Nuclear Export of Human Papillomavirus Type 31 E1 Is Regulated by Cdk2 Phosphorylation and Required for Viral Genome Maintenance

The initiator protein E1 from human papillomavirus (HPV) is a helicase essential for replication of the viral genome. E1 contains three functional domains: a C-terminal enzymatic domain that has ATPase/helicase activity, a central DNA-binding domain that recognizes specific sequences in the origin of replication, and a N-terminal region necessary for viral DNA replication in vivo but dispensable in vitro. This N-terminal portion of E1 contains a conserved nuclear export signal (NES) whose function in the viral life cycle remains unclear. In this study, we provide evidence that nuclear export of HPV31 E1 is inhibited by cyclin E/A-Cdk2 phosphorylation of two serines residues, S92 and S106, located near and within the E1 NES, respectively. Using E1 mutant proteins that are confined to the nucleus, we determined that nuclear export of E1 is not essential for transient viral DNA replication but is important for the long-term maintenance of the HPV episome in undifferentiated keratinocytes. The findings that E1 nuclear export is not required for viral DNA replication but needed for genome maintenance over multiple cell divisions raised the possibility that continuous nuclear accumulation of E1 is detrimental to cellular growth. In support of this possibility, we observed that nuclear accumulation of E1 dramatically reduces cellular proliferation by delaying cell cycle progression in S phase. On the basis of these results, we propose that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.Human papillomaviruses (HPV) are small double-stranded DNA viruses that infect keratinocytes of the differentiating epithelium of the skin or mucosa (reviewed in references 4 and 63). Of more than 150 different HPV types identified thus far, about 25 infect the anogenital region (9). The low-risk types, such as HPV11 and HPV6, are associated with the development of genital warts, while the high-risk types, such as HPV16, -18, and -31, cause high-grade lesions that can progress to invasive cervical carcinoma (17, 38, 61).The HPV life cycle is coupled with the differentiation program that keratinocytes undergo in the epithelium. After infection of the basal cell layer of the epithelium, the virus establishes and maintains its genome as an extrachromosomal element (episome) in the nucleus of infected cells. While the viral episome is maintained at low levels in basal cells, its amplification to a high copy number is trigged in the upper layers of the epithelium by the action of the viral oncogenes E6 and E7 and the differentiation of the infected keratinocytes (reviewed in reference 21). Replication of the HPV genome relies on the viral proteins E1 and E2 and the host DNA replication machinery. Viral DNA replication is initiated by the binding of E2 to specific sites on the viral origin where it facilitates the recruitment and assembly of E1 into a double hexamer that is required to unwind DNA ahead of the bidirectional replication fork (3, 14, 15, 31, 33, 36, 43-45, 52, 60). In addition to its helicase activity, E1 interacts with several cellular replication factors, including polymerase α-primase, replication protein A (RPA), and topoisomerase I, to replicate the viral episome (5, 6, 19, 32, 35, 39).E1, which belongs to helicase superfamily III (SF3) (22, 26), can be divided into three functional regions. Its C-terminal domain has ATPase and helicase activity and can self-assemble into hexamers. It is also this domain that is contacted by E2 to recruit E1 at the origin (50, 57, 58). The middle portion of E1 encompasses the origin-binding domain (OBD) that binds and dimerizes on specific sequences in the origin (55, 56). We and others previously found that a fragment of E1 containing only the C-terminal enzymatic domain and the OBD is capable of supporting viral DNA replication in vitro but is inactive in vivo (2, 51). This suggested that the N-terminal region of E1 plays an essential regulatory function in vivo. As such, it has been shown for HPV11 E1 that this region contains a cyclin E/A-Cdk2 (cyclin-dependent kinase 2) binding motif (CBM), a bipartite nuclear localization signal (NLS) and an CRM1-dependent nuclear export signal (NES), which together regulate the nucleocytoplasmic shuttling of the protein (10, 30, 34). Specifically, it has been shown that phosphorylation of HPV11 E1 on three serine residues within its N-terminal region inhibits its nuclear export (10, 62). Interestingly, bovine papillomavirus (BPV) E1 was also shown to shuttle between the nucleus and the cytoplasm in a phosphorylation-dependent manner. In this case, however, Cdk2 phosphorylation was found to promote, rather than inhibit, the export of the viral helicase (24). This apparent discrepancy between HPV11 and BPV E1 prompted us to examine the regulation of a third E1 protein, specifically that of the high-risk HPV31.We report here that HPV31 E1 also shuttles between the nucleus and the cytoplasm through its conserved NLS and NES. We determined that nuclear export of HPV31 E1 is dependent on the CRM1 export pathway and is inhibited by Cdk2 phosphorylation of serines 92 and 106. We also found that nuclear export of E1 is not required for transient viral DNA replication and thus investigated its role in viral genome maintenance and amplification in immortalized keratinocytes. In contrast to the wild type (WT), a mutant genome carrying a defective E1 NES was poorly maintained and progressively lost upon cell division, indicating that nuclear export of E1 is required for long-term maintenance of the viral episome. Because nuclear export of E1 is not required for viral DNA replication per se but needed for episomal maintenance over several cell divisions, we investigated the possibility that continuous accumulation of E1 into the nucleus is detrimental to cellular proliferation. In support of this possibility, we found that the accumulation of E1 at high levels in the nucleus impedes cellular proliferation by delaying cell cycle progression in the S phase. In addition, we found that this delay was alleviated when nuclear export of E1 was increased. Altogether, these results suggest that nuclear export of E1 is required, at least in part, to limit accumulation of this viral helicase in the nucleus in order to prevent its detrimental effect on cellular proliferation.     

Differential Requirements for Conserved E2 Binding Sites in the Life Cycle of Oncogenic Human Papillomavirus Type 31

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062–3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.     

人乳头瘤病毒E2蛋白及其相关疫苗

人乳头瘤病毒（human papilloma virus,HPV）感染在全球范围内颇为常见,其与肛门生殖器疣、生殖器肿瘤的发生关系密切。研究发现,乳头瘤的形成与HPVE2蛋白密不可分,该蛋白质涉及到病毒生命周期的各个阶段,与病毒的有丝分裂、其他早期蛋白的转录及细胞凋亡有关。近年来,各国学者利用E2蛋白的特性研制出E2相关疫苗,分别使用不同的重组病毒来传输E2,或是使用纯化的E2蛋白或E2融合蛋白,运输至体内的HPV转化细胞和／或HPV感染细胞中,以期达到防治HPV感染相关疾病的目的。     

The Carboxyl-Terminal Region of the Human Papillomavirus Type 16 E1 Protein Determines E2 Protein Specificity during DNA Replication

The mechanism of DNA replication is conserved among papillomaviruses. The virus-encoded E1 and E2 proteins collaborate to target the origin and recruit host DNA replication proteins. Expression vectors of E1 and E2 proteins support homologous and heterologous papillomaviral origin replication in transiently transfected cells. Viral proteins from different genotypes can also collaborate, albeit with different efficiencies, indicating a certain degree of specificity in E1-E2 interactions. We report that, in the assays of our study, the human papillomavirus type 11 (HPV-11) E1 protein functioned with the HPV-16 E2 protein, whereas the HPV-16 E1 protein exhibited no detectable activity with the HPV-11 E2 protein. Taking advantage of this distinction, we used chimeric E1 proteins to delineate the E1 protein domains responsible for this specificity. Hybrids containing HPV-16 E1 amino-terminal residues up to residue 365 efficiently replicated either viral origin in the presence of either E2 protein. The reciprocal hybrids containing amino-terminal HPV-11 sequences exhibited a high activity with HPV-16 E2 but no activity with HPV-11 E2. Reciprocal hybrid proteins with the carboxyl-terminal 44 residues from either E1 had an intermediate property, but both collaborated more efficiently with HPV-16 E2 than with HPV-11 E2. In contrast, chimeras with a junction in the putative ATPase domain showed little or no activity with either E2 protein. We conclude that the E1 protein consists of distinct structural and functional domains, with the carboxyl-terminal 284 residues of the HPV-16 E1 protein being the primary determinant for E2 specificity during replication, and that chimeric exchanges in or bordering the ATPase domain inactivate the protein.     

Multimer Formation Is Not Essential for Nuclear Export of Human T-Cell Leukemia Virus Type 1 Rex trans-Activator Protein

The Rex trans-regulatory protein of human T-cell leukemia virus type 1 (HTLV-1) is required for the nuclear export of incompletely spliced and unspliced viral mRNAs and is therefore essential for virus replication. Rex is a nuclear phosphoprotein that directly binds to its cis-acting Rex response element RNA target sequence and constantly shuttles between the nucleus and cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced viral RNA. Three protein domains which mediate nuclear import-RNA binding, nuclear export, and Rex oligomerization have been mapped within the 189-amino-acid Rex polypeptide. Here we identified a different region in the carboxy-terminal half of Rex which is also required for biological activity. In inactive mutants with mutations that map within this region, as well as in mutants that are deficient in Rex-specific multimerization, Rex trans activation could be reconstituted by fusion to a heterologous leucine zipper dimerization interface. The intracellular trafficking capabilities of wild-type and mutant Rex proteins reveal that biologically inactive and multimerization-deficient Rex mutants are still efficiently translocated from the nucleus to the cytoplasm. This observation indicates that multimerization is essential for Rex function but is not required for nuclear export. Finally, we are able to provide an improved model of the HTLV-1 Rex domain structure.     

Production of Human Papillomavirus Type 16 E7 Protein in Lactococcus lactis

The E7 protein of human papillomavirus type 16 was produced in Lactococcus lactis. Secretion allowed higher production yields than cytoplasmic production. In stationary phase, amounts of cytoplasmic E7 were reduced, while amounts of secreted E7 increased, suggesting a phase-dependent intracellular proteolysis. Fusion of E7 to the staphylococcal nuclease, a stable protein, resulted in a highly stable cytoplasmic protein. This work provides new candidates for development of viral screening systems and for oral vaccine against cervical cancer.     

Evidence of Diversifying Selection in Human Papillomavirus Type 16 E6 But Not E7 Oncogenes

Human papillomavirus type 16 is a common sexually transmitted pathogen capable of giving rise to cervical intraepithelial neoplasia and invasive carcinoma through the expression and activity of two adjacent oncogenes: E6 and E7. Naturally occurring amino acid variation is commonly observed in the E6 protein but to a much lesser extent in E7. In order to investigate the evolutionary mechanisms involved in the generation and maintenance of this variation, we examine 42 distinct E6-E7 haplotypes using codon-based genealogical techniques. These techniques involve estimation of the ratio of nonsynonymous to synonymous substitutions (dn/ds) and allow testing for directional (positive) natural selection. Positive selection was detected for four codon sites within the E6 oncogene but not in any E7 codons. The amino acid compositions and locations of selected sites are described. Possible sources of natural selection including antiviral immune pressure and polymorphism of host cellular proteins are discussed.