Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera

The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.     

Human papillomavirus type 16 E1circumflexE4 contributes to multiple facets of the papillomavirus life cycle

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1(circumflex)E4 is a viral protein that has been ascribed multiple biochemical properties of potential biological relevance to the viral life cycle. To identify the role(s) of the viral E1(circumflex)E4 protein in the HPV life cycle, we characterized the properties of HPV type 16 (HPV16) genomes harboring mutations in the E4 gene in NIKS cells, a spontaneously immortalized keratinocyte cell line that when grown in organotypic raft cultures supports the HPV life cycle. We learned that E1(circumflex)E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1(circumflex)E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1(circumflex)E4 mutant HPV16 genomes there were alterations in the frequency of suprabasal cells supporting DNA synthesis, the levels of viral DNA amplification, and the degree to which the virus perturbs differentiation. Interestingly, the comparison of the phenotypes of various mutations in E4 indicated that the E1(circumflex)E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1(circumflex)E4 is a multifunctional protein and that the different properties of E1(circumflex)E4 contribute to different processes in both the early and late stages of the virus life cycle.     

Human papillomavirus type 31 E5 protein supports cell cycle progression and activates late viral functions upon epithelial differentiation

The function of the E5 protein of human papillomaviruses (HPV) is not well characterized, and controversies exist about its role in the viral life cycle. To determine the function of E5 within the life cycle of HPV type 31 (HPV31) we first constructed HPV31 mutant genomes that contained an altered AUG initiation codon or stop codons in E5. Cell lines were established which harbored transfected wild-type or E5 mutant HPV31 genomes. These cell lines all maintained episomal copies of HPV31 and revealed similar phenotypes with respect to growth rate, early gene expression, and viral copy number in undifferentiated monolayer cultures. Following epithelial differentiation, genome amplification and differentiation-dependent late gene expression were observed in mutant cell lines, but at a rate significantly reduced from that observed in cells containing the wild-type genomes. Organotypic raft cultures indicated that E5 does not effect the expression of differentiation markers but does reduce expression of late viral proteins. Western analysis and immunofluorescence staining for cyclins during epithelial differentiation revealed a decreased expression of cyclin A and B in E5 mutant cells compared to HPV wild-type cells. Using a replating assay, a significant reduction in colony-forming ability was detected in the absence of E5 expression when cells containing wild-type or E5 mutant HPV genomes were allowed to proliferate following 24 h in suspension-induced differentiation. This suggests that HPV E5 modifies the differentiation-induced cell cycle exit and supports the ability of HPV31-positive keratinocytes to retain proliferative competence. In these studies, E5 was found to have little effect on the levels of the epidermal growth factor receptor (EGFR) or on its phosphorylation status. This indicates that EGFR is not a target of E5 action. Our results propose a role for high risk HPV E5 in modulation of late viral functions through activation of proliferative capacity in differentiated cells. We suspect that the primary target of E5 is a membrane protein or receptor that then acts to alter the levels or activities of cell cycle regulators.     

Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle

Human papillomaviruses (HPVs) are small circular DNA viruses that cause warts. Infection with high-risk anogenital HPVs, such as HPV type 16 (HPV16), is associated with human cancers, specifically cervical cancer. The life cycle of HPVs is intimately tied to the differentiation status of the host epithelium and has two distinct stages: the nonproductive stage and the productive stage. In the nonproductive stage, which arises in the poorly differentiated basal epithelial compartment of a wart, the virus maintains itself as a low-copy-number nuclear plasmid. In the productive stage, which arises as the host cell undergoes terminal differentiation, viral DNA is amplified; the capsid genes, L1 and L2, are expressed; and progeny virions are produced. This stage of the viral life cycle relies on the ability of the virus to reprogram the differentiated cells to support DNA synthesis. Papillomaviruses encode multiple oncoproteins, E5, E6, and E7. In the present study, we analyze the role of one of these viral oncogenes, E5, in the viral life cycle. To assess the role of E5 in the HPV16 life cycle, we introduced wild-type (WT) or E5 mutant HPV16 genomes into NIKS, a keratinocyte cell line that supports the papillomavirus life cycle. By culturing these cells under conditions that allow them to remain undifferentiated, a state similar to that of basal epithelial cells, we determined that E5 does not play an essential role in the nonproductive stage of the HPV16 life cycle. To determine if E5 plays a role in the productive stage of the viral life cycle, we cultured keratinocyte populations in organotypic raft cultures, which promote the differentiation and stratification of epithelial cells. We found that cells harboring E5 mutant genomes displayed a quantitative reduction in the percentage of suprabasal cells undergoing DNA synthesis, compared to cells containing WT HPV16 DNA. This reduction in DNA synthesis, however, did not prevent amplification of viral DNA in the differentiated cellular compartment. Likewise, late viral gene expression and the perturbation of normal keratinocyte differentiation were retained in cells harboring E5 mutant genomes. These data demonstrate that E5 plays a subtle role during the productive stage of the HPV16 life cycle.     

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates epithelial differentiation. There are many uses for this system. For instance, the ability to grow three-dimensional organotypic raft cultures of keratinocytes has been an important milestone in the study of human papillomavirus (HPV)¹. The life cycle of HPV is tightly linked to the differentiation of squamous epithelium². Organotypic epithelial raft cultures as demonstrated here reproduce the entire papillomavirus life cycle, including virus production^3,4,5. In addition, these raft cultures exhibit dysplastic lesions similar to those observed upon in vivo infection with HPV. Hence this system can also be used to study epithelial cell cancers, as well as the effect of drugs on epithelial cell differentiation in general. Originally developed by Asselineau and Prunieras⁶ and modified by Kopan et al.⁷, the organotypic epithelial raft culture system has matured into a general, relatively easy culture model, which involves the growth of cells on collagen plugs maintained at an air-liquid interface (Figure 1A). Over the course of 10-14 days, the cells stratify and differentiate, forming a full thickness epithelium that produces differentiation-specific cytokeratins. Harvested rafts can be examined histologically, as well as by standard molecular and biochemical techniques. In this article, we describe a method for the generation of raft cultures from primary human keratinocytes. The same technique can be used with established epithelial cell lines, and can easily be adapted for use with epithelial tissue from normal or diseased biopsies⁸. Many viruses target either the cutaneous or mucosal epithelium as part of their replicative life cycle. Over the past several years, the feasibility of using organotypic raft cultures as a method of studying virus-host cell interactions has been shown for several herpesviruses, as well as adenoviruses, parvoviruses, and poxviruses⁹. Organotypic raft cultures can thus be adapted to examine viral pathogenesis, and are the only means to test novel antiviral agents for those viruses that are not cultivable in permanent cell lines.     

Interactions with pocket proteins contribute to the role of human papillomavirus type 16 E7 in the papillomavirus life cycle

Human papillomaviruses (HPVs), most commonly the HPV16 genotype, are the principle etiological determinant for cervical cancer, a common cancer worldwide resulting in over 200,000 deaths annually. The oncogenic properties of HPVs are attributable in part to the virally encoded protein E7, best known for its ability to bind to and induce the degradation of the retinoblastoma tumor suppressor, pRb, and related "pocket proteins" p107 and p130. Previously, we defined a role for E7 in the productive stage of the HPV16 life cycle, which takes place in stratified squamous epithelia. HPV perturbs the normal processes of cell growth and differentiation of stratified squamous epithelia. HPVs reprogram cells to support continued DNA synthesis and inhibit their differentiation in the suprabasal compartment of the epithelia, where cells normally have withdrawn from the cell cycle and initiated a well-defined pattern of terminal differentiation. These virus-induced perturbations, which contribute to the production of progeny HPVs, are dependent on E7. In this study, we define the mechanism of action by which E7 contributes to the productive stage of the HPV16 life cycle. We found that the ability of HPV16 to reprogram suprabasal cells to support DNA synthesis correlates with E7's ability to bind pocket proteins but not its ability to induce their degradation. In contrast, the ability of HPV16 to perturb differentiation correlated with both E7's binding to and degradation of pocket proteins. These data indicate that different hallmarks of the productive stage of the HPV16 life cycle rely upon different sets of requirements for E7.     

Expression of the HPV16 E7 gene generates proliferation in stratified squamous cell cultures which is independent of endogenous p53 levels.

Monolayer cultures of human foreskin and ectocervical epithelial cells were infected with retroviral vectors expressing HPV16 oncogenes, selected for G418 resistance, and cultured organotypically so that they reformed the fully differentiated, stratified squamous tissues from which they were originally derived. Expression of HPV16 E7 prevented cell cycle withdrawal in the suprabasal layers of these stratified cultures but had no effect on terminal differentiation. Cultures expressing E7 alone and those coexpressing E6 and E7 were identical in terms of suprabasal proliferation and terminal differentiation, but they differed in expression of the endogenous tumor suppressor protein p53. Immunohistochemically detectable p53 protein localized to the proliferative compartment in normal and E7-containing cultures but was undetectable in those cultures which coexpressed E6 and E7. This result suggests that E7-induced suprabasal proliferation is independent of the steady-state level of p53.     

Induction of the upstream regulatory region of human papillomavirus type 31 by dexamethasone is differentiation dependent

Glucocorticoids have been shown to play a role in the transforming abilities of human papillomaviruses (HPVs), and glucocorticoid response elements (GREs) have been identified in the upstream regulatory regions (URRs) of various HPV types. These findings have made glucocorticoids potential therapeutic targets for HPV infection. We have previously shown that the URR of HPV type 31 (HPV31) is insensitive to induction by the synthetic glucocorticoid dexamethasone (dex) in monolayer culture, despite the identification of three potential GREs in the 5' region of the URR. Due to the fact that the HPV life cycle is intimately linked to the differentiation of the host tissue, we chose to determine whether the URR of HPV31 was inducible by dex under differentiating conditions. Upon suspension of cells in a semisolid medium of methylcellulose, we found that the URR of HPV31 was inducible by dex. The three GREs appear to play roles as independent repressors of this inducibility. By 5' deletion analysis, the element(s) responsible for this induction was localized to nucleotides (nt) 7238 to 7557. Furthermore, we found that the region between nt 7883 and 7900 appears to act as a repressor of dex inducibility. These findings indicate that epithelial differentiation has a profound effect on the action of dex on the URR of HPV31, suggesting that glucocorticoids play an important role in the differentiation-dependent life cycle of HPV.     

Bap31 is a novel target of the human papillomavirus E5 protein

The E5 proteins of human papillomaviruses (HPVs) are small hydrophobic proteins that are expressed in the early and late stages of the viral life cycle; however, their role in HPV pathogenesis is not clearly understood. In this study, a split-ubiquitin yeast (Saccharomyces cerevisiae) two-hybrid system was used to identify B-cell-associated protein 31 (Bap31) as a binding partner of HPV E5 proteins. The association of these proteins was confirmed by coimmunoprecipitation of complexes of Bap31 with either HPV type 16 (HPV16) or HPV31 E5. In addition, Bap31 and E5 were found to colocalize in perinuclear patterns consistent with localization to the endoplasmic reticulum. Mutational analysis of E5 identified amino acids in the extreme C terminus as important for stabilizing the interaction with Bap31. Deletion of these C-terminal amino acids of E5 in the context of complete HPV31 genomes resulted in impaired proliferative capacity of HPV-positive keratinocytes following differentiation. When small interfering RNAs were used to reduce the levels of Bap31, the proliferative ability of HPV-positive keratinocytes upon differentiation was also reduced, implicating Bap31 as a regulator of this process. These studies identify a novel binding partner of the high-risk HPV E5 proteins and provide insight into how the E5 proteins may modulate the life cycle in differentiating cells.     

The E7 open reading frame acts in cis and in trans to mediate differentiation-dependent activities in the human papillomavirus type 16 life cycle

Human papillomaviruses (HPVs) are the causative agents of several important genital and other mucosal cancers. The HPV16 E7 gene encodes a viral oncogene that is necessary for the continued growth of cancer cells, but its role in the normal, differentiation-dependent life cycle of the virus is not fully understood. The function of E7 in the viral life cycle was examined using a series of mutations of E7 created in the context of the complete HPV16 genome. The effect of these E7 mutations on key events of the viral life cycle, including immortalization, episomal maintenance, late promoter activation, and infectious virion synthesis, was examined. Our studies show that the pRb binding domain is indispensable for early viral activities, whereas the C-terminal zinc finger domain contributed primarily to very late events. Mutations of the casein kinase II phosphorylation site caused a complex phenotype involving both the function of E7 protein and a cis element necessary for the activation of the late promoter, identifying for the first time a promoter element important for late promoter function in the context of the viral genome. All mutant genomes tested showed reduced viral titers following growth in organotypic raft cultures. These studies clarify the role of E7 as a regulator of late events in the differentiation-dependent HPV life cycle.