona genome and its immune system seems to represent the genetic lull before the storm of colonization that is associated with the evolution of the adaptive immune system. However, because we know very little concerning autonomous or endogenous viruses of jawless vertebrates, the likely candidates for such colonization are unknown.

201


Lamprey Cellular Identity and the Variable Lymphocyte Receptor System Lampreys and hagfish have evolved lymphocytes that can also recognize foreign cells.85,86 They have a unique system that rearranges DNA to express variable lymphocyte receptors (VLRs, leucine-rich surface binding proteins). To do this, a germline copy of the VLR DNA is subjected to somatic DNA rearrangement in the lymphocyte. The process occurs by insertion of a leucine-repeat region into an incomplete VLR gene copy. Then, DNA amplification along with recombination during cellular differentiation form an expressing lymphocyte cell population. The process clearly has much logical resemblance to CTL development in the adaptive immune system of jawed vertebrates. In addition, it uses a developmental mechanism that has been called a suspended apoptosis and can potentially express up to 1014 distinct VLRs. The evolutionary origin of this system, however, is mysterious because the needed components were not present in representative ancestral genomes, such as Ciona. Hagfish also express receptors on leukocytes that resemble TCR (V-domain) that are membrane glycoproteins. It seems possible that the characteristics of the VLR system could have originated following colonization by genetic parasites, especially DNA elements. For example, amplification of specific DNA regions, the site-specific invasive recombination and apoptosis are all features associated with DNA virus of invertebrates. However, none of these VLR components are similar to those adaptive immune genes in the jawed vertebrates (e.g., RAG1/2, MHC I, II, III). Thus, it appears lampreys underwent a distinct type of genetic colonization that resulted in their VLR system. They did not, however, undergo the major genomic DNA rearrangements of jawed vertebrates, although they were colonized by specific chromovirus (gypsy-like ERVs). Although no retroviruses or other viruses have yet been reported for lampreys they are known to support some herpesviruses.13

Sharks: A Basal Virus/Host Transition The nurse shark has been considered to represent the earliest vertebrate to have all the hallmark genes of adaptive immunity: RAG1/2, T-cell receptor, antigen processing, and presentation genes (TAP, LMP; combined MHC I/II).87–89 The absence of these subcomponents in ancestral genomes suggests that all these interacting components of adaptive immune systems were acquired together, essentially en toto. In addition, the emergence of the immune system is also associated with the development of calcified jaws in sharks and subsequent calcified bones in fish. As mentioned, calcification is a common defense response by invertebrate blood cells to wall off foreign cells. In bone, the internal marrow cells provide the progenitors for the entire adaptive immune system. Bone marrow thus provides the site of proliferation and differentiation of the hematopoetic system resulting in the differentiation of diverse leukocyte types. This development is via antigen regulated apoptosis in leukocyte selection and differentiation. How then did the simple ancestral phagocytic blood cells of oceanic animals acquire such complex, transformative genetic programming? This system does not seem to be more effective against pathogens. Nor is there evidence of a selective sweep by pathogens of jawless vertebrate species prior to the evolution of adaptive immunity. It has been proposed that the adaptive immune system represents a highly sophisticated self-identity system, whose initial selective pressure now seems obscure and only later did it evolve to oppose pathogens.82 Furthermore, there is a clear association between acquisition of adaptive immunity and major genome colonization, especially the large scale ERV colonization (e.g., gypsy-like chromoviruses). An expansion of RT dependent elements (LINES, SINES), large scale duplication of the genome, and a large scale elimination and/or inactivation of Tc1and other DNA elements also occurred. Such genomic changes mark major alterations mediated by viruses and genetic parasites. I

202

suggest that such extensive colonization provided major changes in the genetic identity of the host; shifting the virus-host relationships and establishing transformed blood cells with antiviral activity. Because this system was the product of virus colonization, the immune system itself became able to support many viruses, including autonomous retroviruses, herpesviruses, baculoviruses, parvoviruses, many +RNA viruses, bunyaviruses, and especially the negative strand rhabdoviruses.13 Accordingly, below I present a “virus-first” perspective on the origin and evolution of all the components of the adaptive immune system. The Chromovirus Colonization In the jawed vertebrates we see a large increase in chromoviral-derived ERVs. Although chromoviral (gypsy) related elements were found in sea urchins, starfish, and tunicates, including Ciona (i.e., the 100 c/c of well-conserved CsRn1), the copy numbers of these ancestral elements were low. Yet tunicate ERVs differ from those of these invertebrates in having been colonized by larger numbers of specific types of chromovirus. In addition, the type-specific chromoviral gag encodes a capsid gene (with a clearly conserved structural domain). The chromoviral gag also shows a clear relationship to MLV gag, hence it can be considered as a relative of the gammaretrovirus and related to the numerous elements found in mammalian genomes. Also, these chromoviral elements were not conserved in the genomes of mammals.90,91 Clearly there seems to have been a shift in chromoviral-host relationship near the time jawed vertebrates evolved. Historically, the large increase in chromoviral diversity as well as in related LTR elements observed in bony fish DNA has been dismissed as simply being the product of overactive selfish DNA that had little functional consequence. But the selfish DNA hypothesis does not explain why they expanded (and


were maintained) in fish,plants, and insects but were subsequently displaced from mammalian genomes. These were not trivial evolutionary events. Indeed, all fish species examined to date have 30 novel ERV families (defined via pol/RT sequence similarity).92 Mart and Jule elements are better known examples of the chromovirus found in fish.93 They are absent from mammals and birds, except for one conserved uncorrupted copy. The Gypsy elements as they are commonly (historically) known are LTR and RT containing relatives of chromoviruses but they typically lack gag similarity (hence are defective).94,95 There is a considerable difference in the size range of fish genomes. The pufferfish (Tetraodon nigroviridis) represents the most compact of fish genomes, and it has been suggested that it underwent an evolutionary loss of unneeded DNA.96 Yet, from an ERV perspective, pufferfish DNA closely resembles the genomes of other fish. The pufferfish has conserved most (although not all) of the fish ERV families.97 Pufferfish have 25 LTR containing retrotransposon elements that includes the five major groups. This level of ERV diversity is very large compared to other animals and is also not seen in mammals. Such conservation in a compact genome suggests some crucial conserved role. In addition, most of these fish ERV elements are transcribed, especially in reproductive tissue and thymus.98 What might be the selecting pressure that led to the colonization and maintenance of so many ERVs in fish genomes? Because virus colonization and persistence tends to provide the selective pressure to resist similar and other viral competition and to exclude exogenous versions of related retroviruses, this massive ERV colonization is proposed to have provided the basic elements to recognize and exclude other virus infected cells. Indeed, complex autonomous retroviruses of bony fish are well known (see below) but are not a characteristic virus that infects jawless fish or any oceanic invertebrate,13 although some invertebrate neoplasias might be due to retrovirus (see below).


Retrovirus and ERV Role in Evolution of Bony Fish Adaptive Immunity Let us now consider the possible role of viruses in the evolution of the immune system as seen in jawed fish. Previously, explanations that proposed a viral role in adaptive immunity simply suggested that the host coopted and used viral functions during the evolution of adaptive immunity (such as integrase).99 However, as I have previously asserted, successful virus colonization requires highly specific and often complex strategies, such as addiction modules. These colonizations have strong effects on group identity and immunity (especially against related genetic parasites) that underlie new host identity (immunity) systems. As these are also potentially transmissive agents, they can affect survival of host group as well. The zebrafish germ line, in contrast to that of mammals, is much more susceptible to infection and colonization by modified exogenous retroviruses. In mammals, germ cell lines suppress (methylate) DNA of new MLV-like retroviruses. In fish, such a retrovirus activity provides a currently used technology to construct transgenic fish using retroviral vectors.100 Here, the vesicular stomatitis virus (VSV) envelope protein has been used to coat MLV recombinant retrovirus for infecting reproductive cells. Such mixed virus can efficiently infect differentiating spermatogonia, establishing a sperm with multicopy integration of MLV. However, although fish genomes have high levels of LTR elements, natural germ line retrovirus infections do not appear to be very frequent as most fish LTR transposons are functionally silent. Because this MLV capsid (gag) is similar to chromoviral capsid found in fish, there seems to be no fundamental barriers to retrovirus integration into fish germ lines. An example in zebrafish is an 11.2 kb ERV with intact env and LTRs that also has gag, pol and env sequences.98 This ERV is related to salmon swimbladder sarcoma virus (SSSV), an autonomous retrovirus that causes bladder sarcomas in juvenile At-

203

lantic salmon. Curiously, this element is one of the very few fish ERVs that is also conserved in the human genome. Indeed, this is an MLV-like virus whose lineage is distantly related to the human endogenous retrovirus (HERV)-Ks that are abundantly found in the human genome.101 However, in striking contrast to the numerous human HERV-Ks, which lack any known source of autonomous virus, autonomous retrovirus related to this fish ERV do exist (see below). Besides SSSV, there is a second and prevalent and related autonomous retrovirus that infects chinook salmon (plasmacytoid leukemia retrovirus, discussed below). This second virus is especially a problem for pen-reared fish, but it is most interesting in that differentiating leukocytes (not derma) are the target of this virus infection and cellular transformation. Thus, unlike invertebrates, both lytic and endogenous versions of related retroviruses infect fish. The autonomous retroviruses of fish have some very interesting distinctions with those of mammals, especially regarding their regulatory complexity. Walleye dermal sarcoma virus (WDSV) is perhaps the best studied of these and is a 13 kd retrovirus that encodes several additional genes.102 It has a large gag (unrelated to MLV) with additional domains of unknown function as well as an unusual location for its env gene. One additional gene includes a viral cyclin D, which is involved in inducing proliferation of infected cells (neoplastic dermal and mononuclear inflammatory cells).103 WDSV also encodes Orf C, which targets mitochondria and affects apoptosis, associated with seasonal tumor regression.104 Infection often results in benign seasonal neoplasia (skin tumors) as has been seen in Lake Oneida, NY. These viral induced skin tumors appear to provide a tissue site for reemerging viral persistence. Thus, WDSV is prevalent in natural settings, specific to fish and induces complex regulated neoplasias associated with viral persistence. In terms of the evolution of WDSV, the viral cyclin gene are clearly unlike any host cyclin genes. The presence of such a

204

unique gene, clearly indicates that the virus did not “steal” a host proto-oncogene (cyclin) in order to induce cellular proliferation as is often believed when viruses have host-like genes.105 In addition, this cyclin is not only a unique viral version, it is also highly conserved in this viral lineage.102 Thus, WDSV presents an example of the first fully autonomous retrovirus known to infect oceanic animals. Previously, others had proposed that autonomous retroviruses likely evolved from chromosomal LINE-like retrotransposon elements, because these are found in early animal genomes.106 This was based on the existence of DIRS1 elements in genomes of various early eukaryotes, and they have distinct subregions with similarity to retroviral RT. It has been argued that such elements predate ERVs.101 However, we now know of no examples in which any autonomous retrovirus has such a LINEtype origin. All emergent retroviruses have been characterized to have emerged from endogenous or often recombinant autonomous viruses. In addition, as mentioned above, earlier examples of chromoviruses are found in essentially all eukaryotes, including several basal eukaryotic lineages that lack any LINE or DIRS1 elements.107 Indeed, as I have previously asserted, it appears more likely that LINE elements themselves may have evolved from a recombination between the RT of chromovirus and a clearly phage-like integrase domain. Thus, it is quite clear that WDSV (or chromoviruses) did not emerge from any LINES (or other non-LTR RT encoding elements) present in the ancestors to fish genome. Indeed, there is much more compelling evidence that autonomous retroviruses have undergone endogenization to become host genetic elements. In direct support of this idea, a close relative of WDSV appears to have undergone lineage specific endogenization in amphibians. Xen-1 is a complete copy endogenous retrovirus specific to African clawed toad (Xenopus laevis).108 This highly unusual and very large ERV has retained the additional complex genes characteristic of WDSV.


Another fish retrovirus is WEHV-1, walleye epidermal hyperplasia (skin growth associated), which also retains a more complex genome.108 This virus is also seasonal, observed during the spring spawning run. Almost all susceptible fish eventually get infected with it so it is also highly prevalent. Sea bass retrovirus is another fish retrovirus that affects blood cells.109 Additionaly, SSSV is a retrovirus that infects juvenile Atlantic salmon and poses a major commercial problem.110 Chinook salmon are also subjected to infection with a similar plasmacytoid leukemia retrovirus (especially penreared salmon). Thus, autonomous retroviruses are of considerable natural and commercial importance to fish populations and have big impacts on their host. However, although all these viruses may persist in individual hosts, they do not normally establish germ line infections. Yet WDSV is clearly related to both fish chromovirus or gypsy-ERVs via gag/pol/env, a similarity as seen in zebrafish.102 As mentioned, this noteworthy ERV is the only fish ERV that is known that has been conserved in the human genome101 is expressed in immune tissue98 and is linked with sex determination and Y chromosome evolution in fish.111 These are all hallmarks of possible involvement in fish identity systems. Origin of TCR, a Likely Viral Receptor Let us now turn to a most basic element of the adaptive immune system so that we might systematically consider possible viral roles. The T-cell receptor is a membrane-spanning, signal-transducing, surface glycoprotein that undergoes gene specific recombination, then somatic selection to evolve into an antigen specific binding molecule needed to initiate the adaptive nature of the immune response. The TCR is a member of a large family of Ig-like proteins that are composed of two chains from distinct loci, heavy and light.112,113 The light locus has multiple V, J, and C domains that


undergo site-specific recombination via the action of RAG to generate a combination of VJC light chains. The heavy chain undergoes similar domain recombination but has a D domain between the V and J domain to generate a VDJC heavy chain. The heavy chain is the more basal element of the Ig molecule and has been used to trace gene trees.87 It has been assigned into five groups (A through E). Of these, the E group is the most basal as it is found both in bony fish and cartilaginous fish (sharks, skates). Group D is found only in bony fish. Phylogenetic analysis suggests that the most basal members of all the TCR-related genes are the JAM/CTX/PVR related receptors as all have simplified Ig-like structure with only one V domain and one C domain, followed by transmembrane and cytoplasmic domains. This set of receptors has also been called the CTX-like family of viral receptors.70 Specific members include SLAM, CAR, PRV, JAM, and CD155. These receptors support many virus interactions and are often used as specific virus receptors. For example, JAM is a reovirus receptor, PVR is a poliovirus receptor. From this we can propose that the origin of the TCR was from viral sources. Variable Viral Receptors Originate from Viral Sources: The Bordetella Phage Exemplar As presented in my book, viruses are often the evolutionary source of host-expressed virus receptors, especially when such receptors show high diversity.1 Indeed, the retroviruses are known to have evolved the greatest diversity of env (receptor) genes of all the viruses of animals. In bacteria, such a viral receptor shift involves the familiar mechanism of phage mediated host conversion. In prokaryotes, various phage systems have also evolved phage specific molecular strategies that generate gene diversity of the expressed viral receptor, usually in association with tropism switching. The most relevant of these phage systems is found in a lysogenic phage of Bordetella,114 related to

205

T7 and P22. In an amazingly relevant process, this phage encodes a reverse transcriptase that introduces sequence variation to the receptor (variable region 1), generating diversity via the action of a viral encoded reverse transcriptase. This is a site-directed process of RTmediated mutagenesis and gene integration.115 In another amazingly relevant issue, the receptor protein for the phage (Mtd) has a C-type lectin fold that is able to tolerate tremendous amino acid sequence variation needed to generate diverse surface binding.116 Outside of the immunoglobulin fold, this is one of the very few protein folds that can tolerate such sequence diversity. Numerous other lysogenic phages of Bordetella that have elements of this variable receptor system have also been observed. However, in keeping with predictions of virus mediated addiction hypothesis, it was reported that lysogenic harboring Bordetella were significantly better able to resist competition and habitat invasion by similar Bordetella that did not harbor phage.117 With regard to the CTX TCR family of receptors (which are both antigen and virus receptors), there is another interesting implication regarding the consequences of viral binding. When JAM binds virus, it directly induces cellular apoptosis. Because apoptosis and the inhibition of apoptosis is a crucial feature of the TCR response to antigen and to initiate the adaptive immune response, this simpler viral response could represent an early version of an immune response. CTV is a basal viral receptor that has the V domain. However, the CTV receptor gene is also present in the genomes of protochordates, Branchiostoma and Ciona, but here it is unknown if it has any viral role. These organisms do show variable CTV expression. Given, however, that viruses of these organisms are so poorly characterized, these issues may be difficult to evaluate. It has been asserted, however, that along with the emergence of the TRC, the link to gene rearrangment and RAG, must also have occurred.118 In addition, the emergence of TRC also seems linked to MHC acquisition.113 Thus, most evidence supports

206


the idea that a complex and linked set of functions were simultaneously acquired from nonancestral sources. Viruses would be well suited for such a symbiogenic role. Origin of Lymphocyte Apoptosis: Invertebrate Metamorphosis and Shrimp Virus Although phagocytic blood lymphocytes are found in all invertebrate oceanic animals, none is known to be virus specific nor do they undergo clonal expansion or apoptosis during their development. If we examine shrimp regarding the issue of apoptosis, we see that in adult shrimp, apoptotic tissue is mostly associated with sex cell development (oocyte nurse cells, sperm). However, during larval development, apoptosis is prominent and associated with metamorphosis in that most of the preexisting larval cells will die by apopotosis.119 Shrimp apoptosis is distinct from that of simpler animals and uses additional genes similar to those in vertebrates, such as caspase-3, cytochrome c release, and bcl 2 proteins (all absent in C. elegans). With C. elegans, apoptosis was mainly observed during neuron development and was not a prominent aspect of their sex cell development. However, virus infection of shrimp is common in natural settings, and some infections can induce high-level apoptosis in lymphocytes. In particular, WSSV infection of larval (but not adult) shrimp induces pathology in shrimp lymphocytes by inducing a massive apoptotic response, which can be visualized by staining for the cleaved DNA.120 Note that such DNA fragmentation during apoptosis is similar to that seen in higher animals. Why would WSSV (a large DNA virus) induce lymphocyte apoptosis? Could this be of significance for the evolution of vertebrate lymphocytes? WSSV Exemplar WSSV is a well-characterized 300 kb dsDNA virus that has many distinctive genes

as well as a distinct ovoid virion resembling baculovirus.121 The genomes of natural isolates range from 293 to 312 kbp, due mainly to variation in one general polymorphic loci, corresponding to a 54 bp repeat element and two polymorphic ORFs (ORFs 14/15, 23/24). This satellite-like 54 bp repeat pattern can even be “pond” specific as seen in freshwater WSSV isolates.122 Curiously, the most pathogenic version of WSSV is also the one with the smallest genome.123,124 Furthermore, phylogenetic analysis support the idea that the larger genome is ancestral to the smaller, more pathogenic genome. WSSV has a wide host range and can be isolated throughout the world. WSSV was initially discovered due to large population crashes that it caused in young animals in commercial shrimp farms.125–127 However, it can often be isolated from adult host (shrimp) as asymptomatic persistent infections. WSSV can also infect 20 species of marine crabs, but some species are resistant to disease. WSSV replication is tightly linked to development and larval stage of its host. Larval development of Penaeus monodon, a major WSSV host, is among the most complicated of all crustaceans. However, early developmental stages nauplius, protozoa, and mysis larvae show no viral disease even when WSSV is clearly present. WSSV disease is mostly seen in late postlarvae and in juveniles; pathology is fast (3–7 days) and complete (100% mortality). In such acute WSSV infections, high levels of hemocyte apoptosis results in large (10fold) decreases in cell numbers.120 And ROS (oxidative stress) is also strongly induced in infected hemolymph.128 These reactive apoptotic lymphocytes can be visualized via caspase-3 increase or DNA fragmentation assays.129,130 Yet, curiously, infected lymphocytes are not virus-producing cells. Thus this pathology is not resulting from virus replication but from viral induced self-destruction of host phagocytes. Because these affected cells are neither making virus nor providing a clear antiviral defensive function, neither the ROS nor the apoptosis appears to be protective against WSSV.130 Curiously, similar to C. elegans, shrimp also have an


effective antiviral response mediated by small interfering RNA (siRNA) that is also effective against WSSV. There is a sharp transition between invertebrate and vertebrate hemocyte biology with respect to virus. But why would WSSV induce such a profound, damaging, and developmentally specific apoptosis in larval hemocytes that are not even replicating virus? I suggest such damaging reactions are consistent with a toxic reaction of a possible viral T/A addiction module. Because WSSV is prevalent, stable, and persistent in specific host, it is clear the hemocytes of such colonized host are not susceptible to viral induced apoptosis (a virus-mediated A state). Hosts that were not virus colonized (or other species), however, are strongly susceptible to a virus induced and highly pathogenic lymphocyte-killing response (a T state). In such hosts, WSSV appears to specifically trigger a self-destructive lymphocyte killing, which is not relevant to its own viral replication. Similar pathology is seen in other marine crustacean species, including various marine and freshwater crabs, copepods, and prawns, so this process is not unique to shrimp species. Those specific species that are refractive to all WSSV disease (including some marine crabs) are likely to have coevolved with WSSV, therefore are specific, persistent, and asymptomatic WSSV hosts.131 Because WSSV codes for 3 LATs (latency associated transcripts), first described in herpesvirus-ganglion persistence, these latency genes must control any destructive viral response. In the herpesvirus, similar LATs are anti-apoptotic genes (also similar to antiapoptotic Epstein–Barr virus encoded nonpolyadenylated RNAs of Epstein–Barr virus). WSSV also encodes anti-apoptotic ORFs.132 This clearly suggests WSSV has a persistence mechanism that controls apoptosis133 and if this persistence is not established, such as during acute replication, hemocyte apoptosis and death will result. How might such virus observations relate to the origin of apoptosis in vertebrate lymphocytes? Because viral persistence is often

207

attained by the use of addiction modules that can oppose similar and other related viruses, the ability of invertebrate viruses to control and induce lymphocyte apoptosis could provide the general basis of an acquired host identity module. This is conceptually similar to an E. coli colony that is colonized by P1 virus that can be induced to self-destruct when other viruses infect the same host or if the host is cured the persistent P1 (such as during sexual reproduction). Here, P1 functions as a host acquired identity module. Because such destructive lymphocytes are virus controlled, this development would also create the potential for lymphocytes to further evolve into a viral derived host antiviral identity system. But for this process to become part of the germ line, such an exogenous gene system would need to integrate into and function within the host chromosome. Unlike prokaryotes (and some brown algae), there are no known DNA viruses for any animal species that integrate as a normal part of its life strategy. Nor are any large DNA viruses known to code for integrases. Although algal DNA viruses (i.e., PBCV-1) do have a diverse set of homing integrases and show some relation to baculoviruses and herpesviruses, all known animal DNA viruses lack such genes. Yet viral integration is exceedingly common in vertebrate animals, including their germ lines, but such integration is mediated by retroviruses, not DNA viruses. Thus we might consider if a large DNA could have acted in concert with retroviruses to originate the adaptive immune system? Indeed, it has been noted for some years that herpesviruses and retroviruses appear to have broadly affected each other’s evolution.134–136 Various large DNA viruses of invertebrates and vertebrates indeed have encoded entire retroviruses as pathogenic loci for prevalent infections (discussed below). Even more relevant, many of the basal elements of the adaptive immune system appear to have retroviral-like origins. In addition, the adaptive immune system is the most highly dynamic genetic locus and its evolution toward greater complexity has been

208

mediated directly by the action of retroviruses as will be presented below. Retroviral Induced Clonal Lymphocyte Proliferation and Cellular Destruction Antigen (or virus) specific expansion of lymphocytes is a basic feature of the adaptive immune system, absent from invertebrate lymphocytes. How did this cellular transformation evolve? Both large DNA viruses and retroviruses are well known for their ability to transform cells and induce clonal cellular proliferation, seen in both invertebrate and jawed vertebrate lymphocytes. Bivalve hemocytes are normally nonproliferative phagocytic cells that wall off invaders (with calcium deposits) but are prone to tumors (leukemias).137 Herpesvirus is also noted for its ability to induce nervous tumors in bivalves. Although autonomous invertebrate retroviruses appear to be rare (essentially undescribed) in aquaculture, an important exception to this is known. A transmissible leukemia-like disease with massive mortality of bivalves (disseminated neoplasia of soft shell clams) has been observed.33 Here, an uncharacterized retrovirus that produces many particles is associated with these tumors.138,139 The disease involves heavy hamocyte infiltration (inflammation) with fiber bounded foci of cells and local tissue destruction. These infiltrates also show cytoplasmic paracrystalline arrays of viral-like particles as well as a highly modified endoplasmic reticulum structure, lined with layer of single virus particles containing RT activity. Thus, both proliferation and cellular destruction appear to be viral induced in these normally nonproliferative invertebrate blood cells. Could a similar retrovirus infection lead to the evolution of proliferating antiviral immune cells? Some invertebrates clearly harbor endogenous retroviruses that can be developmentally induced to be apoptotic and self-destructive.140,141 Thus a germ line retrovirus colonized blood cell could


provide the gene set needed for the origin of proliferating adaptive lymphocytes with antiviral activity. Such germ line persistence would likely require a virus-based addiction of T/A module (most likely involving apoptosis). This module would be normally silenced during viral persistence but would also induce protective self-destruction of cells newly infected with other virus. Thus, such cells could also kill cells that are not addicted by the same virus, providing the basis of a leukocyte-based antiviral response. Because defective virus is a common way to attain persistence, the large-scale ERV and ERV defective colonization associated with origin of adaptive immunity could also beexplained. To identify foreign cells and express a cell-based toxicity against these uncolonized or foreign colonized host cells, the host only needs to maintain the same viralmediated killing process noted above for the bivalve retrovirus. However, what might still be needed for a full-fledged adaptive immune response would be a mechanism to better recognize adjacent addicted and nonaddicted cells (via surface antigen presentation), and to select for those receptors. In addition, some form of regulation of this induced proliferation (apoptosis) would be needed, allowing reversion to silent persistent states. Combined Role for Retrovirus/DNA Virus Autonomous retroviruses of fish are well established, diverse, and prevalent in natural settings, and they tend to induce cellular proliferation, often in skin. Also common is lymphocyte proliferation that can be induced by various large DNA viruses of fish. We know that retroviral-induced leukemias are common in birds, amphibians, placental, and marsupial mammals. Also, resistance to retroviralinduced leukemia is mostly associated with ongoing “endogenization” of the same autonomous virus (e.g., see mouse and koala retrovirus below). Thus, it is highly interesting to

209


note that when MLV transforms mouse lymphocytes prior to leukemia induction, it acts on and transforms the basal hemopoetic stem cell that is the precursor to all the cell types of the adaptive immune system.142,143 Furthermore, when MLV integrates into hemopeotic cells, it can trigger a nonmalignant clonal expansion of the cellular decedents by altering transcriptional regulation resulting from the insertion into regulatory regions.142,144–146 Thus, these retroviruses have the inherent capacity to reprogram lymphocyte proliferation and specificity. Both DNA and retroviruses of avian species (Marek’s disease virus [MDV], reticuloendothelial virus [REV], lymphoid leukosis virus [LLV]) show an especially strong general tendency to cause proliferative lymphoid and hematopoetic disease in domestic bird flocks136,147 Indeed, this has been one of the major and growing sources of losses in the poultry industry and REV has become a major problem in the recent period.148 Yet REV in wild bird populations is much less pathogenic and although infected birds can be viremic, they are normally asymptomatic. Infection of wild young birds leads to tolerance, with no antibody production. Thus REV can also alter immune education and does so by expressing v-rel, a highly efficient NFKB B family member and central regulator of the core immune response. Clearly, in the case of REV, there is compelling evidence that large DNA viruses and retroviruses can act in concert to affect viral success in their host. Various other large DNA viruses can also incorporate retroviruses into their genomes.149 With REV, there has been a recent and large expansion of epidemics in turkeys. Yet this is not due to REV acting alone, but rather REV disease is mediated by fowlpox virus (FPV). This pathogenic version of FPV has incorporated REV as a new locus into its DNA that encodes the entire retroviral genome. REV is thus a pathogenic locus in FPV. Nor is this DNA-retrovirus relationship unique to FPV. Turkey herpesvirus is also pathogenic to turkey flocks and also has a REV locus that contributes to its pathogenicity. In

herpes, the REV locus is a defective copy, and young bird infection is needed for pathogenicity to develop. It is thus curious that avians are distinct from placental mammals regarding the presence of ERVs and their relationship to exogenous viruses. Avian genomes have many fewer ERV and pseudogene copies then do mammals. Yet, ironically, avian genomes are also known for using pseudogene copies of Ig domains (products of RT) to generate antigen diversity in their B cells. However, the above examples make one strong point, large persisting DNA viruses can indeed act in concert with retroviruses to reprogram host immune cell function. Mouse Retrovirus and Clonal Transformation of Hematopoetic Stem Cells The ability of exogenous and endogenous retroviruses to induce leukemias in mammals has long been recognized and well studied in wild and lab populations of mice. In particular, the endogenous MLV of both lab and wild Mus populations can induce lymphomas, but patterns of disease induction and resistance are highly species specific.150,151 Both lab strains and wild Mus populations show highly restricted patterns of MLV-induced leukemias. And the restriction of MLV pathology is generally due to endogenous (often defective) versions of MLV, such as Mus spicilegus resistance to MLV-like ecotropic viruses, or Asian wild mouse (Mus castaneus) resists mouse gammaretrovirus via XPR1 locus (a surface receptor for P-MLV, and the interfering X-MLV env gene).152,153 As will be described below, retroviruses are also mediating the recent evolution of both mouse and primate MHC locus as well as broader evolutionary patterns.154 For example, the koala bear population in Australia is currently undergoing a large-scale population crash (resembling an extinction event) due to retroviral induced leukemia.155,156 Although most infected koalas succumb to viral induced pathology, some do survive. However, the

210

survivors are undergoing large-scale endogenization of this virus as it colonizes their genomes and appears to provide resistance to disease. It now seems clear that surviving populations of koalas will have become endogenized by this virus. Clearly, survivors will have modified their lymphocytes or immune system biology to control lethal leukemias. This virus, however, does not appear to originate from any marsupial species as it is due to an MLV-like gammaretrovirus that likely evolved from an endogenous mouse retrovirus (MDEV, M. dunni terricolor 157 ). However, env of this virus is distinct from those normally found in MLV. Instead it resembles the env as found in Xen1 (xenopus), a relative of fish WDSV that was described above. The ability of DNA and retroviruses to affect the fundamental biology of lymphocytes is thus clear, ancient, and ongoing. Both largescale apoptosis, clonal proliferation, and control of differentiation are well-established consequences of such virus infection. It is also clear that different viral lineages (DNA/RNA) can act in concert and impart combined selective capacity in natural settings. These viral agents can then define some of the basic characteristics that would have been necessary to initiate the origin of the adaptive immune system. As these components were absent from ancestral organisms, it seems much more likely that they were exogenously derived from a viral (or mixed viral) colonization event. This persistent colonization would have resulted in the acquisition of a complex system of cellular identity being acquired en toto, whose main selective pressure was to resist other related and competing viruses with destruction. What is still needed regarding the origin of adaptive immunity is to account for the origin of surface and antigen variability and specificity from a viral perspective. Rag1/2: A Mu Family Integrase The gene specific recombination of the Ig locus is an essential feature the adaptive immune


system needed to generate diverse (and anticipatory) binding specificity. This recombination is restricted to lymphocytes and is mediated by the two recombinase proteins, RAG1 and RAG2 that bind the DNA of Ig domains, and bring them together for recombination. The ensuing reaction is a conservative DNA strand transfer (cut and paste). Two mechanisms for such a transfer reaction are known in biology, one involving a covalently bound protein intermediate (i.e., cre, flp) and the other involves a direct transesterification (e.g., phage mu, HIV integrase). This latter reaction is called a Tn7 like transposase, of which RAG1 and 2 are members. RAG1/2 are of the TnsB transposase family that have conserved the DDE metal binding motif and have striking structural similarity to the the RNAse H fold of retroviral integrases.99 Although this similarity might suggest retroviral involvement in the origin of RAG, that RAG is IS4-like (“cut and paste” transposon) has been used to argue against possible retroviral involvement. The most basal and by far most active version of any cut-and-paste transposase is that of phage Mu (an IS4 family), because this virus depends on transposition for its DNA replication.158,159 Transposons can also be classified by complexity of their action, classified into simple proteins (i.e., Tn5, hermes) and complex proteins that are also site specific (Tn7, transib, IS4, RAG). Some exceptions, such as IS917, are distinct in being RCR transposons. For use in Ig gene variation, RAG must be highly specific to the Ig gene, thus it is unlike most transposons that lack such gene specificity. However, homing introns (transposons), are abundant and conserved in crucial viral genes, such as DNA polymerase, and can clearly have a gene specific interruption capacity. The RAG1/2 transposase also resembles the Tc1 family transposase (with DDE motif) in that they both have inverted repeats as target sites, a transposon that is common in invertebrates, but uncommon in vertebrates.99,160 One percent of fish and amphibian genomes are related to this Tc1, but all fish copies are inactive, although many are


transcribed. Because neither RAG1 or 2 is found in ancestors to the jawed vertebrates, it seems clear that the RAG system is of external origin. In addition, the two genes are always tightly clustered, lack introns, and have the LTR-like repeats (RSS, target of transposition) and are thus consistent with an exogenous origin. RAG has a 600 amino acid core region that contains the catalytic DDE motif. This domain is also similar to the Transib element found in fruitfly and hydra genomes, but the version found in fungi appear to be the phylogenetically most basal. The sea urchin genome also has Sp Rag 1L and Sp Rag 2L versions that are coexpressed during early development.83,161 The sea urchin ORF1 has 25–30% identity to RAG 1 in N-terminal core. However, there is no evidence that these sea urchin genes have any immune function so their purpose is unclear. A common view regarding the exogenous origin of RAG1/2 is that it entered the vertebrate genomes by lateral gene transfer as a DNA transposon from another lineage of eukaryotic cell and was then usurped by the host to evolve adaptive immunity. However, such a scenario is neither plausible (mechanistically) nor are the selective pressures for creating a gene specific transposon into the Ig gene system apparent. Because all DNA transposons are silent or defective in ancestral host vertebrate genomes and because transposons are not known to move between species, the transposon transfer itself is problematic. It is also commonly assumed that the ancestor to the Iglike receptor was a nonrearranging version of V gene to which the RAG recombination became somehow specificity associated. But here too, no such gene is known. A much more plausible scenario would involve viral agents (both DNA and retro) as the originators of adaptive immunity that acted in concert to encoded sitespecific recombination proteins for Ig receptors. In addition, as noted above, the numerous other interacting basal genes (RAG1, RAG2, TCR, MHC) must have entered the genome together because none is found in the ancestral organisms. Thus, numerous problems with

211

existing explanations for the origin of the RAGIg system arise if we do not invoke the role of persisting virus colonization. What viral candidates might have been present and able to colonize vertebrate genomes and supply a complex system able to generate receptor diversity en toto? The large DNA viruses of lower eukaryotes and of bacteria provide the most diverse and active source of site specific integrase genes.162 These include such genes as the DNA site-specific endonuclease of algal viruses (PBCV-1).45 Furthermore, phage frequently utilize gene-specific and sitespecific integration to preclude and interrupt related genes from other competing phage. Because, as mentioned above, some DNA phage are known to use reverse transcriptase to generate diverse versions of viral receptors, it seems clear that viruses have all the needed components to create the foundations of adaptive immunity and also use these systems to compete with other viruses. The Bordetella phage is a T7-like system that uses a viral-encoded reverse transcriptase to generate gene-specific surface receptor variability (in variable region 1), clearly reminiscent of Ig variation.114,163 Such receptor expression is an example of lysogenic host conversion by a prophage (integrating DNA virus). Other prophages, such as SF370.1 of streptococcus, can also encode superantigens (exotoxin C and antitoxins) as part of their lysogenic conversion and host addiction.164 Thus, viruses have established the capacity to generate somatic variation in specific surface proteins (viral receptors) and alter cell recognition. Such capacity involves a preselected set of highly specific, complex interacting genes, encoded by the genetic parasite, not the ancestral host. Thus the assumption that there once existed a nonrecombining version of V gene prior to vertebrate entry would be wrong if it is virally derived. The variable V gene and RAG recombination system would have entered together as part of a viral addiction/identity module. Terrestrial invertebrates (insects) also provide some interesting examples regarding the

212

role of DNA viruses and transposons in host evolution. Hymenoptera species are the most numerous of all insect species (estimated at 200,000). Many thousands of species are known to harbor persistent infection by various types of genomic and extragenomic DNA viruses. These viruses can manipulate host hormonal systems, developmental patterns, and immune systems (hemolymph) and can be transmitted by both sexual means (e.g., ascoviruses, distant iridovirus relatives) or integrated into host germ lines (e.g., polydnaviruses, distant ascovirus relatives). Indeed, one invertebrate baculovirus (granulovirus cydia pomonella) encodes a Tc1-like transposase element (TCP3.2) that closely resembles the V(D)J recombination system.165,166 Furthermore, this element, when present, is used to preclude and outcompete similar viruses that lack the element.167 Recall that Tc-1 like elements are prevalent in the genomes of C. elegans and jawed fish, but lost in tetrapods. Although many of these viruses can clearly have persistent life strategies, they use unknown mechanisms to attain biological stability. It is interesting however, that ascoviral genomes encode caspase, cathepsin, and other apoptosis-modifying genes, suggesting persistent strategies. Iridoviruses are common to both fish and amphibians but especially numerous in fish. In both hosts, viral-induced tissue proliferation is also common. Although some DNA viruses encode DNA and RNA transposons, it has long been puzzling that herpesvirus genes lack introns, in contrast to several ancestral DNA viruses (e.g., PBCV-1). Some have suggested an extensive role of retroviruses in herpesvirus evolution.135,148,168,169 Indeed herpesvirus transcription is otherwise very much like host transcription except for this lack of splicing.170 Thus, it seems quite plausible that mixed retrovirus/DNA virus colonization of host DNA may have occurred during herpesvirus evolution. If so, an RNAse H-like retroviral ancestor could explain the high structural similarity to RAG1/2, while still acting like a DNA cut-and-paste element in the context of a DNA


virus. Such a colonization would not only be able to supply the complex, sequence-specific RAG1/2 function linked to gene specific (Ig) receptor targets, but also other complex and preselected interacting genes and functions (i.e., apoptosis and inhibitors) that are not found in ancestral genomes. RAG as One of Several T/A Sets Persistence of viruses is asserted to need addiction modules or strategies that promote stability but also provide identity or immunity systems. Clearly, various aspects of the adaptive immune system have the characteristics of addiction modules. The RAG genes should be genotoxic if expressed. Normally, to be protected from the destructive toxic activity (T) the ongoing suppression of toxicity (A) is needed. Thus the endonuclease/recombinase activities of RAG 1 and 2 should inherently be highly genotoxic and mutations in either gene indeed cause severe disease.21 Normally, RAG1 is bound to RAG2 and is inactive in vitro when bound together. This bound state can be considered as an inactive T/A set and would provide protection against an otherwise inherently genotoxic endonuclease activity. The site specificity of binding of RAG 1 and 2 to RSS heptamer and nonamer repeat elements which flanks V, D, J. This can also be thought of a T/A system designed to interrupt similar (competing or related) genes (receptors), which clearly resembles both homing endonucleases and receptor diversity systems of bacterial viruses described above. Indeed, homing endonucleases and restriction endonucleases are both used by phage and parasitic plasmids as T of a T/A addiction module, see example.171 Similarly, the T-cell apoptosis can be considered as a general T/A system that is necessary for the development of immune recognition, but its inherently self-destructive function is held in check by the proper surface receptor signaling and inhibitors of apoptosis. Loss or gain of improper signals will induce self-destruction. Indeed, the

213


entire adaptive immune system, which must undergo education early in development in order to set self-identity and prevent self-destruction (acquire tolerance, avoid autoimmunity) is also behaving like one extensive addiction module. An exogenous viral origin for this system could explain why RAG was never present in common ancestors to jawed vertebrates. Its presence in echinoderm and other invertebrates, but absence from ancestral C. elegans, would indicate a related but separate genetic colonization event that occurred in sea urchins from a related infectious source. This colonization, however, does not provide adaptive immunity for sea urchins. MHC Locus The MHC contains TCR as well as a other rearrangeable Ig superfamily genes that also provides cellular identity. The human MHC locus is a gene rich loci (>200 genes) with a high density (40%) of immunity associated genes and is found in all jawed vertebrates.172,173 It also has within it the genes that are involved in antigen processing and presentation. Class I and II MHC genes are also found in all jawed vertebrates. Numerically, most genes are Ag presentation genes. Class I genes are responsible for proper folding, translocation and peptide processing, ER transport, and MHC assembly (e.g., TAP1/2, beta2 microglobulin). Class II binds and presents Ag but is not ER processed and therefore employs a distinct pathway. Only a few of these genes can be identified in the tunicate genome. The MHC I loci besides being gene dense also lacks introns, which suggesting an external origin. These I/II loci are separated from each other by about 700 kb which is called the class III region. This intragenic region is also very dense region and has about 60 genes. The particular makeup of these genes is less broadly conserved across vertebrate species, but they are very similar between mouse and human MHC. However, this more complex I/II/III organization as seen in mam-

mals is simplified in fish.87 In fish, the simpler conserved core region still encodes glycoproteins that deliver peptides to the cell surface and initiates signal transduction to promote cellular proliferation and differentiation. As a result, T cells are developed via MHC presentation that provide a system of self-identification or histocompatibility (tissue rejection). MHC Evolution The MHC loci is the most polymorphic loci in both fish and humans.16 It shows a complicated evolutionary history of rearrangements, duplication, insertions, and deletions which are often genus specific. In tetrapods, MCH locus is more highly clustered than that of fish and includes odor receptor (OR) genes, involved in offspring and mate identity, linked to MHC I genes and peptide (pheromone) presentation in olfactory neurons.174 In addition, in fish, many genes found in the mammalian MHC are unlinked (i.e., OR genes), although Ag presentation genes (IA) remain linked in fish.175 Because the perspective of this presentation is to consider viral roles in the origin of identity systems, before further considering the evolutionary history of the MHC locus, it is necessary to define the viral genomic parameters that are seen in vertebrate immune loci. As we will focus on mammals, especially primates, we need to understand the ERV situation in these vertebrates. Distinct Waves of ERV Colonization Leading to Mammals The ERVs of bony fish were mostly derived from the diverse chromovirus family (Gypsy-related)95,176 and such elements were also retained in amphibians.90 Mammalian ERVs are distinct, expanded MLVrelated ERVs, and SINES.177,178 But mammalian genomes have thousands more copies of less diverse endogenous retroviruses. Yet most

214


LTR-retrotransposons are nonfunctioning. These genomic patterns in mammals can be taken as evidence of large-scale colonization and displacement by new versions of persisting virus (mostly MLV-related ERVs) that displaced prior viral colonizers of the host genome (chromoviral, DNA transposon) assisted by the concerted action of ERV defectives and hyperparasites (alus, SINES, LINES). For such colonization to succeed, each wave of new virus must superimpose a new version of an addiction module or identity system that operates with or incapacitates prior identity systems. Such action will result in ever increasing overlays of self-identity systems in surviving hosts leading to ever more complex immune genes. If we now consider the transition from fish to tetrapods and eventually mammals, we see that there has been serial waves of distinct ERV and retroposon colonization as well as an incremental increase in the complexity of their immune system. MHC Evolution in Mammals is ERV Mediated Fish genomes have a distinctly simpler MHC loci. Sharks have a single chromosome locus MHC I of about 100 Kb. This can be compared to only the alpha block subregion of the human MHC 1, which is 298–310 kb by itself. The human MHC locus is thus much more complex then that of fish, consistent with the overall pattern of MHC evolution from simple to complex.87,88,172,175 The reasons for this increasing complexity are not obvious. Recall that in their water habitat, teleost fish are immersed in a highly virus-rich environment and diverse and pathogenic versions of fish virus are well established, ubiquitous, and they pose major problems for fish farms and wild populations. Given this, the retention of a relatively simple MHC locus in fish seems odd. It is often assumed that MHC complexity is associated with better immunity. Comparative genomics clearly establishes that the MHC re-

gion is the most rapidly evolving and diverse genetic loci in the entire vertebrate genome and it has become increasingly complex during evolution of vertebrates to mammals.174,179 Such studies also make clear that overall, MHC evolution occurs by a process of duplication of “frozen” gene blocks (alpha, beta), followed by their diversification, not by convergent evolution.180,181 Thus, it appears that these MHC I blocks are ancestral to MHC II.173 MHC III has a distinct evolutionary history as described below. The MHC I/II regions are also unusual in being densely colonized by ERVs and retroelements and these have directly contributed to MHC evolution.182–184 Phylogenetic analysis has made clear that in primates the basic unit of MHC gene block duplication includes an ERV (HERV-16 in the case of human genomes).185 Thus, it appears that the “preduplicated” amplicon has a PERB11 gene and an HERV-16.186 Clearly ERV colonization and mobilization is a most basic element of MHC evolution.182 The Link Between MHC and OR in Mammals: Identity, Immunity, and Pheromones In mammals and tetrapods, many OR genes also became associated with the MHC locus.187,188 Indeed in the mouse genome, one dense cluster of mouse OR genes are just 5 to the TCR genes. This cluster has 59 OR genes with open ORFs, as well as another 20% pseudogenes. In the same loci of human DNA, 25 OR genes are found with 50% pseudogenes. The OR genes, however, don’t seem to undergo somatic recombination as do the TCR genes. Furthermore, another distinct type but much smaller class of odor receptor, the vomeronasal receptor (VNO), is also linked to the MHC loci. Because the VNO receptor binds to pheromones and affects family and sexual behavior in all nonprimate tetrapods, and is used for the purpose of group identity (offspring, mate identification), its association


with MHC would be logical if we assume that the locus is under selection as a system of identity. If so, it is especially curious to note that in humans, all 5 the VNO ORFs within the human MHC I locus (and throughout the genome) have been converted to pseudogenes (via alu interruption).189 No other ORF family within the human MHC loci shows this feature, but it may be common to African primates.190 This link between the MHC locus, OR, and VNO receptors might therefore be understood if they are all components of an identification system initially invented by the jawed vertebrates. However, their loss during primate evolution suggests a significant shift in the evolution of primate identity systems. As primates no longer depend on pheromones or many odorants for family or sex identification, perhaps the emergence of their large social brain of primates is of some relevance to this. Possible Viral Sources of the Gene Dense MHC III Loci MHC III is the most gene dense loci in the human genome and is about 700 kb, 14% coding with 72% transcribed.191 The MHC III region is involved in various aspects of antigen processing, presentation, and regulation. Unlike MHC I and II loci, MHC III does not share an evolutionary history with these core genes. MHC III also has a much less genetic polymorphism relative to MHC I/II.192 Antigen presentation is a complex process of cell biology that involves various aspects of membrane associated assembly. Although some of the genes involved in processing can be found in vertebrate ancestors, most cannot. Thus many of these genes appear to be novel and vertebrate specific. Also, as mentioned, this gene dense loci also has limited RNA splicing. These features could suggest that most MHC III genes are also of a external (DNA viral) origin. Chemokines are essential small signaling proteins (8–12 kd) that bind specific receptors of various immune cells,193 and some can be found in this loci.

215

DNA viruses also encode many chemokines and manipulate essentially all other aspects of the MHC functions.194,195 Because some of these viral chemokine genes can show high similarity to their human gene counterparts, it is commonly thought that viruses acquire chemokine and receptor genes from hosts by a process of genetic piracy.196,197 Indeed, this has been the prevalent assumption by medical researchers regarding the origin of all such viral genes. However, I suggest that viruses are originating most such regulatory genes, and it is more likely they can sometimes provide them to the host via colonization. Viruses, especially large DNA viruses are well known for their diverse and often simplified immune regulatory genes. In addition, many of these genes are more phylogenetically basal and structurally simpler then their host gene counterparts. This is seen with cytomegalovirus (CMV)198,199 and poxvirus.200 CMV like most all human herpesviruses appear to evolve mostly by recombination with other herpesviruses.201 However, the herpesvirus lineage appears to have ancient bacterial phage ancestors.202,203 Thus, this viral lineage was present long before the origin of adaptive immunity and is found to infect many invertebrates. Interferon (IFN) is considered to represent one of the earliest cytokines used by both innate and adaptive immune response and is found in fish.72 However, tumor necrosis factor has a 10 stranded jelly role structure that appears to have originated from the PRD phage and adenovirus capsid structure.204 Many of the above viral immuno-regulatory genes are novel to the particular virus lineage and clearly have no host counterparts.205 One prominent example are viral versions of the IL-10 receptor, found in some herpesviruses, such as human CMV (HCMV).206 This viral version will affect immune cells without MHC presentation.207 Although the neurotropic alpha herpesviruses do not generally encode any such chemokines or their receptors, all the immunotropic beta herpesviruses (i.e., CMV) do encode such genes, and in the case of IL10, three distinct lineages can be observed.208 It

216

thus appears that viral versions of IL10 receptor have independently originated on at least three occasions. IL10 is generally thought to suppress immune responses (cellular infiltration). However, during HCMV latency, both suppressive (anti-apoptotic) and proapoptotic viral genes are expressed,209 an apparent T/A module. Other viral immuno-regulatory genes that have no similarity to host genes homologues include several broad spectrum CC chemokine receptors, such as US28 from HCMV210 and the CXC ORF74 chemokine (from HHSV8211 ). ORF74 is unusual and active via the MAP kinase pathway.201 The US28 is interesting in this regard for its novel ability to undergo constitutive endocytosis and recycling to the plasma membrane and also for activating several different signal transduction pathways. Some viral genes, such as EBV BILF1, are also constitutively active, requiring no ligand and have a simpler and more basal protein structure.212 Genes related to BILF1 are conserved in all gamma herpesviruses, indicating an ancient origin and a highly selected viral function.213 As BILF1 shows no similarity to any known cellular chemokine receptor, it appears to be a unique viral creation. In fact, EBV seems to control G protein-coupled receptor (GPCR) settings at many levels for the purpose of immune evasion during viral persistence, and also for the purpose of virus dissipation. Other viral genes, such as latently expressed vGPCR of Kaposi’s sarcoma-associated herpesvirus, also appear to be crucial for inhibition of cellular apoptosis.214 Other families of DNA viruses also express basal immune regulatory genes. In terms of TCR-like molecules, European fowlpox encodes both a V-type Ig domain protein as well a GPCR.215 Lumpy skin disease virus (LSDV) encodes a G-coupled CC chemokine receptor, not found in other poxviruses.216 Because phylogenetic analysis does not place any of these viral genes at the tips of host gene clades (such as CCR1), this also suggests that they are not derived from host genes but are all old viral creations. Vaccinia virus can also control immune response


at numerous points,217 including synthesizing immune regulatory steroid hormones (3betaHSD). We can recall the ability of WSSV to induce high-level apoptosis in hemocytes of shrimp, a blood cell that doesn’t normally proliferate nor replicate WSSV. Other DNA invertebrate viruses (TnBV1), an immunoregulatory polydnavirus of parasitoid wasps, also induce programmed apoptosis in host immune cells.218 Thus, persisting DNA viruses that infect invertebrates also have many regulatory genes, often active during persistence, that could provide the numerous basal functions needed for a simplified early MCH locus of the adaptive immune response. Because, like MuLV, various lineages of large DNA viruses (e.g., HCMV) appear to target hemopoetic stem cells for persistent infection (latency) and transformation,219,220 they have the needed mechanisms to reprogram cell fates. The usual result is that virus production becomes linked to immune cell activation,221 and this requires numerous virus regulatory genes. Because such transformation and regulation involves essentially all aspects of the immune response, these features could have originated from viral sources to provide host immune function rather then being derived from host genes. Thus, the MHC III region itself could then have originated by ERV-mediated DNA viral colonization and transformation of the host chromosome. It is worth noting that MHC III also encodes the C4 complement gene. This complement protein is used to create toxic pores that kill foreign cells. However, C4 expressing cells are protected from its toxicity by the RCA complex, essentially a C4 binding structure that plugs the hole. This presents a clear T/A module situation. C4 is interesting in that it represents one of the few genes within the adaptive immune locus that was also found in ancestral genomes (such as tunicates). It thus appears to represent an older element of cellular identity that predates adaptive immunity. However, the human C4 gene shows some interesting and recent genetic variation as a size polymorphism. Both

217


long and short versions of C4 gene exist in human populations due to difference in intron 9 length. This difference is the consequence of a recent HERV-K (C4) colonization.222 As discussed below, this specific HERV also appears able to express an antisense RNA in that LTR has alu in reverse orientation, suggesting complex regulatory control has resulted from this recent HERV insertion. The ERV Colonization Pathway to Primates HERV-K genomic colonization is especially associated with the evolution of African primates. However, this is but the most recent wave of ancient ERV colonization that we can describe in vertebrate genomes. Earlier, we mentioned vertebrate ERV colonization of the tetrapods associated with the displacement and loss of ancestral chromovirus ERVs (Ty-1 related), elements prevalent in fish. Later, we see evidence of a large scale ERV-L colonization (foamy related virus), as elements characteristic of the genomes of all placental species. However, HERV-L elements were initially present at low copy number but underwent a large expansion in primate and some rodent (Mus) lineages. For example, Old World simians expanded from 10–30 c/c of HERV-L to about 200 c/c.177 With primate placentals, we can also see that HERV-FRD and HERVW both colonized these genomes and contribute directly, as membrane env fusion genes, to host placental biology.223,224 HERV-FRD is present in all simians. HERV W is found in all catarrhines (Old World apes), including env genes.225,226 The HERV-W env (Syncytin 1) is a single complete open reading frame on chromosome 7 and is mainly trophoblast expressed (as a paternal gene).227 HERV-W predated New-Old World split but was not conserved (became inactive) in New World primates, presumably due to relaxed selective pressure. Thus, many HERVs were fixed into the genomes of Old World monkeys immediately after separa-

tion from New World primates (about 35 million ybp).228–230 The burst of HERV colonization that is also related to SINE and alu retrotransposition and amplification characterizes in Old World primates.228,231–233 Curiously, much of this genetic ERV material appears to have maintained for unknown reasons in hominids as testis-specific transcribed RNA.234,235 On a broad scale, we can note that there are relatively few genes (ORFs) that can be used to distinguish human from chimpanzee genomes. However, in spite of this, comparison between their sequenced genomes indicate that they have about 400,000 indel differences. Indels are insertion/deletion events that have mostly occurred into noncoding regions and account for the majority of differences between these genomes.236,237 The bulk of indels correspond to LTR element differences that average about 300 bp in length.238 Clearly, such differences are mostly mediated by the reverse transcriptase characteristic of retroviruses, and the Y chromosomes are especially affected by such events as presented below. Morphologically, modern humans are only about 150,000 years old. Yet even within this short time span, we can see evidence of recent human specific HERV activity affecting the immune locus as presented below.239 The Great Human HERV Colonization As mentioned above, the most dramatic ERV colonization and expansion in the Old World great ape genomes involved various HERVK elements (mouse mammary tumor virus [MMTV]-related human ERVs).240,241 Some intact copies of HERV-K (including env) are found only in human, chimpanzee, and gorilla genomes,101,242 although these often differ in a species-specific way. In addition, these HERVK elements conserve a functional integrase gene, consistent with selection for some activity. Within the great apes, human evolution in particular shows specific colonization by HERV-K

218

family members as well as an associated expansion or replacement of LINE and alu elements. SINE elements can be used to closely mark recent great ape and human evolution (such as SINE-R.C2, found in their C2 complement component of the MHC III locus228 ). This element is absent from gibbons (lesser apes). As discussed below, this element also traces the recent evolution of MHC genes (and identity) in the hominids. And as also discussed below, primates have specifically acquired MLV-resistant genes (such as APOBEC3)243 that seems coincident with HERV-K colonization/expansion and resistance to foamy viruses.244 In total, humans have 30–50 HERV families, all of which appear to have resulted from independent HERV colonizations. This was truly a great primate-specific viral invasion. In addition, various ERV colonizations appear to have been roughly coincident with each other, such as HERV-I, ERV-L, and HERV-K. Yet all represent distinct retroviral lineages that cannot result from a common ancestor. HERV-Ks also appear to evolve from distinct exogenous viruses. HERV-Ks are present in 10 distinct families. Of all the primate ERVs, only HERVK has continued to be active into recent human evolution. The HERV-K primate lineage is also distinct from other ERVs in that they also contain and conserve a dUTPase gene,245 an enzyme that can promote cytoplasmic retrovirus DNA synthesis. With one possible exception, phylogenetic analysis suggests that all HERVK families may have initially encoded a dUTPase gene. Within the human HERV-K clade, K11l (HML8) appears to be the most phylogenetically basal member.241 One well-studied HERV-K group is the HERV-K(10), which is considered also as the parent of several other HERV-Ks. HERV-K(10) has six HML groups (all with dUTPase). All HERV-K groups seem to be monophyletic, including the older HML5 (via dUTPase analysis).246 However, they are not monophyletic via LTR sequence analysis. Clearly mixed (recombinant) lineages apply even to specific HERV-K families. Although the HML5 group seems older than other clades, it


does not appear to be ancestral to the more recent HML2 group. Together, these observations support the concept of independent (exogenous) ERV colonizations, such as by HML5 and HML2 ERVs. Yet, in spite of all the ERV evolutionary activity in primates, human ERVs don’t show genetic polymorphism at integration sites to any significant extent. Although integration sites don’t vary much, there is, however, some population variation in the presence of specific HERVs. Possibly the most evolutionary recent HERVs to enter the human genome are the full length HERV-K113 and HERV-K115.247 HERV-K113 is found in only 20% of the human population (distributed African, Asian, Polynesian) but almost absent from people from the United Kingdom.248 HERV-K115 is found only in 15% of the human population mostly in sub-Saharan Africa. It has been estimated that HERV-K113 entered the human genome about 100,000–200,000 ybp, near the time modern humans and Neanderthals emerged. Did Rodents Provide HERV-K? If they were absent from ancestral primate genomes and are not active endogenous viruses of primates, what then was the source of all these African primate-specific HERVs? Based on phylogenetic analysis, an argument can be made that that various rodent-like endogenous and exogenous viruses (within the broader MMTV clade) were ancestral to primate HERV-Ks. The HERV-K clade is monophyletic, but within this dendogram, MMTV is the only exogeneous virus but is also the basal member of the clade. This is consistent with the idea that an MMTV-like rodent virus originally provided the exogenous virus to form new endogeneous states in primate species. The implication is that hominids and their immune systems underwent sweeps of lymphoproliferative retroviral disease that led to new states of primate-virus persistence via ERV endogenization. Such retroposon colonization and


activation significantly added to immune system regulatory complexity and increasingly complex virus-host identity. Indeed, an endogenous retrovirus of hamsters, with a distant sequence relationship to interacisternal A particle of mice, were used to clone the original HERVK of humans.249–251 Such a scenario is very much like that currently happening to koala bears in Australia due to rodent derived retroviruses as described below. Recent Evolution of Primate MCH via Retrovirus The MHC locus can be thought to function as an identity system, originally directed against virus-infected self, with a main aim to generate T cells active against infection. Accordingly, various researchers have suggested that selective sweeps by viruses and virushost coevolution was especially involved in the recent evolution of primate MHC.252,253 Mutations within the MHC locus are also often associated with autoimmune disease.254 The human MHC locus is the most polymorphic of all human gene regions and differs in several aspects from that of other primates, including chimpanzee.181,255 Distinct MHC differences are also apparent more broadly, between New and Old World primates.256 Like other vertebrates, most of the human MHC changes have also been mediated by the action of ERVs, LINES, and alu elements. Clearly, retroviral activity within the MHC locus has been extensive and ongoing. In keeping to the pattern with other vertebrates, the overall evolution of the MHC locus in the primates is also from simple to complex, with humans being the most complex.257 As noted, MHC I appears to be ancestral to MHC II, but MHC II evolution is also mainly driven by ERVs and retroposon activity.258 MHC I genes provides the best trace of human MHC evolution. Chimpanzee MHC I differs from human in that it has a large deletion (95 kb within beta block). This deletion has lost HERV-L and HERV-16,184,185 elements which

219

are thought to be important for the fact that chimps don’t develop AIDS from HIV-1 infection. Of special interest in this is that HERV16 here seems to be in an antisense orientation and should express and anti RT pol RNA, suggesting complex transcriptional regulation can result from retroposon activity. MHC III seems to represent a latter addition during evolution, along with origin of placentals, but has remained as most gene dense and less polymorphic. However, the human MHC III, although less polymorphic, does show C4 gene variation that can also differentiate humans from chimpanzees.259 This change is due to the HERV-K (C4) insertion as noted above.222 Chimpanzees and gorillas both have a short C4 locus. Overall, the human MHC l locus is organized into three regions, (class I,II, III) plus some extended (x) regions.260 Humans have 19 HLA class II genes (11 coding 8 pseudogenes, which are often reverse oriented). In the class II locus, human and chimps also differ with human class II being more complex. The most basal MHC I locus of human has about 18 HLA class I genes (6 coding 12 pseudogenes) containing three recognizable gene blocks: beta, kappa, and alpha. Within HLA1, humans have six coding genes (-A, -B, -C, -E, -F, and -G) and 12 pseudogenes (found in the above three distinct blocks). The human MHC1 alpha block is between 289–310 kb and has 10 duplicated MHC I genes and these genes are shared in primates. The genes within the beta block are less shared with primates. The C block is found only in gorilla, chimp, and human and not in other Old World primates. In contrast, Asian rhesus macaque has a markedly different MHC I with an additional 20 genes in the alpha block and 17 in the beta block. Curiously, wild Indian Ocean macaques (island isolated) have a surprisingly simple MHC haplotype makeup but are susceptible to SIV infection suggesting some relationship of retroviruses to MHC complexity.261,262 As mentioned, the human MHC I locus is surprisingly dense with ERVs.184 Human MHC I alone has 16 HERVs (mostly HERV-16,

220

but also a basal HERV-L). Many of the HERV16s are found in both the alpha and beta block. However, in the HLA-b or HLA-c regions, few if any HERV-16s are found. Instead HERVI is present here (which colonized Old World primates at the New/Old World split). Thus HLA-b/c seems to represent an older but distinct wave of ERV colonization associated with the earlier evolution of this locus. The MHC alpha and beta block can be considered to have evolved via the action of a duplicon.263,264 The alpha block has 10 genes and 3 gene fragments that appear to have been grouped together during this duplication.186 Each duplicon also has a PERB II (MHCI) region along with an HERV 16 (ERV9).185 ERV9 appears to be a basal element of the duplicon and also surrounds the HLA-A locus. Alu elements (and LINES) are also active in MHC locus evolution and have been used to classify duplicons and infer ancestral relationships.263 Although the MHC is also variable and dense with LINES, SINES, and alu elements, some blocks seem frozen in evolution. Yet even within these frozen regions, ERV16 is most often found.185 Interestingly, even though the MHC locus is dense with ERVs, most of these ERVs are interrupted by alu inserts and only HERV-K91 (in HLA-C) has conserved its pol and protease ORFs. This HERV is also the most recent insertion into the class I locus. Because the biggest difference between the chimpanzee and human MHC I locus also involves both ERVK9 and L1 (human genome has an ERVK9 insertion), it is clear ERVs continue to affect evolution of immunity. In contrast to primates, in the mouse MHC locus, MuERV-L is found at the position of the primate ERVK9 but has also conserved gag, pol, and dUTPase, unlike the human locus. Besides being interrupted by alus, many MHC ERVs (such as HERV-L) have themselves interrupted LINE-1 elements via insertion, such as L1MD1 and the primate-specific L1PA2. Thus, it is striking that all LINES in MHC-I are fragmented by the action of LTRs and alu elements.265 Human MHC also encodes the multicopy gene family of P5 that is specifically


transcribed in immune cells. As P5 is related to HERV-L and HERV-16, and because P5 is transcribed into an mRNA that is RT pol antisense, it seems clear that retroviruses (ERVs) and retroposons (alus/LINES) have been most responsible for shaping the regulation and evolution of the MHC locus, including the human locus.266,263 Thus, it appears clear that most MHC genes have evolved by the action of RT and duplication, but that the specific ERV involved is species specific. MHC Changes: Pseudogenes and OR Genes In terms of consequences to host genes, LINES (especially L1) and RT encoding ERVS are thought to generate the majority of pseudogenes. Mammalian genomes (human) have about 15,000 pseudogenes. But neither humans nor rodents use pseudogenes to generate diversity of the adaptive immune system and it appears clear that the RAG Ig-based recombination system used by fish, humans, and rodents evolved prior to the pseudogene Ig system of birds and some mammals. Clearly, ERVs and retroposons were much involved in the origin and more recent evolution of this immune system element in avians and farm animals but the selective reasons favoring such a major molecular transition remain obscure. In terms of pseudogenes within the MHC locus, humans (and hominids) differ significantly from the other nonprimate mammals. All the human VNO receptors genes within the MHC locus have become pseudogenes.189,190,267,268 In addition, many of the OR receptor genes within the human MHC locus have also become pseudogenes. The human OR cluster on 17p13.3 has a high proportion of pseudogenes (60%), in which interruption by LINE elements predominate. Human MHC locus has 34 OR genes with 14 coding and 20 pseudogene (all LINE interrupted). In contrast the mouse xMHC I region has 59 OR genes of which only 20% are pseudogenes. Within the


human class I loci, OR genes are in a single extended cluster (17 OR sequences; 10 intact, 6 pseudogenes, with 1 dimorphic pseudogene, intact in some individuals). In this same cluster, chimpanzees have 15 OR genes (as do other apes), including 4 pseudogenes conserved in apes (two related to human).181 The reasons for the presence of OR or VNO genes within the MHC locus has been rationalized as being associated with mate selection via MHC peptide orders. Thus, in the mouse (and most terrestrial tetrapods) the MHC locus conserves the VNO and OR gene cluster and these genes are used for social identification (via peptide pheromone binding).269,270 Indeed these mouse MHC I OR genes were co-duplicated with the evolution of mouse MHC I genes.187 But the mate selection theory is problematic for numerous reasons, such as the use for offspring identification by mouse dams.271 Instead, the OR-MHC linkage seems associated with and selected for its function as an identity system. However, its relative loss in the human genome suggests that humans (and African primates) underwent a significant ERV mediated shift in their identity-immunity systems relative to other mammals (i.e., decreased olfaction dependence for this purpose). But this too seems to have resulted from genetic colonization.

221

tolerance functions.273 MMTV, which can also be an endogenous virus is a rodent virus, expresses sag, which binds to MHC II molecules and strongly affects central tolerance via the action of its genes on immune cells in that viral superantigens are able to induce B cell differentiation in an antigen independent way.274 This function may be associated with viral persistence.275 MMTV stimulates Vbeta7CD4 T cells such that 103 to 105 more T cells are induced compared to conventional antigens. Such stimulation is via selective binding of viral antigen to Vbeta chains, thus they are not dependent on their antigen specificity. Such an induction process seems to represent a more basal control of T-cell development but also seems essential for the control of the normal life cycle of some B cells. In the human genome, HERV-K18 also encodes a superantigen with a broad capacity to stimulate the immune system.276 HERV-K18 also stimulates Vbeta7 and Vbeta13 T cells and is constitutively expressed in thymus, resulting in central tolerance. This HERV-K18 superantigen and tolerance is also associated with the establishment of EBV persistence.277,278 Thus, both HERV-K18 and MMTV can stimulate T cells via receptors without antigen. Indeed, such a less specific activity for T-cell stimulation and growth control might better represent an early event in the evolution of T-cell antigen specificity.

Tolerance and Viral Superantigens The education of the immune system is essential to set self-identity and prevent autoimmunity by the adaptive immune system.272 This education occurs via the development of tolerance which involves negative selection (via apoptosis) and MHC mediated selfpresentation of lymphocytes early in the development of thymus of the adaptive immunity. However, here too, there is evidence that retroviruses may have contributed. The study of central tolerance was very much assisted by the study of retroviruses, especially the MMTV envelope superantigen, which aided in working out much of our understanding of how central

Recent Human and Marsupial Evolution and Retroviruses It seems quite clear that recent human evolution has been much affected by retroviruses. Indeed, humans differ in several aspects regarding their relationship to primate retroviruses. For example, African monkeys all harbor species specific apathogenic foamy viruses (related to ERV-L), as well as SIV.279,280 But these are both absent from humans, expect for occasional nontransmitting zoonosis from primate to human281 and the recent emergence of HIV from recombinant SIV sources. Indeed,

222

it appears that humans have undergone some recent adaptations that affect their general ability to support retroviruses. Most notable is the acquisition of and expansion of APOBEC3C related genes, which have clear antifoamy virus activity.244 However, mostly, these new human antiretroviral genes are especially active against MLV-like (HERV-K-related) viruses. Such antiviral expansion strongly supports the idea that humans did indeed undergo a selective sweep mediated by HERV-K-related and possibly other retroviruses.7,282 A related sweep by retroviruses may be currently ongoing in other species. As mentioned, there is currently an epidemic of lethal lymphomas sweeping both captive and wild populations of koala bears in Australia that threatens this population.283,284 This natural epidemic is caused by an MLV-related virus (GALV-like), similar to and possibly derived from an endogenous viruses of Asian rodents mentioned above.155,156 However, it is apparent that some koala bears will be likely survive this epidemic, but it is already clear that such survivors have been massively colonized by various genetic variants of the same virus.154,285 And it is clear that the immune cells of these survivors will also be modified, most likely by ERVs and their defectives to resist leukemic transformation. The surviving koalas will be able to host this virus without succumbing to disease. Most likely, various immune alterations will be needed that allow virus persistence. However, this will generate a new genetic variant of doala bear whose immune cells are now resistant to this same virus. But this new variant will also likely pose a serious infectious risk to those koala populations that have not yet been exposed to the virus. Indeed, an isolated koala population in Kangaroo Island has remained free of this virus and would be threatened if exposed to persistently infected koalas. I suggest that these surviving koalas will have acquired a new viralmediated group identity that distinguishes them from ancestral species provides them a distinct survival advantage (via viral addiction)


relative to their ancestral species. These koalas will also need to adjust the regulation of their adaptive immune system to insure that this new endogenous virus does not initiate a destructive immune response. This virus will have become self. Thus the koala immune system will be under strong selection by this virus to acquire additional complexity. A similar scenario would likely explain why and how retroviruses were involved in the divergence of human and chimpanzee genome and MHC locus. Conclusions: A Summary of Evidence and Arguments Although the idea that viruses may have contributed to the evolution of the adaptive immune system has previously been suggested, in all those proposals the role of viruses was simply to provide a vehicle for the lateral transfer of some functions (genes) that were assumed to have originated in other cellular organisms. Viruses in these proposals were simply being usurped to transport useful elements to the host. In this article, however, I examine the role of viruses in adaptive immunity from a very different perspective, as originators of new systems of identity and as originators of most of the components of adaptive immunity itself. Based on previously published arguments and evidence, I have asserted that stable viral persistence is a major life strategy of viruses (and related genetic parasites) which provides stability on an evolutionary time scale. Stable persistence is often attained by viral-mediated strategies involving addiction modules, and in some cases, the lethal action of the virus itself can be the toxic element of such an addiction module. Here, virus persistence itself provides protection against lethal virus infection. From this foundation, I have examined all the elements of the adaptive immune system to consider evidence that relates to possible viral origins. We have thus considered the origin of white blood cells that recognize and kill viruses infected cells, the origin of the T-cell receptor, the origin of the rearrangable RAG system,


the origin of cellular apoptosis, the origin of proliferating and differentiating lymphocytes, and the origin and evolution of the MHC locus, including antigen presentation and modification genes all from a viral perspective. In all cases, clear evidence can be found to support a plausible viral role early in immune evolution. In many cases, viruses indeed provide the best evidence for originators of the needed genes or functions (i.e., TCR, RAG). From the very origins of adaptive immunity in jawed fish to the most recent differences between human and chimpanzee immune systems, we can also see strong evidence as viral footprints having been left on their genomes. Such evidence, taken as a whole, provides a strong argument for a most counterintuitive perspective. Immunity (identity) was likely to have been invented by genetic parasites. Thus, when we examine ongoing epidemic and pandemic events from this perspective, we can see the initial steps in an evolutionary process that can result in greater host identity and immune complexity. The HIV pandemic currently in sub-Saharan Africa, for example, is lowering human life expectancy in those countries that lack resources. If left unchecked by modern culture, cooperation, and medicine, this pandemic would be expected to select for human nonprogressors (as already seen in some sex workers) similar to the koala epidemic. Clearly, survivors of such viral sweeps would also need to modify the regulation of their adaptive immune system as well. It can no longer be denied that viruses were part of our human ancestors. It seems clear that these ancestral viruses participated directly in the evolution of our adaptive immunity and provide new sources of self. Conflicts of Interest

The author declares no conflicts of interest. References 1. Villarreal, L.P. 2005. Viruses and the Evolution of Life. ASM Press. Washington, DC.

223 2. Lehnherr, H. et al. 1993. Plasmid addiction genes of bacteriophage P1: doc, which causes cell death on curing of prophage, and phd, which prevents host death when prophage is retained. J. Mol. Biol. 233: 414–428. 3. Lehnherr, H. & M.B. Yarmolinsky. 1995. Addiction protein Phd of plasmid prophage P1 is a substrate of the ClpXP serine protease of Escherichia coli. Proc. Natl. Acad. Sci. USA 92: 3274–3277. 4. Yarmolinsky, M.B. 2004. Bacteriophage P1 in retrospect and in prospect. J. Bacteriol. 186: 7025– 7028. 5. Yarmolinsky, M.B. 1995. Programmed cell death in bacterial populations. Science 267: 836–837. 6. Villarreal, L.P. 2007. Virus-host symbiosis mediated by persistence. Symbiosis 43: 1–10. 7. Ryan, F.P. 2007. Viruses as symbionts. Symbiosis 44: 11–21. 8. Pancer, Z. & M.D. Cooper. 2006. The evolution of adaptive immunity. Annu. Rev. Immunol. 24: 497– 518. 9. Cooper, M.D. & M.N. Alder. 2006. The evolution of adaptive immune systems. Cell 124: 815–822. 10. Flint, S.J. 2000. Principles of Virology: Molecular Biology, Pathogenesis, and Control. ASM Press. Washington, DC. 11. Muroga, K. 2001. Viral and bacterial diseases of marine fish and shellfish in Japanese hatcheries. Aquaculture 202: 23–44. 12. Betts, A.M. et al. 2003. Emerging vesiculo-type virus infections of freshwater fishes in Europe. Dis. Aquat. Organ. 57: 201–212. 13. Essbauer, S. & W. Ahne. 2001. Viruses of lower vertebrates. J. Vet. Med. B 48: 403–475. 14. Hoffmann, B. et al. 2005. Fish rhabdoviruses: Molecular epidemiology and evolution. World Rhabdoviruses 292: 81–117. 15. Lumsden, J.S. et al. 2007. Mortality event in freshwater drum Aplodinotus grunniens from Lake Ontario, Canada, associated with viral haemorrhagic septicemia virus, type IV. Dis. Aquat. Organ. 76: 99–111. 16. Hughes, A.L. & M. Yeager. 1997. Molecular evolution of the vertebrate immune system. Bioessays 19: 777–786. 17. Alder, M.N. et al. 2005. Diversity and function of adaptive immune receptors in a jawless vertebrate. Science 310: 1970–1973. 18. Shintani, S. et al. 2000. Do lampreys have lymphocytes? The Spi evidence. Proc. Natl. Acad. Sci. USA 97: 7417–7422. 19. Kurstak, E. 1991. Viruses of Invertebrates. M. Dekker. New York. 20. Loker, E.S. et al. 2004. Invertebrate immune systems—not homogeneous, not simple, not well understood. Immun. Rev. 198: 10–24.

224 21. Camargo, M.M. & L.A. Nahum. 2005. Adapting to a changing world: RAG genomics and evolution. Hum. Genomics 2: 132–137. 22. Barstead, R. 2001. Genome-wide RNAi. Curr. Opin. Chem. Biol. 5: 63–66. 23. Bagasra, O. & K.R. Prilliman. 2004. RNA interference: The molecular immune system. J. Mol. Histol. 35: 545–553. 24. Vastenhouw, N.L. & R.H. Plasterk. 2004. RNAi protects the Caenorhabditis elegans germline against transposition. Trends Genet. 20: 314–319. 25. Murakami, M. et al. 2005. Inhibitory effect of RNAi on Japanese encephalitis virus replication in vitro and in vivo. Microbiol. Immunol. 49: 1047–1056. 26. Schott, D.H. et al. 2005. An antiviral role for the RNA interference machinery in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 102: 18420–18424. 27. Wilkins, C. et al. 2005. RNA interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature 436: 1044–1047. 28. Nelstrop, A.E., G. Taylor & P. Collard. 1968. Studies on phagocytosis. 3. Antigen clearance studies in invertebrates and poikilothermic vertebrates. Immunology 14: 347–356. 29. Tripp, M.R. 1970. Defense mechanisms of mollusks. J. Reticuloendothel. Soc. 7: 173–182. 30. Hildemann, W.H. & A.L. Reddy. 1973. Phylogeny of immune responsiveness: Marine invertebrates. Fed. Proc. 32: 2188–2194. 31. Robalino, J. et al. 2007. Double-stranded RNA and antiviral immunity in marine shrimp: Inducible host mechanisms and evidence for the evolution of viral counter-responses. Dev. Comp. Immunol. 31: 539–547. 32. Ward, M.E., J.D. Shields & C.L. Van Dover. 2004. Parasitism in species of Bathymodiolus (Bivalvia: Mytilidae) mussels from deep-sea seep and hydrothermal vents. Dis. Aquat. Organ. 62: 1–16. 33. Elston, R. 1997. Bivalve mollusc viruses. World J. Microbiol. Biotechnol. 13: 393–403. 34. Renault, T. & B. Novoa. 2004. Viruses infecting bivalve molluscs. Aquat. Living Resour. 17: 397–409. 35. Vigneron, V. et al. 2004. Detection of ostreid herpesvirus 1 (OsHV-1) DNA in seawater by PCR: Influence of water parameters in bioassays. Dis. Aquat. Organ. 62: 35–44. 36. Davison, A.J. et al. 2005. A novel class of herpesvirus with bivalve hosts. J. Gen. Virol. 86: 41–53. 37. Renault, T., C. Lipart & I. Arzul. 2001. A herpeslike virus infects a non-ostreid bivalve species: Virus replication in Ruditapes philippinarum larvae. Dis. Aquat. Organ. 45: 1–7. 38. Burge, C.A., F.J. Griffin & C.S. Friedman. 2006. Mortality and herpesvirus infections of the Pacific oyster Crassostrea gigas in Tomales Bay, California, USA. Dis. Aquat. Organ. 72: 31–43.

Annals of the New York Academy of Sciences 39. Arzul, I., T. Renault & C. Lipart. 2001. Experimental herpes-like viral infections in marine bivalves: Demonstration of interspecies transmission. Dis. Aquat. Organ. 46: 1–6. 40. Lipart, C. & T. Renault. 2002. Herpes-like virus detection in infected Crassostrea gigas spat using DIGlabelled probes. J. Virol. Methods 101: 1–10. 41. Barbosa-Solomieu, V. et al. 2005. Ostreid herpesvirus 1 (OsHV-1) detection among three successive generations of Pacific oysters (Crassostrea gigas). Virus Res. 107: 47–56. 42. Batista, F.M. et al. 2007. Detection of ostreid herpesvirus 1 DNA by PCR in bivalve molluscs: A critical review. J. Virol. Meth. 139: 1–11. 43. Van Etten, J.L. 2003. Unusual life style of giant chlorella viruses. Annu. Rev. Genet. 37: 153–195. 44. Kang, M. et al. 2004. Genetic diversity in chlorella viruses flanking kcv, a gene that encodes a potassium ion channel protein. Virology 326: 150–159. 45. Yamada, T., H. Onimatsu & J.L. Van Etten. 2006. Chlorella viruses. Adv. Virus Res. 66: 293–336. 46. Mehmel, M. et al. 2003. Possible function for virus encoded K+ channel Kcv in the replication of chlorella virus PBCV-1. FEBS Lett. 552: 7–11. 47. Neupartl, M. et al. 2008. Chlorella viruses evoke a rapid release of K +from host cells during the early phase of infection. Virology 372: 340–348. 48. Muroga, K., Y. Inui & T. Matsusato. 1999. Workshop “Emerging diseases of cultured marine molluscs in Japan.” Fish Pathol. 34: 219–220. 49. Hooper, C., P. Hardy-Smith & J. Handlinger. 2007. Ganglioneuritis causing high mortalities in farmed Australian abalone (Haliotis laevigata and Haliotis rubra). Aust. Vet. J. 85: 188–193. 50. Tan, J. et al. 2008. Purification of a herpes-like virus from abalone (Haliotis spp.) with ganglioneuritis and detection by transmission electron microscopy. J. Virol. Methods 149: 338–341. 51. Harada, T. et al. 1993. Tumors in nervous tissues of abalones, Nordotis discus. J. Invertebr. Pathol. 62: 257– 261. 52. Volff, J.N., U. Hornung & M. Schartl. 2001. Fish retroposons related to the Penelope element of Drosophila virilis define a new group of retrotransposable elements. Mol. Genet. Genomics 265: 711–720. 53. Leaver, M.J. 2001. A family of Tc1-like transposons from the genomes of fishes and frogs: Evidence for horizontal transmission. Gene 271: 203–214. 54. Abrusan, G. & H.J. Krambeck. 2006. Competition may determine the diversity of transposable elements. Theor. Popul. Biol. 70: 364–375. 55. Marin, I. et al. 1998. Evolutionary relationships among the members of an ancient class of non-LTR retrotransposons found in the nematode Caenorhabditis elegans. Mol. Biol. Evol. 15: 1390–1402.

225

Villarreal: Viral Origin of Adaptive Immunity 56. Bowen, N.J. & J.F. McDonald. 1999. Genomic analysis of Caenorhabditis elegans reveals ancient families of retroviral-like elements. Genome Res. 9: 924–935. 57. Ganko, E.W., K.T. Fielman & J.F. McDonald. 2001. Evolutionary history of Cer elements and their impact on the C. elegans genome. Genome Res. 11: 2066– 2074. 58. Xu, Z. et al. 1999. Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome. Anim. Genet. 30: 150–156. 59. You, E.M. et al. 2008. Microsatellite and mitochondrial haplotype diversity reveals population differentiation in the tiger shrimp (Penaeus monodon) in the Indo-Pacific region. Anim. Genet. 39: 267–277. 60. Williams, S.T. & N. Knowlton. 2001. Mitochondrial pseudogenes are pervasive and often insidious in the snapping shrimp genus Alpheus. Mol. Biol. Evol. 18: 1484–1493. 61. Maningas, M.B. et al. 2008. Essential function of transglutaminase and clotting protein in shrimp immunity. Mol. Immunol. 45: 1269–1275. 62. de Luca di Roseto, G., G. Bucciarelli & G. Bernardi. 2002. An analysis of the genome of Ciona intestinalis. Gene 295: 311–316. 63. Dehal, P. et al. 2002. The draft genome of Ciona intestinalis: Insights into chordate and vertebrate origins. Science 298: 2157–2167. 64. Holland, L.Z. & J.J. Gibson-Brown. 2003. The Ciona intestinalis genome: When the constraints are off. Bioessays 25: 529–532. 65. Permanyer, J., R. Gonzalez-Duarte & R. Albalat. 2003. The non-LTR retrotransposons in Ciona intestinalis: New insights into the evolution of chordate genomes. Genome Biol. 4: R73. 66. Britten, R.J. et al. 1995. Gypsy/Ty3-class retrotransposons integrated in the DNA of herring, tunicate, and echinoderms. J. Mol. Evol. 40: 13–24. 67. Volff, J.N. et al. 2004. Retroelement dynamics and a novel type of chordate retrovirus-like element in the miniature genome of the tunicate Oikopleura dioica. Mol. Biol. Evol. 21: 2022–2033. 68. de Boer, J.G. et al. 2007. Bursts and horizontal evolution of DNA transposons in the speciation of pseudotetraploid salmonids. BMC Genomics 8: 422. 69. Du Pasquier, L., I. Zucchetti & R. De Santis. 2004. Immunoglobulin superfamily receptors in protochordates: Before RAG time. Immunol. Rev. 198: 233–248. 70. Du Pasquier, L. 2004. Speculations on the origin of the vertebrate immune system. Immunol. Lett. 92: 3–9. 71. Khalturin, K. et al. 2004. Recognition strategies in the innate immune system of ancestral chordates. Mol. Immunol. 41: 1077–1087. 72. Magor, B.G. & K.E. Magor. 2001. Evolution of ef-

73.

74.

75. 76. 77.

78.

79.

80.

81.

82. 83.

84.

85.

86.

87.

88.

89.

fectors and receptors of innate immunity. Dev. Comp. Immunol. 25: 651–682. Marino, R. et al. 2002. Complement in urochordates: Cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis. Immunogenetics 53: 1055–1064. Nonaka, M. & F. Yoshizaki. 2004. Primitive complement system of invertebrates. Immunol. Rev. 198: 203–215. Klein, J. 2006. The grapes of incompatibility. Dev. Cell. 10: 2–4. Litman, G.W. 2006. How Botryllus chooses to fuse. Immunity 25: 13–15. De Tomaso, A.W. & I.L. Weissman. 2003. Initial characterization of a protochordate histocompatibility locus. Immunogenetics 55: 480–490. De Tomaso, A.W. et al. 2005. Isolation and characterization of a protochordate histocompatibility locus. Nature 438: 454–459. Khalturin, K. et al. 2003. Urochordates and the origin of natural killer cells: Identification of a CD94/NKR-P1-related receptor in blood cells of Botryllus. Proc. Natl. Acad. Sci. USA 100: 622–627. Ballarin, L., F. Cima & A. Sabbadin. 1998. Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri. Dev. Comp. Immunol. 22: 479– 492. Ballarin, L. et al. 2002. Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 133: 411–418. Rinkevich, B. 2004. Primitive immune systems: Are your ways my ways? Immunol. Rev. 198: 25–35. Fugmann, S.D. et al. 2006. An ancient evolutionary origin of the Rag1/2 gene locus. Proc. Natl. Acad. Sci. USA 103: 3728–3733. Hibino, T. et al. 2006. The immune gene repertoire encoded in the purple sea urchin genome. Dev. Biol. 300: 349–365. Pancer, Z. et al. 2005. Variable lymphocyte receptors in hagfish. Proc. Natl. Acad. Sci. USA 102: 9224– 9229. Rogozin, I.B. et al. 2007. Evolution and diversification of lamprey antigen receptors: Evidence for involvement of an AID-APOBEC family cytosine deaminase. Nat. Immunol. 8(6): 647–656. Marchalonis, J.J. et al. 1998. Antibodies of sharks: Revolution and evolution. Immunol. Rev. 166: 103– 122. Hashimoto, K. et al. 1999. Conservation and diversification of MHC class I and its related molecules in vertebrates. Immunol. Rev. 167: 81–100. Flajnik, M.F. et al. 1999. Insight into the primordial MHC from studies in ectothermic vertebrates. Immunol. Rev. 167: 59–67.

226 90. Miller, K. et al. 1999. Identification of multiple Gypsy LTR-retrotransposon lineages in vertebrate genomes. J. Mol. Evol. 49: 358–366. 91. Volff, J.N. 2005. Genome evolution and biodiversity in teleost fish. Heredity 94: 280–294. 92. Herniou, E. et al. 1998. Retroviral diversity and distribution in vertebrates. J. Virol. 72: 5955–5966. 93. Volff, J.N. et al. 2001. Jule from the fish Xiphophorus is the first complete vertebrate Ty3/Gypsy retrotransposon from the Mag family. Mol. Biol. Evol. 18: 101–111. 94. Gorinsek, B., F. Gubensek & D. Kordis. 2004. Evolutionary genomics of chromoviruses in eukaryotes. Mol. Biol. Evol. 21: 781–798. 95. Gorinsek, B., F. Gubensek & D. Kordis. 2005. Phylogenomic analysis of chromoviruses. Cytogenet Genome Res. 110: 543–552. 96. Jaillon, O. et al. 2004. Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature 431: 946– 957. 97. Poulter, R. & M. Butler. 1998. A retrotransposon family from the pufferfish (fugu) Fugu rubripes. Gene 215: 241–249. 98. Shen, C.H. & L.A. Steiner. 2004. Genome structure and thymic expression of an endogenous retrovirus in zebrafish. J. Virol. 78: 899–911. 99. Dreyfus, D.H., J.F. Jones & E.W. Gelfand. 1999. Asymmetric DDE (D35E)-like sequences in the RAG proteins: Implications for V(D)J recombination and retroviral pathogenesis. Med. Hypotheses 52: 545–549. 100. Kurita, K., S.M. Burgess & N. Sakai. 2004. Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm. Proc. Natl. Acad. Sci. USA 101: 1263–1267. 101. Villesen, P. et al. 2004. Identification of endogenous retroviral reading frames in the human genome. Retrovirology 1: 32. 102. Zhang, Z. et al. 1996. Phylogenetic and epidemiologic analysis of the walleye dermal sarcoma virus. Virology 225: 406–412. 103. Poulet, F.M. et al. 1995. In-situ hybridization and immunohistochemical study of walleye dermal sarcoma-virus (Wdsv) nucleic-acids and proteins in spontaneous sarcomas of adult walleyes (Stizostedion vitreum). Vet. Pathol. 32: 162–172. 104. Nudson, W.A. et al. 2003. Walleye dermal sarcoma virus Orf C is targeted to the mitochondria. J. Gen. Virol. 84: 375–381. 105. Monier, A., J.M. Claverie & H. Ogata. 2007. Horizontal gene transfer and nucleotide compositional anomaly in large DNA viruses. BMC Genomics 8: 456. 106. Xiong, Y. & T.H. Eickbush. 1990. Origin and evolu-


107.

108.

109.

110.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

tion of retroelements based upon their reverse transcriptase sequences. EMBO J. 9: 3353–3362. Khan, H. et al. 2007. Retrotransposons and tandem repeat sequences in the nuclear genomes of cryptomonad algae. J. Mol. Evol. 64: 223–236. Kambol, R., P. Kabat & M. Tristem. 2003. Complete nucleotide sequence of an endogenous retrovirus from the amphibian, Xenopus laevis. Virology 311: 1–6. Pinto, R.M. et al. 1995. Retroviral properties inherent to viral erythrocytic infection in sea bass. Arch. Virol. 140: 721–735. Paul, T.A. et al. 2006. Identification and characterization of an exogenous retrovirus from atlantic salmon swim bladder sarcomas. J. Virol. 80: 2941– 2948. Volff, J.N. & M. Scharlt. 2002. Sex determination and sex chromosome evolution in the medaka, Oryzias latipes, and the platyfish, Xiphophorus maculatus. Cyt. Gen. Res. 99: 170–177. Marchalonis, J.J., S.F. Schluter & A.B. Edmundson. 1997. The T-cell receptor as immunoglobulin: Paradigm regained. Proc. Soc. Exp. Biol. Med. 216: 303–318. Kurosawa, Y. & K. Hashimoto. 1997. How did the primordial T cell receptor and MHC molecules function initially? Immunol. Cell Biol. 75: 193– 196. Liu, M. et al. 2002. Reverse transcriptase-mediated tropism switching in Bordetella bacteriophage. Science 295: 2091–2094. Doulatov, S. et al. 2004. Tropism switching in Bordetella bacteriophage defines a family of diversitygenerating retroelements. Nature 431: 476–481. McMahon, S.A. et al. 2005. The C-type lectin fold as an evolutionary solution for massive sequence variation. Nat. Struct. Mol. Biol. 12: 886–892. Joo, J. et al. 2006. Bacteriophage-mediated competition in Bordetella bacteria. Proc. Bio. Sci. 273: 1843– 1848. Galaktionov, V.G. 2004. [Evolutionary development of the immunoglobulins super family]. Izv. Akad. Nauk. Ser. Bio 1133–1145. Jiravanichpaisal, P. et al. 2007. Expression of immune-related genes in larval stages of the giant tiger shrimp, Penaeus monodon. Fish Shellfish Immunol. 23: 815–824. Hameed, A.S.S. et al. 2006. Quantitative assessment of apoptotic hemocytes in white spot syndrome virus (WSSV)-infected penaeid shrimp, Penaeus monodon and Penaeus indicus, by flow cytometric analysis. Aquaculture 256: 111–120. van Hulten, M.C. et al. 2001. The white spot syndrome virus DNA genome sequence. Virology 286: 7–22.

Villarreal: Viral Origin of Adaptive Immunity 122. Wongteerasupaya, C. et al. 2003. High variation in repetitive DNA fragment length for white spot syndrome virus (WSSV) isolates in Thailand. Dis. Aquat. Organ. 54: 253–257. 123. Lan, Y., W. Lu & X. Xu. 2002. Genomic instability of prawn white spot bacilliform virus (WSBV) and its association to virus virulence. Virus Res. 90: 269– 274. 124. Marks, H. et al. 2005. Fitness and virulence of an ancestral white spot syndrome virus isolate from shrimp. Virus Res. 110: 9–20. 125. Lo, C.F. & G.H. Kou. 1998. Virus-associated white spot syndrome of shrimp in Taiwan: A review. Fish Pathol. 33: 365–371. 126. Chen, L.L. et al. 2000. Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata. Dis. Aquat. Org. 40: 157–161. 127. Sanchez-Martinez, J.G., G. Aguirre-Guzman & H. Mejia-Ruiz. 2007. White spot syndrome virus in cultured shrimp: A review. Aquac. Res. 38: 1339– 1354. 128. Rameshthangam, P. & P. Ramasamy. 2006. Antioxidant and membrane bound enzymes activity in WSSV-infected Penaeus monodon fabricius. Aquaculture 254: 32–39. 129. van de Braak, C.B. et al. 2002. Preliminary study on haemocyte response to white spot syndrome virus infection in black tiger shrimp Penaeus monodon. Dis. Aquat. Organ. 51: 149–155. 130. Wu, J.L. & K. Muroga. 2004. Apoptosis does not play an important role in the resistance of ‘immune’ Penaeus japonicus against white spot syndrome virus. J. Fish Dis. 27: 15–21. 131. Wu, Y. et al. 2007. Inhibition of white spot syndrome virus in Litopenaeus vannamei shrimp by sequencespecific siRNA. Aquaculture 271: 21–30. 132. Wang, Z. et al. 2004. ORF390 of white spot syndrome virus genome is identified as a novel antiapoptosis gene. Biochem. Biophys. Res. Commun. 325: 899–907. 133. Khadijah, S. et al. 2003. Identification of white spot syndrome virus latency-related genes in specificpathogen-free shrimps by use of a microarray. J. Virol. 77: 10162–10167. 134. Kung, H.J. & C. Wood. 1994. Interactions between Retroviruses and Herpesviruses. World Scientific. Singapore; River Edge, NJ. 135. Brunovskis, P. & H.J. Kung. 1995. Retrotransposition and herpesvirus evolution. Virus Genes 11: 259– 270. 136. Witter, R.L. 1997. Avian tumor viruses: Persistent and evolving pathogens. Acta Vet. Hung. 45: 251–266. 137. Cooper, E.L. 1976. Immunity and neoplasia in mollusks. Isr. J. Med. Sci. 12: 479–494.

227 138. Romalde, J.L. et al. 2007. Evidence of retroviral etiology for disseminated neoplasia in cockles (Cerastoderma edule). J. Invertebr. Pathol. 94: 95–101. 139. House, M.L., C.H. Kim & P.W. Reno. 1998. Soft shell clams Mya arenaria with disseminated neoplasia demonstrate reverse transcriptase activity. Dis. Aquat. Organ. 34: 187–192. 140. Mondy, W.L. & S.K. Pierce. 2003. Apoptotic-like morphology is associated with annual synchronized death in kleptoplastic sea slugs (Elysia chlorotica). Invertebr. Biol. 122: 126–137. 141. Pierce, S.K. et al. 1999. Annual viral expression in a sea slug population: Life cycle control and symbiotic chloroplast maintenance. Biol. Bull. 197: 1–6. 142. Chung, S.W. et al. 1991. Leukemia initiated by hemopoietic stem cells expressing the v-abl oncogene. Proc. Natl. Acad. Sci. USA 88: 1585–1589. 143. Tumas-Brundage, K.M. et al. 1996. Murine leukemia virus infects early bone marrow progenitors in immunocompetent mice. Virology 224: 573– 575. 144. Mager, D.L. & A. Bernstein. 1985. Induction of clonogenic and erythroleukemic cells by different helper virus pseudotypes of Friend spleen focusforming virus. Virology 141: 337–341. 145. Green, P.L., D.A. Kaehler & R. Risser. 1987. Clonal dominance and progression in Abelson murine leukemia virus lymphomagenesis. J. Virol. 61: 2192– 2197. 146. Cho, S.K. et al. 1999. Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc. Natl. Acad. Sci USA 96: 9797–9802. 147. Davidson, I. & R.F. Silva. 2008. Creation of diversity in the animal virus world by inter-species and intra-species recombinations: Lessons learned from poultry viruses. Virus Genes 36: 1–9. 148. Davidson, I. & R. Borenstein. 1999. Multiple infection of chickens and turkeys with avian oncogenic viruses: Prevalence and molecular analysis. Acta Virol. 43: 136–142. 149. Hertig, C. et al. 1997. Field and vaccine strains of fowlpox virus carry integrated sequences from the avian retrovirus, reticuloendotheliosis virus. Virology 235: 367–376. 150. Hiai, H. 1996. Genetic predisposition to lymphomas in mice. Pathol. Int. 46: 707–718. 151. Levy, J.A. 1978. Xenotropic type C viruses. Curr. Top. Microbiol. Immunol. 79: 111–213. 152. Wu, T., Y. Yan & C.A. Kozak. 2005. Rmcf2, a xenotropic provirus in the Asian mouse species Mus castaneus, blocks infection by polytropic mouse gammaretroviruses. J. Virol. 79: 9677–9684. 153. Yan, Y., R.C. Knoper & C.A. Kozak. 2007. Wild mouse variants of envelope genes of xenotropic/ polytropic mouse gammaretroviruses and their

228

154. 155.

156. 157.

158. 159.

160.

161.

162. 163.

164.

165.

166.

167.

168.

169.

Annals of the New York Academy of Sciences XPR1 receptors elucidate receptor determinants of virus entry. J. Virol. 81: 10550–10557. Denner, J. 2007. Transspecies transmissions of retroviruses: New cases. Virology 369: 229–233. Tarlinton, R.E., J. Meers & P.R. Young. 2006. Retroviral invasion of the koala genome. Nature 442: 79– 81. Stoye, J.P. 2006. Koala retrovirus: A genome invasion in real time. Genome Biol. 7: 241. Bonham, L., G. Wolgamot & A.D. Miller. 1997. Molecular cloning of Mus dunni endogenous virus: An unusual retrovirus in a new murine viral interference group with a wide host range. J. Virol. 71: 4663–4670. Mit’kina, L.N. 2003. [Transposition as a way of existence: Phage Mu]. Genetika 39: 637–656. Harshey, R.M. & M. Jayaram. 2006. The Mu transpososome through a topological lens. Crit. Rev. Biochem. Mol. Biol. 41: 387–405. Kapitonov, V.V. & J. Jurka. 2005. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons. PLoS. Biol. 3: e181. Hemmrich, G., D.J. Miller & T.C.G. Bosch. 2007. The evolution of immunity: A low-life perspective. Trends Immun. 28: 449–454. Nolan, J.M. et al. 2006. Genetic diversity among five T4-like bacteriophages. Virol. J. 3: 30. Liu, M. et al. 2004. Genomic and genetic analysis of Bordetella bacteriophages encoding reverse transcriptase-mediated tropism-switching cassettes. J. Bacteriol. 186: 1503–1517. Desiere, F. et al. 2001. Comparative genomics reveals close genetic relationships between phages from dairy bacteria and pathogenic Streptococci: Evolutionary implications for prophage-host interactions. Virology 288: 325–341. Jehle, J.A. et al. 1997. Identification and sequence analysis of the integration site of transposon TCp3.2 in the genome of Cydia pomonella granulovirus. Virus Res. 50: 151–157. Arends, H.M. & J.A. Jehle. 2002. Homologous recombination between the inverted terminal repeats of defective transposon TCp3.2 causes an inversion in the genome of Cydia pomonella granulovirus. J. Gen. Virol. 83: 1573–1578. Kundu, J.K. et al. 2003. Polymerase chain reaction assay for Cydia pomonella granulovirus detection in Cydia pomonella population. Acta Virol. 47: 153–157. Isfort, R. et al. 1992. Retrovirus insertion into herpesvirus in vitro and in vivo. Proc. Natl. Acad. Sci. USA 89: 991–995. Isfort, R.J. et al. 1994. Integration of multiple chicken retroviruses into multiple chicken herpesviruses: Herpesviral gD as a common target of integration. Virology 203: 125–133.

170. Wagner, E.K. 1984. Regulation of HSV transcription. J. Invest. Dermatol. 83: 48s–52s. 171. Sano, Y. & M. Kageyama. 1993. A novel transposon-like structure carries the genes for pyocin AP41, a Pseudomonas aeruginosa bacteriocin with a DNase domain homology to E2 group colicins. Mol. Gen. Genet. 237: 161–170. 172. Flajnik, M.F. & M. Kasahara. 2001. Comparative genomics of the MHC: Glimpses into the evolution of the adaptive immune system. Immunity 15: 351– 362. 173. Danchin, E. et al. 2004. The major histocompatibility complex origin. Immunol. Rev. 198: 216–232. 174. Ohta, Y. et al. 2006. Ancestral organization of the MHC revealed in the amphibian Xenopus. J. Immunol. 176: 3674–3685. 175. Bartl, S. 1998. What sharks can tell us about the evolution of MHC genes. Immunol. Rev. 166: 317– 331. 176. Kordis, D. 2005. A genomic perspective on the chromodomain-containing retrotransposons: Chromoviruses. Gene 347: 161–173. 177. Benit, L. et al. 1999. ERV-L elements: A family of endogenous retrovirus-like elements active throughout the evolution of mammals. J. Virol. 73: 3301–3308. 178. Weiss, R.A. 2006. The discovery of endogenous retroviruses. Retrovirology 3: 67. 179. Matsuo, M.Y. & M. Nonaka. 2004. Repetitive elements in the major histocompatibility complex (MHC) class I region of a teleost, medaka: Identification of novel transposable elements. Mech. Dev. 121: 771–777. 180. Hughes, A.L. 1991. Independent gene duplications, not concerted evolution, explain relationships among class I MHC genes of murine rodents. Immunogenetics 33: 367–373. 181. Anzai, T. et al. 2003. Comparative sequencing of human and chimpanzee MHC class I regions unveils insertions/deletions as the major path to genomic divergence. Proc. Natl. Acad. Sci. USA 100: 7708– 7713. 182. Lu, C.C. et al. 1996. Evolutionary origins of retroposon lineages of Mhc class II Ab alleles. Immunogenetics 43: 115–124. 183. Kulski, J.K. et al. 1998. The evolution of MHC diversity by segmental duplication and transposition of retroelements. J. Mol. Evol. 46: 734. 184. Kulski, J.K. et al. 1999. Comparison between two human endogenous retrovirus (HERV)-rich regions within the major histocompatibility complex. J. Mol. Evol. 48: 675–683. 185. Kulski, J.K. et al. 1999. Coevolution of PERB11 (MIC) and HLA class I genes with HERV-16 and retroelements by extended genomic duplication. J. Mol. Evol. 49: 84–97.

229

Villarreal: Viral Origin of Adaptive Immunity 186. Kulski, J.K. et al. 2004. Rhesus macaque class I duplicon structures, organization, and evolution within the alpha block of the major histocompatibility complex. Mol. Biol. Evol. 21: 2079–2091. 187. Amadou, C. et al. 2003. Co-duplication of olfactory receptor and MHC class I genes in the mouse major histocompatibility complex. Hum. Mol. Genet. 12: 3025–3040. 188. Shi, P. & J.Z. Zhang. 2007. Comparative genomic analysis identifies an evolutionary shift of vomeronasal receptor gene repertoires in the vertebrate transition from water to land. Genome Res. 17: 166–174. 189. Kouros-Mehr, H. et al. 2001. Identification of nonfunctional human VNO receptor genes provides evidence for vestigiality of the human VNO. Chem. Senses 26: 1167–1174. 190. Zhang, J. & D.M. Webb. 2003. Evolutionary deterioration of the vomeronasal pheromone transduction pathway in catarrhine primates. Proc. Natl. Acad. Sci. USA 100: 8337–8341. 191. Xie, T. et al. 2003. Analysis of the gene-dense major histocompatibility complex class III region and its comparison to mouse. Genome Res. 13: 2621– 2636. 192. Satta, Y., Y.J. Li & N. Takahata. 1998. The neutral theory and natural selection in the HLA region. Front. Biosci. 3: d459–d467. 193. Laing, K.J. & C.J. Secombes. 2004. Chemokines. Dev. Comp. Immunol. 28: 443–460. 194. Vossen, M.T. et al. 2002. Viral immune evasion: A masterpiece of evolution. Immunogenetics 54: 527– 542. 195. Hengel, H., U.H. Koszinowski & K.K. Conzelmann. 2005. Viruses know it all: New insights into IFN networks. Trends Immunol. 26: 396–401. 196. Davis-Poynter, N.J., M. Degli-Esposti & H.E. Farrell. 1999. Murine cytomegalovirus homologues of cellular immunomodulatory genes. Intervirology 42: 331–341. 197. Kim, E. & Y. Kliger. 2005. Discovering hidden viral piracy. Bioinformatics 21: 4216–4222. 198. Dolan, A. et al. 2004. Genetic content of wild-type human cytomegalovirus. J. Gen. Virol. 85: 1301– 1312. 199. Arav-Boger, R. et al. 2006. Human cytomegalovirus-encoded alpha-chemokines exhibit high sequence variability in congenitally infected newborns. J. Infect. Dis. 193: 788–791. 200. Cameron, C. et al. 1999. The complete DNA sequence of myxoma virus. Virology 264: 298–318. 201. McGeoch, D.J. & A.J. Davison. 1999. The descent of human herpesvirus 8. Semin. Cancer Biol. 9: 201– 209. 202. McGeoch, D.J., F.J. Rixon & A.J. Davison. 2006.

203.

204.

205.

206.

207.

208.

209.

210.

211. 212.

213.

214.

215. 216.

217. 218.

Topics in herpesvirus genomics and evolution. Virus Res. 117: 90–104. Akita, F. et al. 2007. The crystal structure of a viruslike particle from the hyperthermophilic archaeon Pyrococcus furiosus provides insight into the evolution of viruses. J. Mol. Biol. 368: 1469–1483. Merckel, M.C. et al. 2005. The structure of the bacteriophage PRD1 spike sheds light on the evolution of viral capsid architecture. Mol. Cell. 18: 161–170. Haggerty, S.M. & M.R. Schleiss. 2002. A novel CC-chemokine homolog encoded by guinea pig cytomegalovirus. Virus Genes 25: 271–279. Hughes, A.L. 2002. Origin and evolution of viral interleukin-10 and other DNA virus genes with vertebrate homologues. J. Mol. Evol. 54: 90–101. Pepperl-Klindworth, S. et al. 2006. Cytomegalovirus interleukin-10 expression in infected cells does not impair MHC class I restricted peptide presentation on bystanding antigen-presenting cells. Viral Immunol. 19: 92–101. Sissons, J.G., M. Bain & M.R. Wills. 2002. Latency and reactivation of human cytomegalovirus. J. Infect. 44: 73–77. Pleskoff, O. et al. 2005. The human cytomegalovirus-encoded chemokine receptor US28 induces caspase-dependent apoptosis. FEBS J. 272: 4163–4177. Fraile-Ramos, A. et al. 2001. The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling. Mol. Biol. Cell. 12: 1737–1749. Rosenkilde, M.M. et al. 2001. Virally encoded 7TM receptors. Oncogene 20: 1582–1593. Beisser, P.S. et al. 2005. The Epstein–Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase. J. Virol. 79: 441–449. Paulsen, S.J. et al. 2005. Epstein–Barr virus-encoded BILF1 is a constitutively active G protein-coupled receptor. J. Virol. 79: 536–546. Sherrill, J.D. & W.E. Miller. 2008. Desensitization of herpesvirus-encoded G protein-coupled receptors. Life Sci. 82: 125–134. Afonso, C.L. et al. 2000. The genome of fowlpox virus. J. Virol. 74: 3815–3831. Seet, B.T. et al. 2003. Analysis of an orf virus chemokine-binding protein: Shifting ligand specificities among a family of poxvirus viroceptors. Proc. Natl. Acad. Sci. USA 100: 15137–15142. Smith, G.L. et al. 1997. Vaccinia virus immune evasion. Immunol. Rev. 159: 137–154. Lapointe, R. et al. 2005. Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells. J. Gen. Virol. 86: 963–971.

230 219. Sindre, H. et al. 1996. Human cytomegalovirus suppression of and latency in early hematopoietic progenitor cells. Blood 88: 4526–4533. 220. Maciejewski, J.P. & S.C. St Jeor. 1999. Human cytomegalovirus infection of human hematopoietic progenitor cells. Leuk. Lymphoma 33: 1–13. 221. Soderberg-Naucler, C. & J.Y. Nelson. 1999. Human cytomegalovirus latency and reactivation—a delicate balance between the virus and its host’s immune system. Intervirology 42: 314–321. 222. Pani, M.A. et al. 2002. The variable endogenous retroviral insertion in the human complement C4 gene: A transmission study in type I diabetes mellitus. Hum. Immunol. 63: 481–484. 223. de Parseval, N. et al. (2001. Characterization of the three HERV-H proviruses with an open envelope reading frame encompassing the immunosuppressive domain and evolutionary history in primates. Virology 279: 558–569. 224. Blaise, S. et al. 2004. Identification of an envelope protein from the FRD family of human endogenous retroviruses (HERV-FRD) conferring infectivity and functional conservation among simians. J. Virol. 78: 1050–1054. 225. Benit, L., P. Dessen & T. Heidmann. 2001. Identification, phylogeny, and evolution of retroviral elements based on their envelope genes. J. Virol. 75: 11709–11719. 226. Bonnaud, B. et al. 2005. Natural history of the ERVWE1 endogenous retroviral locus. Retrovirology 2: 57. 227. Caceres, M. & J.W. Thomas. 2006. The gene of retroviral origin Syncytin 1 is specific to hominoids and is inactive in Old World monkeys. J. Hered. 97: 100–106. 228. Kim, H.S. et al. 1999. Phylogenetic analysis of a retroposon family in African great apes. J. Mol. Evol. 49: 699–702. 229. Kim, H.S., O. Takenaka & T.J. Crow. 1999. Isolation and phylogeny of endogenous retrovirus sequences belonging to the HERV-W family in primates. J. Gen. Virol. 80: 2613–2619. 230. Huh, J.W. et al. 2006. Molecular evolution of the periphilin gene in relation to human endogenous retrovirus m element. J. Mol. Evol. 62: 730– 737. 231. Kim, H.S. & O. Takenaka. 2001. Phylogeny of SINE-R retroposons in Asian apes. Mol. Cells 12: 262–266. 232. Kim, T.M., S.J. Hong & M.G. Rhyu. 2004. Periodic explosive expansion of human retroelements associated with the evolution of the hominoid primate. J. Korean Med. Sci. 19: 177–185. 233. Zwolinska, K. 2006. [Retroviruses-derived sequences in the human genome. Human endoge-


234.

235.

236.

237.

238.

239.

240.

241.

242.

243.

244.

245.

246.

247.

248.

nous retroviruses (HERVs)]. Postepy Hig. Med. Dosw. (Online) 60: 637–652. Lavie, L. et al. 2004. Human endogenous retrovirus family HERV-K(HML-5): Status, evolution, and reconstruction of an ancient betaretrovirus in the human genome. J. Virol. 78: 8788–8798. Bannert, N. & R. Kurth. 2004. Retroelements and the human genome: New perspectives on an old relation. Proc. Natl. Acad. Sci. USA 101: 14572– 14579. Mills, R.E. et al. 2006. An initial map of insertion and deletion (INDEL) variation in the human genome. Genome Res. 16: 1182–1190. Wetterbom, A. et al. 2006. Comparative genomic analysis of human and chimpanzee indicates a key role for indels in primate evolution. J. Mol. Evol. 63: 682–690. Polavarapu, N., N.J. Bowen & J.F. McDonald. 2006. Identification, characterization and comparative genomics of chimpanzee endogenous retroviruses. Genome Biol. 7: R51. Herrera, R.J. et al. 2006. Ancient retroviral insertions among human populations. J. Hum. Genet. 51: 353–362. Tonjes, R.R. et al. 1996. HERV-K: The biologically most active human endogenous retrovirus family. J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 13(Suppl. 1): S261–267. Zsiros, J. et al. 1998. Evolutionary relationships within a subgroup of HERV-K-related human endogenous retroviruses. J. Gen. Virol. 79: 61–70. Flockerzi, A. et al. 2005. Human endogenous retrovirus HERV-K14 families: Status, variants, evolution, and mobilization of other cellular sequences. J. Virol. 79: 2941–2949. Okeoma, C.M. et al. 2007. APOBEC3 inhibits mouse mammary tumour virus replication in vivo. Nature 445: 927–930. Delebecque, F. et al. 2006. Restriction of foamy viruses by APOBEC cytidine deaminases. J. Virol. 80: 605–614. Mayer, J. & E.U. Meese. 2003. Presence of dUTPase in the various human endogenous retrovirus K (HERV-K) families. J. Mol. Evol. 57: 642– 649. Jern, P., G.O. Sperber & J. Blomberg. 2005. Use of endogenous retroviral sequences (ERVs) and structural markers for retroviral phylogenetic inference and taxonomy. Retrovirology 2: 50. Turner, G. et al. 2001. Insertional polymorphisms of full-length endogenous retroviruses in humans. Curr. Biol. 11: 1531–1535. Moyes, D.L. et al. 2005. The distribution of the endogenous retroviruses HERV-K113 and HERVK115 in health and disease. Genomics 86: 337–341.

Villarreal: Viral Origin of Adaptive Immunity 249. Ono, M. et al. 1980. Sequence organization of cloned intracisternal A particle genes. Cell 21: 465– 473. 250. Ono, M. 1986. Molecular cloning and long terminal repeat sequences of human endogenous retrovirus genes related to types A and B retrovirus genes. J. Virol. 58: 937–944. 251. Ono, M. 1990. Molecular biology of type A endogenous retrovirus. Kitasato Arch. Exp. Med. 63: 77–90. 252. Vogel, T.U. et al. 1999. Major histocompatibility complex class I genes in primates: Co-evolution with pathogens. Immunol. Rev. 167: 327–337. 253. de Groot, N.G. et al. 2002. Evidence for an ancient selective sweep in the MHC class I gene repertoire of chimpanzees. Proc. Natl. Acad. Sci. USA 99: 11748– 11753. 254. Lie, B.A. & E. Thorsby. 2005. Several genes in the extended human MHC contribute to predisposition to autoimmune diseases. Curr. Opin. Immunol. 17: 526–531. 255. Bontrop, R.E. 2006. Comparative genetics of MHC polymorphisms in different primate species: Duplications and deletions. Hum. Immunol. 67: 388–397. 256. Watkins, D.I. 1995. The evolution of major histocompatibility class I genes in primates. Crit. Rev. Immunol. 15: 1–29. 257. Chen, Z.W. et al. 1992. Molecular cloning of orangutan and gibbon MHC class I cDNA. The HLA-A and -B loci diverged over 30 million years ago.. 148: 2547–2554. 258. Doxiadis, G.G., N. de Groot & R.E. Bontrop. 2008. Impact of endogenous intronic retroviruses on MHC class II diversity and stability. J. Virol.82: 6667–6677. 259. Martinez, O.P. et al. 2001. Genetics of human complement component C4 and evolution the central MHC. Front. Biosci. 6: D904–D913. 260. Horton, R. et al. 2004. Gene map of the extended human MHC. Nat. Rev. Genet. 5: 889–899. 261. Wiseman, R.W. et al. 2007. Simian immunodeficiency virus SIVmac239 infection of major histocompatibility complex-identical cynomolgus macaques from Mauritius. J. Virol. 81: 349–361. 262. Pendley, C.J. et al. 2008. MHC class I characterization of Indonesian cynomolgus macaques. Immunogenetics 60: 339–351. 263. Kulski, J.K., S. Gaudieri & R.L. Dawkins. 2000. Using alu J elements as molecular clocks to trace the evolutionary relationships between duplicated HLA class I genomic segments. J. Mol. Evol. 50: 510–519. 264. Kulski, J.K. et al. 2002. Comparative genomic analysis of the MHC: The evolution of class I duplication blocks, diversity and complexity from shark to man. Immunol. Rev. 190: 95–122.

231 265. Fukami-Kobayashi, K. et al. 2005. Genomic evolution of MHC class I region in primates. Proc. Natl. Acad. Sci. USA 102: 9230–9234. 266. Kulski, J.K. & R.L. Dawkins. 1999. The P5 multicopy gene family in the MHC is related in sequence to human endogenous retroviruses HERV-L and HERV-16. Immunogenetics 49: 404–412. 267. Giorgi, D. et al. 2000. Characterization of nonfunctional V1R-like pheromone receptor sequences in human. Genome Res. 10: 1979–1985. 268. Liman, E.R. & H. Innan. 2003. Relaxed selective pressure on an essential component of pheromone transduction in primate evolution. Proc. Natl. Acad. Sci. USA 100: 3328–3332. 269. Olson, R. et al. 2005. Structure of a pheromone receptor-associated MHC molecule with an open and empty groove. PLoS Biol. 3: e257. 270. Spehr, M. et al. 2006. Essential role of the main olfactory system in social recognition of major histocompatibility complex peptide ligands. J. Neurosci. 26: 1961–1970. 271. Yamazaki, K. et al. 2000. Parent-progeny recognition as a function of MHC odortype identity. Proc. Natl. Acad. Sci. USA 97: 10500–10502. 272. del Guercio, P. 1993. The self and the nonself: Immunorecognition and immunologic functions. Immunol. Res. 12: 168–182. 273. Lobo-Yeo, A. & J.R. Lamb. 1993. Superantigens as immunogens and tolerogens. Clin. Exp. Rheumatol. 11: S17–S21. 274. Huber, B.T., U. Beutner & M. Subramanyam. 1994. The role of superantigens in the immunobiology of retroviruses. Ciba Found. Symp. 187: 132–40; discussion 140–143. 275. Ross, S.R. 1998. Mouse mammary tumor virus and its interaction with the immune system. Immunol. Res. 17: 209–216. 276. Meylan, F. et al. 2005. Negative thymocyte selection to HERV-K18 superantigens in humans. Blood 105: 4377–4382. 277. Sutkowski, N. et al. 2004. Epstein–Barr virus latent membrane protein LMP-2A is sufficient for transactivation of the human endogenous retrovirus HERV-K18 superantigen. J. Virol. 78: 7852– 7860. 278. Sutkowski, N. et al. 2001. Epstein–Barr virus transactivates the human endogenous retrovirus HERVK18 that encodes a superantigen. Immunity 15: 579– 589. 279. Switzer, W.M. et al. 2005. Ancient co-speciation of simian foamy viruses and primates. Nature 434: 376– 380. 280. Murray, S.M. & M.L. Linial. 2006. Foamy virus infection in primates. J. Med. Primatol. 35: 225– 235.

232 281. Jones-Engel, L. et al. 2005. Primate-to-human retroviral transmission in Asia. Emerg. Infect. Dis. 11: 1028–1035. 282. Sverdlov, E.D. 2000. Retroviruses and primate evolution. Bioessays 22: 161–171. 283. Canfield, P.J., J.M. Sabine & D.N. Love. 1988. Virus particles associated with leukaemia in a koala. Aust. Vet. J. 65: 327–328.

Annals of the New York Academy of Sciences 284. Hanger, J.J. et al. 2000. The nucleotide sequence of koala (Phascolarctos cinereus) retrovirus: A novel type C endogenous virus related to Gibbon ape leukemia virus. J. Virol. 74: 4264– 4272. 285. Fiebig, U. et al. 2006. Transspecies transmission of the endogenous koala retrovirus. J. Virol. 80: 5651– 5654.

The Source of Self - Log in to Wiley Online Library

The Source of Self - Log in to Wiley Online Library

Suggest Documents

Safety of folic acid - Log in to Wiley Online Library

Alternative therapies for GERD - Log in to Wiley Online Library

PACAP, VIP, and PHI - Log in to Wiley Online Library

Biodiversity and evolutionary history - Log in to Wiley Online Library

Open source in cachexia? - Wiley Online Library

ImageCL: Language and source-to-source ... - Wiley Online Library

Uncertainties in the global source distribution of ... - Wiley Online Library

XIIntention and the Self - Wiley Online Library

Managing Impressions Online: Self ... - Wiley Online Library

Source and Delivery of Nutrients to Receiving ... - Wiley Online Library

Our sense of self - Wiley Online Library

log-ratio compositional data analysis in ... - Wiley Online Library

Learning to practise the Guided Self‐ ... - Wiley Online Library

Flaxseed Lignans: Source, Biosynthesis ... - Wiley Online Library

Lithospheric instability and the source of the ... - Wiley Online Library

A new source of water in seismogenic ... - Wiley Online Library

Modulation and Source of Procalcitonin in ... - Wiley Online Library

Source locations of secondary microseisms in ... - Wiley Online Library

'source occurrence data to identify patterns and ... - Wiley Online Library

Carbon source control of the phosphorylation ... - Wiley Online Library

Tracing the source of ENSO simulation ... - Wiley Online Library

The source of infrasound associated with ... - Wiley Online Library

Source analysis of quartz from the Upper ... - Wiley Online Library

The effect of moisture source and synoptic ... - Wiley Online Library