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4 The Differentiation-Dependent Life Cycle of Human Papillomaviruses in Keratinocytes Choogho Lee and Laimonis A. Laimins Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

4.1.

Introduction

Human papillomaviruses (HPVs) are small DNA viruses that induce a variety of hyperproliferative lesions in epithelial tissues. More than 100 different HPV types have been identified and classified on the basis of their nucleotide sequence (Howley and Lowy, 2001). One group of HPVs induces warts on hands or feet and exhibits a high tropism for cutaneous tissues. Another group of HPVs primarily targets genital and oral mucosa. Among these mucosotropic HPVs, types 6 and 11 induce benign lesions and are called “low-risk” HPVs due to their lack of association with cancers. In contrast, HPV types 16, 18, 31, and 45 are referred to as “high-risk” types, since they induce lesions that can ultimately progress to cervical and other cancers (Laimins, 1993; Lowy et al., 1994; zur Hausen, 2002). Despite the differences in oncogenic potential of these various HPV types, it is likely that they share common mechanisms during their productive life cycles.

4.2.

Life Cycle of HPVs

The productive life cycle of HPVs is closely linked to the differentiation program of the host epithelial tissue (Fig. 4.1) (Howley and Lowy, 2001). The cellular receptor for viral entry is still unknown although heparin sulfate seems to facilitate virus attachment in some cell types (Joyce et al., 1999; Giroglou et al., 2001). The targets of initial infection are thought to be basal epithelial cells, which become exposed as a result of microwounds of the stratified epithelia (Howley and Lowy, 2001). Following virus binding and entry, virions migrate to the nucleus and establish their genomes as multi-copy extrachromosomal plasmids, which are maintained at approximately 20 to 100 copies per infected basal cell (Stubenrauch and Laimins, 1999). The replication of viral genomes 45

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Choogho Lee and Laimonis A. Laimins

Mature virions

Suprabasal

Cornified layer Granular layer

Amplify genomes Express late genes

Spinous layer

Maintain genomes Express early genes

Basal layer

Establish genomes

Normal

HPV-infected

Figure 4.1. Tight linkage of the HPV life cycle to the differentiation program of the host epithelial tissue. Drawings show normal (left) and HPV-infected epithelia (right) with various differentiation layers associated with different stages of the HPV life cycle.

occurs in the S-phase of the cell cycle through the action of the viral E1 and E2 proteins in cooperation with cellular replication proteins (Lambert, 1991; Ustav and Stenlund, 1991). Following viral genome replication and cell division, one of the daughter cells migrates away from the basal layer and starts a program of differentiation. Normal uninfected keratinocytes exit the cell cycle after leaving the basal layer, and the nuclei are degraded in many of these differentiating cells. In contrast, HPV-infected cells undergo differentiation but remain active in the cell cycle (Stubenrauch and Laimins, 1999). This allows highly differentiated suprabasal cells to re-enter S-phase and support high-level productive viral DNA replication. The ability of differentiated cells to undergo cell cycle progression is mediated largely through the action of the viral E7 protein (Chen et al., 1995; Flores et al., 1999; Halbert et al., 1992). Differentiation-dependent amplification of viral genomes in suprabasal cells coincides with activation of the late viral promoter (Chen et al., 1995; Flores et al., 1999; Halbert et al., 1992; Hummel et al., 1992). The late viral transcripts encode the capsid proteins L1 and L2 as well as two additional mediators of late viral functions, E1∧ E4, and E5 (Hummel et al., 1992). Progeny virions are assembled in highly differentiated cells and then released to the extracellular environment (Frattini et al., 1996; Howley and Lowy, 2001; Meyers et al., 1992).

4.3.

Methods to Study the HPV Life Cycle

Study of the papillomavirus life cycle has been hampered by the inability of these viruses to replicate to high titer in the laboratory. Initial studies on the life cycle involved descriptive studies of the distribution of viral genomes and gene products in naturally occurring lesions in animals and humans. Cells transformed in culture by bovine papillomavirus were regarded as a model of the early

4. The Life Cycle of Human Papillomaviruses in Keratinocytes

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phase of infection, while the outer epithelial layers of productively infected warts represented the late phase. In recent years, considerable progress has been made in studying the HPV life cycle by using various tissue culture models that include cell lines that stably maintain viral genomes as plasmids and methods to induce epithelial differentiation, which appears to be required for production of progeny virions. In addition, HPV genomes carrying specific mutations have been used to assess the role of viral gene products on the virus life cycle in differentiated epithelial cells. These studies have been supplemented by the analysis of individual viral genes and proteins in cellular and biochemical assays that mirror certain aspects of the virus life cycle, such as cell transformation, transcriptional regulation, and viral DNA replication. A particularly useful cultured epithelial cell system is the organotypic raft culture system initially developed by Asselineau and Prunieras (1984). In this system, undifferentiated keratinocytes are first layered on top of a fibroblast and collagen plug that is then placed on a metal grid at the air–liquid interface (Fig. 4.2). Diffusion of nutrients through the plug allows growth of these cells at this air–liquid interface for about 2 weeks, which induces the differentiation and stratification of the epithelial cells. If the starting cells contain HPV genomes, the resulting differentiated cultures faithfully duplicate the morphology Semisolid media

Undifferentiated keratinocytes

Suspension in methylcellulose for 48 hours

Organotypic raft culture

Undifferentiated keratinocytes

Cells on top of fibroblasts and collagen/grid

Figure 4.2. Methods to induce the epithelial differentiation to study late viral functions. Top: In suspension culture, undifferentiated keratinocytes maintaining HPV genomes are suspended in semisolid media supplemented with 1.6 % methylcellulose for 48 h. Bottom. In organotypic raft culture, undifferentiated keratinocytes maintaining HPV genomes are layered on top of a fibroblast and collagen plug that is then placed on a metal grid at the air–liquid interface for 2 weeks.

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of HPV-induced lesions in vivo (McCance et al., 1988). Furthermore, epithelial differentiation in this system is accompanied by activation of late viral functions, including the focal synthesis of progeny virions in cells in the most highly differentiated layers. These virions are infectious but the titers are low. In some studies, differentiation and virus production is stimulated by agents such as protein kinase C activators (Ozbun and Meyers, 1998b; Sen et al., 2002). A second system for differentiation that utilizes suspension of cells in 1.6 % methylcellulose is useful for studying other aspects of the HPVs life cycle (Flores et al., 1999; Ruesch et al., 1998) (Fig. 4.2). The advantage of suspension culture is that cells progress through differentiation in a coordinate fashion within 48 hours. However, the methylcellulose system obviously does not reproduce the morphologies seen in vivo or in organotypic cultures. Immortalized cell lines derived from biopsy materials that stably maintain viral genomes as plasmids have been studied in both systems. These cell lines include W12 cells, which contain up to 500 copies of the HPV16 genome, and CIN 612 cells, which contain approximately 100 copies of HPV31 DNA (Bedell et al., 1991; Stanley et al., 1989). Both of these cell lines produce HPV virions following differentiation in organotypic raft cultures (Flores et al., 1999; Meyers et al., 1992). The first genetic technique that allowed the analysis of the full papillomavirus life cycle including production of infectious progeny was the introduction of specifically altered cottontail rabbit papillomavirus (CRPV) DNA into rabbit skin (Brandsma and Xiao, 1993; Brandsma et al., 1991). A major technical advance for the study of the HPV life cycle was the development of genetic methods to analyze HPV functions in cell culture (Fig. 4.3) (Frattini et al., 1996).

digest re-ligate pBR322-HPV31

Transfection HPV31

HFKs Neo Neomycin selection

Synthesis of virions

Epithelial differentiation

Stable cell lines

Figure 4.3. Genetic approach to study the life cycle of the HPVs in tissue culture. Wild-type or mutant HPV genome is released from a bacterial plasmid, recircularized and cotransfected with a neomycin resistance gene into human foreskin keratinocytes (HFKs). Transfected cells are subjected to G418 selection, and resistant colonies are pooled to generate stable cell lines maintaining episomal HPV genomes. Epithelial differentiation is induced to study the late stages of the HPV life cycle.


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To perform this analysis, mutations are first introduced into the complete HPV genome contained in a bacterial plasmid. The viral DNA is then excised from vector sequences, circularized, and transfected into normal human keratinocytes along with drug-selectable markers. Following selection and expansion of transfected cells in monolayer culture, effects on the viral life cycle can be studied in either organotypic raft cultures or following suspension in methylcellulose. This method allows analysis of early HPV functions such as establishment and stable replication of viral genomes as well as late functions such as vegetative viral DNA synthesis and late transcription in conditions that mimic the natural situation (Frattini et al., 1996, 1997; Meyers et al., 1997). Such an approach is important because the activities displayed by individual viral proteins in heterologous overexpression assays or in in vitro assays do not necessarily faithfully mimic the actual function of the protein expressed at physiologic levels in the context of other viral proteins in differentiated host epithelial cells. The effects of mutations in HPV types 11, 6, 16, 18 and 31 have been examined using these methods (Flores et al., 1999; Frattini et al., 1996, 1997; Meyers and Laimins, 1994; Meyers et al., 1997; Oh et al., 2004; Thomas et al., 1999).

4.4.

Genome Organization and Gene Products of HPVs

The genetic organizations of all HPV types are similar (Fig. 4.4). The circular HPV DNA genomes are approximately 8 kb in size and consist of early- and latecoding regions as well as sequences that regulate transcription and replication. The major regulatory region is referred to as the upstream regulatory region (URR) or long control region (LCR). HPVs encode six to eight open reading frames that contribute to the productive viral life cycle. P97 polyAL

P742

URR E6

E7

L1

E1

E5

E6 and E7 -Transforming proteins E1 and E2 -Regulators of viral transcription and replication E4 and E5 –Late factors L1 and L2 –Viral structural proteins

E4 E2

L2

polyAE

Figure 4.4. Genomic organization and gene products of high-risk HPV type 31. The early P97 and late P742 viral promoters are shown with arrows. URR stands for the upstream regulatory region. Eight major open reading frames are also indicated with their biological functions.

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The early viral gene products, E6 and E7, play critical roles in the immortalization and transformation of epithelial cells by high-risk HPVs. These proteins bind to and inactivate a number of cellular proteins that inhibit cell cycle progression, creating a cellular environment favorable for viral DNA replication. The E6 protein forms a complex with the tumor suppressor protein p53 and induces its rapid proteosomal degradation (Huibregtse et al., 1991; Scheffner et al., 1990, 1993; Werness et al., 1990). E7 binds to the retinoblastoma (pRb) family of proteins, resulting in the degradation of Rb family members, and thereby facilitates cell-cycle progression by alleviating pRb-mediated repression of genes required for entry into S phase (Cheng et al., 1995; Dyson et al., 1989; Martin et al., 1998; Munger et al., 1989). As discussed in a subsequent section, the E6 and E7 proteins are also important for the maintenance of viral genomes as well as for differentiation-dependent late viral functions (Oh et al., 2004; Thomas et al., 1999). The E1 and E2 proteins regulate viral replication and transcription. Binding of E1 and E2 proteins to their respective recognition sequences in the URR is required for the recruitment of DNA polymerase and other cellular DNA replication proteins to the viral origin of DNA replication (Conger et al., 1999; Frattini and Laimins, 1994; Mohr et al., 1990). The ATPase and helicase activities of E1 protein are also necessary for unwinding of supercoiled DNA to allow efficient viral replication (Seo et al., 1993; Yang et al., 1993), but the binding affinity of the monomeric form of the E1 protein for its recognition sequence is very weak. However, the formation of a complex of the E1 protein with the full-length E2 protein recruits E1 to its recognition sequence and allows it to assemble into hexameric structures with higher affinity for DNA and increased helicase activity (Sanders and Stenlund, 1998; Sedman and Stenlund, 1998). The dimeric E2 protein is required for transcriptional control of viral gene expression as well as for viral DNA replication (Laimins, 1998). Four E2 binding sites consisting of conserved palindromic sequences ACCN6 GGT are found in the URRs of genital papillomaviruses (Laimins, 1998). Through its association with these binding sites, the E2 protein regulates transcription initiated from the early viral promoter, which is located upstream of the E6 open reading frame, and contributes to copy number control (Klumpp and Laimins, 1999). However, the transactivation function of the E2 protein does not appear to be required for the HPV31 life cycle (Stubenrauch et al., 1998a). L1 and L2 are the major and minor capsid proteins, respectively, and are expressed during the late phase of virus life cycle only in the most differentiated epithelial cells where the assembly of HPV virions takes place (Howley and Lowy, 2001). Assembly of L1 and L2 into icosahedral capsids occurs around a single copy of the viral genome associated with cellular histones prior to cell lysis. The most highly expressed HPV protein during productive infection is the E1∧ E4 fusion protein, which consists of the first five amino acids of E1 fused to E4 coding sequences. The E1∧ E4 proteins are synthesized in the late phase of the viral life cycle from spliced transcripts that initiate at the late promoter.


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These transcripts utilize a major splice donor located 15 nucleotides inside the E1 open reading frame and a splice acceptor just upstream of the E4 open reading frame (Doorbar et al., 1996; Nasseri et al., 1987). Because the E4 ORF lacks an ATG, the E1 sequences provide the initiation codon for translation. In the differentiation-dependent life cycle, E1∧ E4 synthesis precedes that of the capsid proteins, L1 and L2, but occurs concurrently with genome amplification (Palefsky et al., 1991; Doorbar et al., 1997; Middleton et al., 2003; Peh et al., 2002; Ruesch and Laimins, 1998). After translation, the E1∧ E4 proteins are processed into several proteolytically cleaved forms that associate into multimeric structures in the cytoplasm and bind to cytokeratins (Sterling et al., 1993; Pray et al., 1995; Roberts et al., 1993, 1997; Wang et al., 2004). In addition, yeast-two hybrid assays showed that E1∧ E4 binds a novel DEAD box-containing RNA helicase (Doorbar et al., 2000). Genetic analysis of E1∧ E4 in the context of complete viral genomes revealed a role in the virus life cycle. Cottontail rabbit papillomaviruses (CRPV) that contained translation termination mutations in the E4 ORF displayed impaired production of progeny virions, indicating an important role for this protein in the productive stage of the life cycle (Peh et al., 2004). Mutations that abrogated HPV31 E4 synthesis had similar effects (Wilson et al., 2005). Cells containing these mutant HPV31 genomes were found to be impaired in differentiationdependent genome amplification and had significantly reduced levels of late viral transcripts. Overexpression of E1∧ E4 in heterologous expression systems can result in the collapse of cytokeratin networks leading to the hypothesis that E1∧ E4 may induce cytokeratin collapse to facilitate viral egress (Doorbar et al., 1991; Roberts et al., 1994). However, only a limited amount of keratin collapse was observed in natural high-risk HPV infections, suggesting that E1∧ E4 may play other roles in the viral life cycle (Pray et al., 1995; Doorbar et al., 1996). Overexpression of HPV16 E1∧ E4 in transient transfection assays also induces G2-M arrest, leading to a model in which the protein facilitates late viral functions by allowing cells to remain in S-phase by blocking progression into mitosis (Davy et al., 2002; Nakahara et al., 2002; Knight et al., 2004). This model is consistent with the recent observation that cyclin B/cdk1 complexes bound to E1∧ E4 were sequestered in the cytoplasm (Davy et al., 2005). Elucidation of the role of E1∧ E4 in the life cycle is likely to yield important insights into the control of late viral functions. The E5 protein is a small hydrophobic protein that is localized to intracellular membranes (Conrad et al., 1993). The BPV1 E5 protein is expressed in both basal keratinocytes and in highly differentiated cells (Burnett et al., 1992), implying that it plays roles in both the early and the late phase of the virus life cycle. However, the absence of a recognizable E5 gene from some HPV genomes suggests that it is not absolutely required for productive virus infection. Removal of the E5 gene from the CRPV genome inhibited but did not abolish papilloma induction in rabbits (Brandsma et al., 1992). The HPV E5 proteins enhance epidermal growth factor (EGF) receptor signaling, apparently through

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upregulation of EGF receptor recycling (Conrad et al., 1993; Straight et al., 1993). E5 also associates with the vacuolar H+ -ATPase and blocks acidification of endosomes, activities which might stimulate receptor recycling (Straight et al., 1995; Conrad et al., 1993).

4.5.

Control of HPV Transcription

Viral gene expression in the high-risk HPV types is mediated by two major promoters and a series of minor promoters (Fig. 4.4). The major early viral promoter, called p97 or p99 in HPV31 and HPV16 and p105 in HPV18, is constitutively active in undifferentiated basal cells as well as most differentiated suprabasal cells (Hummel et al., 1992; Rohlfs et al., 1991). These promoters direct transcription of early viral E6, E7, E1, E2, E1∧ E4, and E5 genes. In the high-risk HPV types, differential expression of E6 and E7 is mediated through a single primary transcript that is alternatively spliced in the E6 gene. The low-risk genital papillomaviruses have two early promoters: one initiates transcription upstream of the E6 open reading frame while the second is within the E7 gene (DiLorenzo and Steinberg, 1995; Smotkin et al., 1989). Most early transcripts terminate at the early polyadenylation (poly A) sequence located downstream of the E5 gene, and inhibitory sequences in the L2 and L1 genes inhibit extension of transcripts that pass through the early poly A sequence in undifferentiated cells (Collier et al., 2002; Oberg et al., 2003). Epithelial differentiation abolishes the inhibitory effect of these elements. HPV early gene expression has been assessed soon after infection of epithelial cells with HPV31 isolated from organotypic cultures. These studies revealed a complex pattern of alternatively spliced transcripts, with E1 and E2 mRNAs being the earliest detectable species (Ozbun, 2002). Upon cellular differentiation, a late viral promoter is activated and drives expression of late RNA with a heterogeneous set of start sites located in the E7 gene (del Mar Pena and Laimins, 2001; Grassmann et al., 1996). The late viral promoter is called p742 in HPV31 and p680 in HPV16 (Grassmann et al., 1996; Hummel et al., 1992) and directs two set of transcripts; one set terminates at the early poly A site while the second set passes through the early poly A site and terminates at the late poly A site located downstream of L1. The first group of late transcripts encodes E1∧ E4, E5, and E1/E2 and the second group of late transcripts encodes the capsid proteins, L1 and L2. Changes in the levels of cellular polyadenylation factors such as CstF64 direct the differentiation-dependent read-through of the early polyadenylation sequence (Terhune et al., 1999, 2001). Additional promoters have also been described (Ozbun and Meyers, 1998b). A variety of cellular and viral transcription factors regulate the activity of the early and late promoters. For the early promoter, cellular transcription factors bind to sequences in the URR and regulate expression of viral genes in a keratinocyte-specific manner (Sen et al., 2002). This cell type-specificity is likely mediated by the cooperative action of multiple constitutively expressed


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keratinocyte-specific transcription factors (Chang et al., 1991). The spectrum of factors that bind to the URR sequences vary between HPV types but all HPV genomes contain binding sites for Ap-1 (Kyo et al., 1995, 1997), Sp-1 (Apt et al., 1996) and TFIID (Tan et al., 1994). Other transcription factors that bind the URR sequences include YY1 (Bauknecht et al., 1995), KRF-1 (Mack and Laimins, 1991), AP-2 (Beger et al., 2001), Oct-1 (Chong et al., 1991), TEF-2 (Chong et al., 1991), TEF-1 (Ishiji et al., 1992), NF-1 (Kyo et al., 1995) and the glucocorticoid receptor (Mittal et al., 1993). The composition of cellular transcription complexes binding to the URR varies during different stages of the HPV life cycle (Sen et al., 2004). In the case of HPV6, that CCAAT displacement protein (CDP) appears to negatively regulate differentiation-specific viral promoters during the early phase of the viral life cycle (Pattison et al., 1997). Little is known about the regulation of late transcription, which is activated upon epithelial differentiation. Changes in chromatin configuration occur in the vicinity of the late HPV 31 promoter, and this is likely to contribute to activation of late viral gene transcription (del Mar Pena and Laimins, 2001). In addition, recent studies using inhibitors of DNA replication have demonstrated that amplification of viral genomes is not necessary for activation of p742 in HPV31 and that differentiation alone is sufficient for its activation (Spink and Laimins, 2005). However, during normal infection, genome amplification does increase template numbers, which also contributes to increased levels of late viral gene expression. The cellular factors that regulate induction of late transcription are currently unknown. In addition, two promoters in the URR appear to be down-regulated during epithelial differentiation (Ozbun and Meyers, 1999). All early HPVs transcripts are synthesized as polycistronic mRNAs that use common start and polyadenylation sites, and alternative splicing is used to regulate expression of viral genes such as E6 and E7 (Fig. 4.5). The E6 protein is translated from unspliced transcripts, whereas the E7 protein is translated from transcripts that have removed the E6 intron, resulting in the generation of an alternative deleted form of E6 called E6∗ (Howley and Lowy, 2001). Many early and late transcripts utilize the E1∧ E4 splice donor and acceptors while those messages encoding E2 use alternate acceptor sequences (Stubenrauch and Laimins, 1999). It is likely that splicing of viral transcripts is regulated in a differentiation-specific manner through the action of splicing enhancers and silencers. The identification of these elements is a difficult but important undertaking. The regulation of HPV transcription through posttranscriptional regulatory mechanisms such as splicing and polyadenylation enables HPVs to fine-tune the expression of early and late viral genes in a differentiation-specific manner. Differential promoter usage may also affect translation. Since HPV translation occurs by a leaky scanning mechanism in which ribosomes bind at the 5 end of messages and scan transcripts in a linear fashion for initiation codons, open reading frames located proximal to 5 ends of mRNA are preferentially translated (Remm et al., 1999). Most early viral transcripts contain E4 and E5 open reading frames, which are the third and fourth open reading frames on these polycistronic messages and hence translated at a low level. In contrast, the E4 and E5 proteins

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P97 0

P742

Poly A early E4

E6 E7

E5

E6

E6

7912

E2

L1

E1 E6

Poly A late

L2

E7

E1

E2

E5

E6, E7, E1, E2, E5

E7

E1

E2

E5

E6*, E7, E1, E2, E5

E7

E2

E5

E6, E7, E2, E5

E7

E2

E5

E6*, E7, E2, E5

E7

E4

E5

E6, E7, E1Ê4, E5

E7

E4

E5

E6*, E7, E1Ê4, E5

E4

E5

E6Ê4, E5

E4

E5

E1Ê4, E5

E2

E5

E2, E2, E5

E2

E5

E1

E4

E5

E2, E5 L2

L1 E1Ê4, L2, L1

E4

L1 E1Ê4, L1

Figure 4.5. Early and late viral transcripts of high-risk HPV type 31. The HPV 31 genome is shown in a linear fashion. The early P97 and late P742 viral promoters are designated with arrows and the early and late polyadenylation sites are identified as Poly A early and Poly A late, respectively. Splice sites are shown by thin lines connecting open reading frames boxes. Potential gene products encoded by each transcript are indicated at the right of the figure.

are the products of the first and second open reading frames on late viral transcripts, allowing their efficient translation in differentiated cells. The E5 protein may also play a role in controlling the switch to late transcription. When the E5 gene is mutated in the context of the complete HPV31 or HPV 16 genomes, the induction of late viral function is impaired, implicating it as a regulator of these activities (Fehrmann et al., 2003; Genther et al., 2003). However, the HPV31 E5 mutant had no effect on the levels of the EGFR or on its phosphorylation status (Fehrmann et al., 2003). Additional studies are needed to further clarify the biological activity of the HPV E5 proteins during the virus life cycle.

4.6. Differentiation-Dependent Mechanisms to Regulate HPV DNA Replication The replication of HPV genomes during the productive life cycle can be divided into three phases: establishment, maintenance, and amplification. During the establishment phase following infection, one HPV genome migrates to the


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nucleus and initiates several rounds of replication to reach 20 to 50 copies per cell. Following establishment, viral genomes replicate on an average of once per cell cycle during S-phase, with the maintenance of a constant copy number for periods ranging up to decades through the regulation of E1 and E2 expression by the E2 protein (Steger and Corbach, 1997; Stubenrauch et al., 1998b). The levels of the E2 proteins regulate early viral transcription. At low genome copy number and levels of E2 expression, E2 activates expression from the early promoter which drives expression of E1 and E2, whereas at high levels, it represses transcription (Stubenrauch et al., 1998b). In this way, expression of the viral replication proteins and viral copy number is believed to be maintained at a stable, optimal level in undifferentiated cells. Upon differentiation, transcription of the replication genes switches from the E2-regulated early promoter to the more active E2-independent late promoter (Klumpp and Laimins, 1999). Highlevel expression of the E1 and E2 replication proteins from this differentiationspecific promoter drives amplification of viral genomes to thousands of copies per cell. Analysis of viral transcript levels during vegetative viral DNA synthesis implies that E1 expression may be the major regulator of viral genome amplification (Ozbun and Meyers, 1998a). In addition, a spliced E8/E2C protein that inhibits transient replication of viral DNA is required for maintenance of episomal HPV31 DNA in keratinocytes (Stubenrauch et al., 2000). It is likely that additional uncharacterized mechanisms regulate viral genome replication.

4.7. Roles of the HPV E6 and E7 Proteins in the Viral Life Cycle The genetic system described above has been used to examine the functions of the E6 and E7 proteins in the viral life cycle. E6 and E7 proteins are encoded by all genital papillomaviruses, suggesting that they provide important functions in viral propagation. In addition, they function as oncoproteins in the high-risk types HPVs. Genetic studies indicate that the E6 and E7 proteins play essential roles in the life cycle of CRPV and the HPVs (Brandsma et al., 1991; Flores et al., 2000; Thomas et al., 1999). High-risk HPV31 genomes that contain translation termination mutations in either E6 or E7 are unable to replicate as plasmids in the stable replication assays (Thomas et al., 1999). The mechanism by which E6 and E7 contribute to the replication and maintenance of viral genomes as plasmids is not yet fully understood. Both the E6 and the E7 proteins bind and alter the activity of cell cycleregulatory proteins (Huibregtse and Beaudenon, 1996; Kubbutat and V.K.H., 1996). Inactivation of the retinoblastoma (pRb) family of proteins is the best characterized function of the E7 protein (Dyson et al., 1989). This interaction sequesters pRb protein away from E2F-pRb complexes and destabilizes the protein, resulting in the constitutive transcription of E2F-responsive S phase-specific genes (Edmonds and Vousden, 1989; Weintraub et al., 1995).

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The low-risk E7 proteins bind to Rb family members with an affinity that is about tenfold lower than that seen with high-risk E7 proteins (Heck et al., 1992). Therefore, high-affinity Rb binding is not required for productive virus growth. This conclusion is consistent with the ability of a pRb binding-deficient CRPV E7 mutant to induce papillomas in rabbits (Defeo-Jones et al., 1993). E7 proteins also bind to histone deacetylases (HDACs), which remove acetyl groups from histones that are in nucleosomes, leading to the formation of heterochromatin that prevents transcription factor binding (Brehm et al., 1999; Longworth and Laimins, 2004). High-risk E7 proteins bind to HDACs through C-terminal sequences that are independent of the Rb binding site (Brehm et al., 1999). The binding to HDACs is indirect and mediated through the Mi2 protein, a component of the mammalian NuRD chromatin remodeling complex. Both pRb and HDAC binding activities are important for the ability of high-risk E7 proteins to immortalize keratinocytes (Longworth and Laimins, 2004). Mutation of the HDAC binding site in E7 leads to an inability to stably maintain viral plasmid genomes, while mutation of the Rb binding domain alters the ability of the E7 protein to maintain differentiating cells active in the cell cycle (Cheng et al., 1995; Flores et al., 2000; Longworth and Laimins, 2004; Thomas et al., 1999). Central to the ability of the E6 protein to immortalize cells is formation of a complex with p53 and E6AP, resulting in rapid p53 ubiquitination and degradation (Huibregtse et al., 1991; Scheffner et al., 1990, 1993; Werness et al., 1990). E6 can also activate expression of hTERT, the catalytic subunit of telomerase (Klingelhutz et al., 1996; Oh et al., 2001) as well as bind to a series of PDZ domain-containing cellular proteins. It is not clear which, if any, of these activities is most important for the viral life cycle, because the low-risk E6 proteins fail to degrade p53, activate telomerase or bind PDZ proteins, yet they propagate efficiently. The binding of E6 to p53 appears to play a role in the replication of highrisk genomes, because E6 mutant HPV31 genomes defective for p53 binding integrated into the host genomes (Thomas et al., 1999). However, E6/p53 binding is required for plasmid maintenance only if the corresponding E7 protein binds Rb with high-affinity (Park and Androphy, 2002). When the Rb binding domain of E7 was mutated to that found in low-risk E7 proteins, the E6 protein/p53 interaction was no longer required for plasmid maintenance. Modulation of the activities of cell cycle regulators such as p53, Rb, HDACs, and other unknown cellular proteins is required to facilitate growth of HPV-infected cells and the maintenance of extrachromosomal viral DNAs. It is speculated that the balance of the activities of these cellular proteins is important and that loss of one function influences the action of other factors. The ability of the E6 protein to bind factors other than p53 is likely to be important in the viral life cycle. Additional cellular proteins that bind to the E6 protein include those involved in calcium signaling (Das et al., 2000), cell adhesion (Tong and Howley, 1997), transcriptional control (Degenhardt and Silverstein, 2001b; Kumar et al., 2002; Patel et al., 1999; Ronco et al., 1998),


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DNA synthesis (Kuhne and Banks, 1998), apoptosis (Filippova et al., 2002; Thomas and Banks, 1998), cell cycle control (Gao et al., 2000), DNA repair (Iftner et al., 2002; Srivenugopal and Ali-Osman, 2002), and small G protein signaling (Gao et al., 1999). As noted above, another set of E6 binding proteins possess multiple copies of a protein–protein interaction domain called PDZ (PSD-95/disc large/ZO-1). PDZ proteins are primarily found at the membrane– cytoskeleton interfaces of cell–cell contact and form multiprotein signaling complexes at the inner surface of the membrane. The primary functions of PDZ proteins involve the regulation of cell growth, cell polarity, and cell adhesion in response to cell contact (Craven and Bredt, 1998; Fanning and Anderson, 1999). In overexpression assays, the E6 protein targets these PDZ proteins for degradation via ubiquitin-proteasome pathway (Gardiol et al., 1999; Glaunsinger et al., 2000; Lee et al., 2000; Nakagawa and Huibregtse, 2000; Pim et al., 2000; Thomas et al., 2002). Mutation of the PDZ-binding motif at the C-terminus of high-risk E6 proteins in the context of the complete HPV 31 genome results in reduced cellular growth rates and low plasmid copy number, which in turn results in reduced virion production upon differentiation (Lee and Laimins, 2004). The PDZ binding motif is therefore important in the life cycle of high-risk HPV types. Several other E6-binding partners contain seven amino acid motifs which form part of an -helical structure that forms a recognition sequence for E6 (Be et al., 2001). -Helix binding partners include E6AP, E6BP (Chen et al., 1995), a calcium-binding protein of the CREC family, paxillin (Tong and Howley, 1997; Vande Pol et al., 1998), a focal adhesion protein, E6-TP1 (Gao et al., 1999), a putative Rap1 GAP protein, and IRF3 (Ronco et al., 1998), a transcriptional regulator involved in interferon response. The role of these interactions in the productive life cycle of HPVs remains to be elucidated. A series of binding partners have also been identified for the low-risk E6 proteins, which do not bind p53. These include zyxin (Degenhardt and Silverstein, 2001a), GPS2 (Degenhardt and Silverstein, 2001b), Bak (Thomas and Banks, 1998), and MCM7 (Kukimoto et al., 1998). As in the case of HPV 31, mutation of E6 in the context of the complete HPV 11 genome leads to loss of the ability to maintain viral plasmids (Oh et al., 2004). Of the identified binding partners of low-risk E6 protein, the association with the cellular replication protein MCM7 is most likely to play a role in mediating plasmid maintenance. It is possible that as-yet-unknown binding partners of E6 that are common to both high- and low-risk types play essential roles in virus replication.

4.8.

Effects of HPVs on Epithelial Differentiation

In normal epithelia, the basal layer contains stem cells as well as transitory amplifying cells, which are the primary generators of cells in stratified epithelia (Zegers et al., 2003). Once transitory amplifying cells divide, one of the daughter

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cells exits the cell cycle and migrates away from the basal layer to begin differentiation. The presence of HPV gene products maintains differentiating cells in the cell cycle and blocks the degradation of nuclei (Stubenrauch and Laimins, 1999). Examination of the expression of markers of epithelial cell differentiation such as involucrin, keratin 10, transglutaminase, and filaggrin indicate that HPVs exert modest effects on cell differentiation during the productive stage of the viral life cycle (Ruesch and Laimins, 1998). During the productive life cycle, there is a close correlation between the expression of filaggrin and the induction of late viral functions (Ruesch and Laimins, 1998). HPVpositive cells activate late viral functions in differentiating cells that have reentered S-phase, and fail to express the intermediate filament associated protein, filaggrin. In contrast, the cells that do not amplify viral DNA nor express late genes remain in G1 and synthesize filaggrin. Since viral late gene expression occurs only upon differentiation, it is likely that differentiation-specific cellular factors regulate expression of the viral genome. Elucidation of these factors should help in understanding how the productive phase of the viral life cycle is controlled.

4.9.

Conclusion

The development of genetic methods to study HPVs functions in the productive life cycle has provided an opportunity to understand the action of viral proteins in the proper context under physiological conditions. Further application of these methods will help us to continue to expand our knowledge of how infection by only certain HPV types leads to malignant transformation and to characterize the mechanisms that regulate viral pathogenesis. Acknowledgements. This work was supported by grants from the National Cancer Institute and National Institute of Allergy and Infectious Diseases to L.A.L.

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