Uncorrected Version. Published on August 21, 2006 as DOI:10.1189/jlb.0306148
HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter nomenclature to define viral phenotypes Maureen M. Goodenow*,1 and Ronald G. Collman†,1 *Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, and †Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia
Abstract: HIV-1 infection of cells is mediated by engagement between viral envelope glycoproteins (Env) and a receptor complex comprising CD4 and one of two chemokine receptors, CCR5 and CXCR4, expressed on the surface of target cells. Most CD4ⴙ-transformed T cell lines express only CXCR4, but primary lymphocytes and macrophages, the main cellular targets for infection in vivo, express both coreceptors. Cell- and viral strain-specific utilization of these coreceptor pathways, rather than coreceptor expression per se, regulates lymphocyte and macrophage entry and tropism. Virus use of coreceptor[s] (R5, X4, or R5 and X4) and its target cell tropism (lymphocytes, macrophages, and/or transformed T cell lines) are related but distinct characteristics of Envs. A comprehensive classification schema of HIV-1 Env phenotypes that addresses both tropism and coreceptor use is proposed. Defining Env phenotype based on both parameters is important in the development of entry inhibitors and vaccines, for understanding changes in Env that evolve over time in vivo, and for discerning differences among viral species that underlie aspects of pathogenesis and transmission. Recognizing how tropism is related to, yet differs from, coreceptor selectivity is critical for understanding the mechanisms by which these viral characteristics impact pathogenesis. J. Leukoc. Biol. 80: 000 – 000; 2006. Key Words: HIV-1 entry 䡠 CCR5 䡠 CXCR4 䡠 host cell tropism 䡠 envelope 䡠 HIV-1 phenotype 䡠 HIV-1 classification 䡠 review
INTRODUCTION The field of HIV research has experienced dramatic changes in how target cell tropism of viruses in vivo and ex vivo is understood and described. Initial focus on viral characteristics in ex vivo assays that measured replicative capacity and syncytium formation has progressed to understanding at the molecular level the interactions between HIV-1 envelope glycoprotein (Env) and CD4 in combination with the chemokine receptors that mediate the multistep process of viral entry into cells. Persistence of research into how HIV-1 enters different types of target cells in vivo and ex vivo attests to the fundamental importance of viral entry for understanding viral patho0741-5400/06/0080-0001 © Society for Leukocyte Biology
genesis and disease progress, discerning the effects of immune surveillance, modeling structural interactions among Env domains, monitoring effectiveness of entry inhibitors, and developing new inhibitors and vaccines to prevent or diminish infection. Although coreceptor use is a principal determinant of target cell tropism, HIV-1 isolates display fundamental differences in their preference for and interactions with coreceptors on different primary cell types. The complex relationship between coreceptor use and cell tropism precludes the use of a singleparameter classification system and, most importantly, indicates that one parameter (coreceptor use) cannot be assumed to be a surrogate for the other (tropism). Both tropism and coreceptor use are critical determinants of pathogenesis and have important implications for the use of coreceptor blocking agents, topics that are discussed comprehensively in recent articles [1, 2]. Therefore, this review is focused on exploring the distinctions between coreceptor use and cellular tropism, how coreceptor use and tropism can be defined, and how celland strain-specific coreceptor use determines tropism for primary target cells relevant to pathogenesis.
TARGET CELL TROPISM VS. CORECEPTOR UTILIZATION Early HIV-1 isolates were typically laboratory adapted and replicated ex vivo only in activated peripheral blood T-lymphocytes and transformed T cell lines [3]. The discovery that a significant proportion of primary viral isolates could replicate in culture in both peripheral blood T lymphocytes and monocyte-derived macrophages, but not T cell lines, led to the dichotomous classification of viral isolates as T cell line (T)tropic or macrophage (M)-tropic [4, 5]. Shortly thereafter, isolates were identified that could replicate in macrophages, T cell lines and primary lymphocytes and were designated dual (D)-tropic (the term dual indicated tropism for both macrophages and T cell lines, previously considered to be mutually
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Correspondence: M. M. G.: Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, 1600 SW Archer Rd., Gainesville, FL 32610-0275. E-mail:
[email protected]; R. B. C.: Department Medicine, University of Florida, 1600 SW Archer Rd., Gainesville, FL 32610-0275. E-mail:
[email protected] Received March 4, 2006; revised June 24, 2006; accepted June 26, 2006; doi: 10.1189/jlb.0306148.
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Copyright 2006 by The Society for Leukocyte Biology.
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exclusive targets, in addition to the “universal” cell target, primary lymphocytes) [6, 7]. The discovery that HIV-1 used CD4 as the primary receptor for entry defined CD4-expressing cells as targets for HIV-1 [8] but failed to provide a basis for differential infection of the spectra of cells that express CD4 [9]. A mechanistic basis for selective cell tropism by viruses was provided a decade ago by the discovery that the chemokine receptors CCR5 and CXCR4 function as the major entry coreceptors used by M-tropic or T-tropic HIV-1 prototypes, respectively, while D-tropic prototypes could use both coreceptors [10 –12]. A classification system that designates viral isolates as R5, X4, or R5X4 based on coreceptor selectivity in a variety of cell line-based assay systems was developed and rapidly accepted in the field [13]. Consequently, the terms R5, X4, and R5X4, which describe coreceptor use, became a surrogate for, and largely replaced, functional characterization of HIV-1 isolates for tropism for various target cells.
CORECEPTOR UTILIZATION IS RELATED TO, BUT DISTINCT FROM, TROPISM The initial observation that M-tropic, T-tropic and dual-tropic prototype strains used CCR5, CXCR4 or both, suggested that tropism would be completely explained by coreceptor selectivity. Significant concordance between CXCR4 use and tropism for transformed CD4⫹ cells (and the consequent phenotype of “syncytia-inducing”) reflects the fact that nearly all CD4⫹ T cell lines express endogenous CXCR4, but not CCR5 (in contrast to indicator cell lines engineered from various cell types to express CD4 plus one or the other coreceptor). Indeed, the identification of viruses as “syncytia-inducing” in T cell lines [14] reflects the tropism of these isolates for T cell lines because of their use of the CXCR4 coreceptor. In contrast to coreceptor use defined by cell lines, however, multiple findings indicate that coreceptor use and cellular tropism for primary lymphocytes and macrophages, which are most relevant to infection in vivo, are distinct albeit related features.
Coreceptor use on primary lymphocytes CD4⫹ T lymphocyte populations expressing CCR5 and CXCR4 are often considered the “universal target” for HIV-1, consistent with CD4⫹ T cells in lymphoid tissues as the main target for replication in vivo. CCR5 is expressed predominantly on a subset of the CD45RO memory CD4⫹ T lymphocytes, while CXCR4 is expressed on CD4⫹ CD45RO and on CD4⫹ CD45RA naive cells [15]. In vitro and in vivo studies confirm that R5 strains of HIV-1 display preferential tropism for CCR5 memory T cells, whereas X4 strains infect CXCR4-expressing CD45RO and CD45RA subsets [16, 17]. Although new infections in individuals are primarily established by strains that use R5, coreceptor preference in vivo frequently evolves over the course of disease progression from R5 to R5X4 and, in some cases, to X4 [18, 19]. In addition to distinct coreceptor expression patterns in lymphocyte subsets, recent data demonstrate important differences in the ability of viruses to use CCR5 or CXCR4 ex2
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pressed on indicator cell lines vs. primary cells. On the basis of results using coreceptor blocking agents, a significant number of R5X4 isolates are unable to use lymphocyte CCR5 for entry and so are restricted to CXCR4, even though these strains are able to use both CCR5 and CXCR4 for entry into macrophages, as well as indicator cell lines [20]. This result indicates the existence of strain- and cell-specific determinants that regulate CCR5 function as a coreceptor on lymphocytes. The patterns of coreceptor use by viruses observed with primary cells in vitro likely reflects the patterns in vivo, since R5X4 viruses are restricted to CXCR4 for infection of human lymphoid tissue ex vivo [21] and HIV/SIV chimeras with R5X4-using Envs appear to function as X4 viruses in infected macaques [22–24]. Because R5X4 strains cannot enter lymphocytes through CCR5, their isolation by culture in peripheral blood lymphocytes clearly results from an ability to use lymphocyte CXCR4. A recent report described HIV-1 Envs that were generated by direct cloning from peripheral blood monocyte/macrophages from infected individuals and found to mediate CCR5-dependent entry into macrophages but not into lymphocytes [25]. These variants could reflect compartmentalized evolution within monocyte/macrophage cells and, if confirmed in additional studies, could be termed macrophagerestricted R5 (Mr-R5). The mechanistic basis for restricted use of lymphocyte CCR5 is currently unknown.
Coreceptor use and tropism for macrophages Shortly after identification of the coreceptors for HIV-1, several groups showed that primary human macrophages expressed CXCR4, as well as CCR5, and by employing coreceptor blocking agents to identify the pathway actually used for entry, found that many primary isolates that used CXCR4 in indicator cells and cell lines (either alone or in conjunction with CCR5) could use macrophage CXCR4, even though prototype T-X4 strains could not [18, 26 –31]. These data indicated that some HIV-1 strains are D-tropic (D-R5X4) due to the ability to use CCR5 and/or CXCR4 in a cell-type specific way to enter primary lymphocytes, macrophages, and standard CD4⫹ cell lines (which express CXCR4 but not CCR5), while other strains are D-tropic (D-X4) because they could use CXCR4 expressed by all three target cell types [18, 30]. Conversely, X4 strains can be classified as either T-X4 based on the ability to use CXCR4 on lymphocytes and cell lines but not macrophages, or D-X4 if they can use CXCR4 expressed by all three cell types for entry. Of note, most prototype X4 strains were extensively passaged in cell lines and have the T-X4 phenotype, while many or perhaps most primary X4 isolates have the D-X4 phenotype. What determines the cell- and strain-specific ability to use macrophage CXCR4 is complex but appears to depend at least in part on an ability to utilize low levels of CXCR4 efficiently in the context of relatively low CD4 expression [32]. Although most CCR5-using strains enter and infect macrophages, several studies found that some primary R5 isolates may not be macrophage-tropic. Indeed, primary isolates display a spectrum in their capacity to use R5 on macrophages, from those that are largely restricted to peripheral blood T lymphocytes to those that enter both T lymphocytes and macrophages efficiently [28, 33–35]. In many cases, entry into macrophages is linked to the ability to use CCR5 in the context http://www.jleukbio.org
of low CD4 levels [34, 35]. The extreme end of the spectrum of macrophage-tropism may be reflected in brain-derived isolates that infect CCR5-positive microglia and brain macrophages, which express very low levels of CD4 [34, 36, 37]. Although no established terminology comparable to D-X4 vs. T-X4 is yet in place for this distinction, R5 strains with the capacity to infect both macrophages and lymphocytes have been termed M-R5, while those that infect lymphocytes but cannot utilize macrophage CCR5 for entry could be termed L-R5 (lymphocytetropic R5). Data from the precoreceptor discovery era showed that initial NSI variants were typically M-tropic [38]. Although evolution from R5 to R5X4 and/or X4 coreceptor preference can occur during disease progression in a large minority of individuals infected by subtype B viruses, advanced disease also develops in the presence of R5 viruses only and increased macrophage-tropism among R5 viruses has been linked to disease progression [28, 33, 39]. Together, these results from studies of peripheral blood lymphocytes and macrophages indicate that coreceptor use as defined on indicator cell lines is an imprecise predictor of ex vivo coreceptor use on primary target cells that are relevant to infection in vivo. Moreover, use of a coreceptor on one type of primary cell may not extend to use of the same coreceptor on all primary cell types.
ENV DETERMINANTS OF CORECEPTOR USE AND TROPISM Virus entry into cells involves a dynamic and ordered series of events that initiate with engagement of CD4, followed by interaction with coreceptor and subsequent fusion of viral and host-cell membranes. The HIV-1 envelope surface glycoprotein gp120 mediates interactions with CD4 and coreceptors on the surface of target cells, whereas the trimeric transmembrane gp41 glycoprotein mediates fusion. Molecular determinants that mediate interactions with CD4 are localized to discontinuous epitopes in gp120. Although CD4 interactions map primarily to a conserved region between hypervariable regions 4 and 5 (V4 and V5) in the carboxy terminal region of gp120, other amino acid residues amino-terminally of V3 also contribute [40]. Early studies identified the V3 hypervariable region as the principal determinant of M- vs. T-tropism [41– 43]. Subsequent studies demonstrated that V3 was also a major determinant of CCR5 or CXCR4 use [44, 45]. Genetic differences between V3 regions provide predictive value for coreceptor use. Envs that use CCR5 usually have V3 regions with low numbers of positively charged amino acids, while CXCR4-using Envs typically have V3 sequences containing an excess of positively charged amino acid residues, most notably at positions 11 and/or 25 [46 – 49]. R5X4 typically have a V3 charge pattern more similar to X4 envs, and the determinants that regulate dual coreceptor use are complex and involve multiple discontinuous regions of env [50 –52]. V3 sequence predictions are generally accurate for aspects of tropism that are tightly linked to coreceptor use, such as T cell line tropism via CXCR4. In contrast, V3 sequence cannot predict tropism for macrophages based on CXCR4 use, because features of the envelope other than V3 charge, and
indeed other than the V3 region, are involved. For example, T-X4 isolates may acquire the ability to use CXCR4 on macrophages (D-X4) by a methionine to isoleucine substitution in V3 that does not change V3 charge, although the converse mutation failed to abrogate macrophage CXCR4 use by D-X4 primary isolates, implicating other complex determinants in Env [18]. Among R5 strains, the use of CCR5 on macrophages (M-R5 vs. L-R5) has been linked to changes at residue 283 in the C2 region of Env, likely through enhanced use of CCR5 in the context of low CD4 levels [53], although V3 determinants have been implicated as well [51]. The determinants that regulate use of lymphocyte CCR5 by R5X4 HIV-1 Env are unknown [20].
MEASURES OF CORECEPTOR USE AND TROPISM Viral isolates vs. Env glycoproteins Although entry is necessary for replication and spread in target cells, restriction of the viral life cycle can occur at a multitude of postentry steps [28, 33, 54], which complicates the evaluation of tropism and cell-type specific coreceptor use for primary HIV-1 isolates based on productive replication. One solution to this problem is to focus on early postentry events in infection, such as PCR detection of early reverse transcription products, which will be less affected by late postentry restrictions [20, 33, 54]. Alternatively, Envs derived from viral isolates or cloned directly from patient cells or plasma, when placed in the background of other viral genes known to support postentry events, can provide an unambiguous assessment of coreceptor use and cell tropism. On the basis of the questions addressed, complete gp160 glycoproteins or gp120/gp41 chimeric Envs can be cloned into appropriate expression vectors and used to pseudotype reference HIV-1 genomes tagged with reporter genes. As some Envs derived from primary isolates may pseudotype poorly, introducing cloned envs into replicationcompetent molecular clones of HIV-1 and measuring coreceptor use in spreading infection increases sensitivity of discerning subtle differences among Envs but is cumbersome for extensive screening of panels of Envs. Entry assays by Env pseudotyped, reporter-viruses engineered for a single cycle of replication are rapid, quantitative, and can be applied to mapping genetic determinants of Env function. A different approach to analyzing individual env clones is an assessment in single-cycle assays of populations of envs in viral swarms [55]. On one hand, quasispecies analysis captures a broader range of species in a swarm than any single env clone. On the other hand, assessment of swarms of env clones may underestimate the range of phenotypic diversity, given an estimated sensitivity of 15-20% [55], as well as an inability to distinguish between Env species that use both pathways (R5X4) and those with mixtures of single pathway species (R5⫹X4), or an inability of R5X4 strains to utilize primary lymphocyte CCR5 [20]. Thus, analysis of multiple individual env clones from a swarm offers combined advantages for evaluating relationships between genotype and function, mapping determinants that confer function, or measuring qualitative and quantitative differences in coreceptor use among distinct variants in a quasispecies.
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Target cells to define coreceptor use Target cells for coreceptor analysis include transiently transfected systems and a large variety of reporter cell lines designed to stably express CD4 and either CCR5 or CXCR4, such as NIH 3T3 murine fibroblasts, canine Cf2Th cells, or U87 human glial cells that express no endogenous human CD4 or coreceptors [56 –58], or HOS cells, GFP-expressing GHOST cells or HeLa cell lines that express endogenous but low levels of CXCR4 [10, 59, 60]. Such cell lines provide a standardized environment for qualitative evaluation of basic CCR5 or CXCR4 coreceptor use primarily by Env pseudotyped, singlecycle viruses and can be used to measure Env sensitivity to neutralizing antibodies or to small molecules that block interactions with CCR5 or CXCR4. Panels of CD4/coreceptor reporter cell lines that express different levels of CD4 and/or coreceptors on the surface or express chimeric coreceptors have been developed for quantitative assessment of Env interactions with receptor complexes [61, 62]. Cell lines have been engineered to express a variety of other chemokine receptors, such as CCR3, CCR2b, CCR8, GPR1, GPR15, and other, to increase the ability to discern differences among R5 or X4 Envs, although the role of these chemokine receptors for infection in vivo remains uncertain. In addition to entry-based assays, coreceptor use can be evaluated in cell-cell fusion assays in which Env is expressed by effector cells that are mixed with CD4/coreceptor-expressing target cells. Coreceptor-dependent, Env-mediated fusion is assessed on the basis of transactivation of a reporter gene due to cell content mixing [11, 12], although Env overexpression in effector cells may lead to differences from Env expressed in the context of virions. In particular, cell-cell fusion assays may overestimate coreceptor utilization when compared with infection, for examTABLE 1A. Coreceptor designation
ple, many minor coreceptors that support HIV-1 cell-cell fusion assays are less active or even inactive in the context of virus infection [63, 64].
Target cell tropism and coreceptor use Target cell tropism reflects the ability of an isolate to replicate in, or for its Env to mediate entry into, particular target cells. Indicator cell lines define the overall capacity of an Env to use a coreceptor, but provide no direct information about the particular coreceptor(s) actually used to mediate entry into a given cell. Thus, comprehensive characterization of tropism must include direct evaluation for ability of Env to mediate CCR5- or CXCR4-dependent infection of primary peripheral blood lymphocytes and monocytederived macrophages. Primary cells present challenges for defining Env tropism for several reasons. Because of donor variability, cells from multiple independent donors must be evaluated. Because primary macrophages and lymphocytes express both CCR5 or CXCR4 (albeit for lymphocytes on somewhat different subsets), identification of the entry pathway(s) employed requires selective inhibition using coreceptor blocking agents and/or use of primary cells lacking CCR5 derived from individuals homozygous for the nonfunctional CCR5 ⌬32 deletion allele [29, 65]. Specificity of coreceptor use for entry into mixed populations of primary cells expressing CCR5 and/or CXCR4 is suited to assays of either cloned Envs or populations of viruses but necessitates entry-blocking experiments. A comprehensive analysis of tropism for X4 Envs that infect lymphocytes, but fail to infect macrophages, may require evaluation for entry into T cell lines.
Proposed Terms for Defining Coreceptor Phenotypes
Description of coreceptor and coreceptor use on indicator cell lines
R5 X4 R5X4
uses CCR5 only uses CXCR4 only defined species that uses both CCR5 and CXCR4
R5⫹X4
quasispecies with a mix of variants that independently use CCR5 or CXCR4 quasispecies with both CCR5 and CXCR4 use, but mixed R5⫹X4 versus single R5X4 species not distinguished
R5/X4
TABLE 1B.
Tropism designation Macrophage (M) T cell line (T) Dual (D) Lymphocyte (L) Macrophage-restricted (Mr)
Relationship to tropism phenotype M or L tropic D or T tropic usually D tropic; may use distinct coreceptor pathways to enter either macrophages or lymphocytes usually D tropic; may use distinct coreceptor pathways to enter macrophages & lymphocytes usually D tropic; may use distinct coreceptor pathways to enter macrophages & lymphocytes
Proposed Terms for Defining Tropism Phenotypes Description of tropism and primary cells and CD4 T cell lines
Relationship to coreceptor use
macrophages & lymphocytes lymphocytes & CD4⫹ T cell lines macrophages, lymphocytes & CD4⫹ T cell lines lymphocytes only macrophages onlya
typically R5 typically X4 may be R5X4 or X4 typically R5 typically R5
a A viral phenotype that uses CCR5 and infects macrophages, but not lymphocytes (macrophage-restricted R5; Mr-R5), was described but warrants further investigation (25).
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Fig. 1. Graphic representation of HIV-1 phenotypes based on both coreceptor utilization and target tropism ex vivo. New infections are almost always initiated by R5 variants, initially identified as replication competent in macrophages (M-R5) [38], although some R5 variants may be limited to lymphocyte tropism (L-R5) [28, 33, 34, 39]. In ⬃40% of infected individuals (at least among those infected with HIV-1 subtype B), disease progression is associated with the emergence of variants that use CXCR4, which confers tropism for and the ability to form syncytia in transformed CD4⫹ T cell lines (syncytia-inducing phenotype). Some X4 strains use CXCR4, in addition to CCR5, and infect macrophages, lymphocytes, and CD4 T cell lines (D-R5X4), although infection of primary cells can be mediated by different coreceptor pathways [20]. Variants that use CXCR4 exclusively also emerge and may coexist with R5 and/or R5X4 species. Most X4 primary isolates can use CXCR4 in macrophages, as well as lymphocytes and CD4⫹ T cell lines (D-X4) [18, 26 –30]. However, variant X4 strains, either primary or passaged in cell lines, fail to use macrophage CXCR4 and are thus restricted to primary lymphocytes and CD4⫹ cell lines ex vivo (T-X4).
TABLE 2B. Phenotype L-R5
M-R5
D-R5X4 D-X4 T-X4
Representative HIV-1 Strains or Envelopes by Phenotype Examples
Source
JRCSF (68) NB13, NB17 (33) L9, L11 (28) NA20 LN8, NA118 LN33 (34) Bal (66) ADA (5) SF162 (67) JRFL YU2 (69) 89.6 (6) DH12 (7) Tybe (30) UG021, UG024 (30)d D01, D02 (18) LAI (LAV/BRU) (70) 3B/HXB (71) NL4-3 (72) SF33 (67) T04 (18)
cerebrospinal fluid/PIa blood/PI blood/PI lymph nodeb lung/LPc in macrophages blood/LP in macrophages cerebrospinal fluid/PI brain/PI brain/PI blood/PI blood/LP in human and chimpanzee PBMC cerebrospinal fluid/PI blood/PI thymus, bloodb lymph node/LP in PBMC lymph node/LP in T cell linesd chimeric clone with env derived from LAI/LP blood/PI thymusb
a
PI (primary isolate) indicates a strain that was cloned directly from tissue source or isolated by minimal passage in vitro in PBMC. b env clone only. c LP (laboratory passaged) indicates a strain that was selected by serial passage in vitro in primary cells other than PBMC or in cell lines. d nonsubtype B (all other strains noted are subtype B). e highly similar and closely related to HIVLAI (73).
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PROPOSED NOMENCLATURE FOR CORECEPTOR USE AND TROPISM In view of the distinctions between coreceptor use and primary target cell tropism, viral phenotype should be described by both characteristics. A combination of the characteristics would provide a basic phenotypic profile for viruses. To distinguish between tropism and coreceptor use, we suggest that the terms for “tropism” currently in use, such as M-, T-, and D-tropic, would specifically describe the phenotype of Envs based on the repertoire of primary target cells and T cell lines competent for entry and infection (Table 1). Coreceptor utilization would be defined based on indicator cell lines and described not as R5- or X4-tropic but rather R5, X4, or R5X4, as outlined previously [13]. R5X4 coreceptor use would be reserved to describe situations in which the use of each coreceptor is determined to be a function of a single viral or Env species, either through biological or molecular clonal analysis; R5⫹X4 would describe a mixture of single coreceptor using Envs within a quasispecies; and R5/X4 should describe swarms that can use both coreceptors but have not been defined at the level of individual viral or cloned Env species (Table 1). Coreceptor use in cell line-based assays must be distinguished from measures of coreceptor use on primary cells (Fig. 1). A dual parameter nomenclature combining coreceptor use and tropism applied to HIV-1 prototypes or reference examples for each group is shown in Table 2. The proposed classification system could be extended to account for novel combinations of tropism in extended cell types, such as microglia or alveolar macrophages, thymocytes, or lymphoid tissues, as long as the target cell type is specified and clear distinction is maintained between coreceptor use on indicator cells and target cell infection profile.
CONCLUSIONS A classification scheme for defining viral Env phenotypes based both on coreceptor use and cellular tropism provides a tool for comparing envs over time within or between infected individuals. Differences between coreceptor use on indicator cell lines and on primary cells and distinctions between tropism and coreceptor selectivity have implications for development of therapeutics and vaccines. Current challenges include identifying the cellular factors that regulate coreceptor utilization in a strain- and cell type-specific manner; understanding consequences of infection by CCR5 or CXCR4 coreceptor pathways; identifying the viral genetic determinants and structural features responsible for cell-specific coreceptor use; and extending findings on primary cell tropism in vitro to tropism for the corresponding cell types in relevant tissues in vivo.
ACKNOWLEDGMENTS The authors receive support for this work from National Institutes of Health Grants AI35502, MH61139, and NS27405 (R. G. C.); HD32259, AI47723, and AI65265 (M. M. G.); Stephany W. Holloway University Chair for AIDS Research (M. M. G.) 6
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