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Animal models of viral pathogenesis are essential tools in human disease research. Human papillomaviruses (HPVs) are a significant public health issue due to their widespread sexual transmission and oncogenic potential. Infection-based models of papillomavirus pathogenesis have been complicated by their strict species and tissue specificity. In this GEM review, we discuss the discovery of a murine papillomavirus, MmuPV1, and how its experimental use represents a major advancement in models of papillomavirus-induced pathogenesis/carcinogenesis, and their transmission. [ABSTRACT FROM AUTHOR]

The family of human papillomaviruses (HPV) includes over 400 genotypes. Genus a genotypes generally infect the anogenital mucosa, and a subset of these HPV are a necessary, but not sufficient, cause of cervical cancer. Of the 13 high-risk (HR) and 11 intermediate-risk (IR) HPV associated with cervical cancer, genotypes 16 and 18 cause 50% and 20% of cases, respectively, whereas HPV16 dominates in other anogenital and oropharyngeal cancers. A plethora of bHPVs are associated with cutaneous squamous cell carcinoma (CSCC), especially in sun-exposed skin sites of epidermodysplasia verruciformis (EV), AIDS, and immunosuppressed patients. Licensed L1 virus-like particle (VLP) vaccines, such as Gardasil 9, target a subset of aHPV but no bHPV. To comprehensively target both a- and bHPVs, we developed a two-component VLP vaccine, RG2-VLP, in which L2 protective epitopes derived from a conserved aHPV epitope (amino acids 17 to 36 of HPV16 L2) and a consensus bHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6D/D or Tmc8D/D) with RG2-VLP induced robust L2-specific antibody titers and protected against b-type HPV5. RG2-VLP protected rabbits against 17 aHPV, including those not covered by Gardasil 9. HPV16- and HPV18-specific neutralizing antibody responses were similar between RG2-VLP- and Gardasil 9-vaccinated animals. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all bHPVs tested. Taken together, these observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent aHPV-driven anogenital, oropharyngeal, and bHPV-associated cutaneous cancers. IMPORTANCE Licensed preventive HPV vaccines are composed of VLPs derived by expression of major capsid protein L1. They confer protection generally restricted to infection by the aHPVs targeted by the up-to-9-valent vaccine, and their associated anogenital cancers and genital warts, but do not target βHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine protective in animal models against known oncogenic aHPVs as well as diverse bHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2. [ABSTRACT FROM AUTHOR]

We previously reported that the cellular transcription factor hypoxiainducible factor 1+ (HIF-1+) binds a hypoxia response element (HRE) located within the promoter of Epstein-Barr virus's (EBV's) latent-lytic switch BZLF1 gene, Zp, inducing viral reactivation. In this study, EBV-infected cell lines derived from gastric cancers and Burkitt lymphomas were incubated with HIF-1+-stabilizing drugs: the iron chelator deferoxamine (Desferal [DFO]), a neddylation inhibitor (pevonedistat [MLN- 4924]), and a prolyl hydroxylase inhibitor (roxadustat [FG-4592]). DFO and MLN-4924, but not FG-4592, induced accumulation of both lytic EBV proteins and phosphorylated p53 in cell lines that contain a wild-type p53 gene. FG-4592 also failed to activate transcription from Zp in a reporter assay despite inducing accumulation of HIF-1+ and transcription from another HRE-containing promoter. Unexpectedly, DFO failed to induce EBV reactivation in cell lines that express mutant or no p53 or when p53 expression was knocked down with short hairpin RNAs (shRNAs). Likewise, HIF-1+ failed to activate transcription from Zp when p53 was knocked out by CRISPR-Cas9. Importantly, DFO induced binding of p53 as well as HIF-1+ to Zp in chromatin immunoprecipitation (ChIP) assays, but only when the HRE was present. Nutlin-3, a drug known to induce accumulation of phosphorylated p53, synergized with DFO and MLN-4924 in inducing EBV reactivation. Conversely, KU-55933, a drug that inhibits ataxia telangiectasia mutated, thereby preventing p53 phosphorylation, inhibited DFO-induced EBV reactivation. Lastly, activation of Zp transcription by DFO and MLN-4924 mapped to its HRE. Thus, we conclude that induction of BZLF1 gene expression by HIF-1+ requires phosphorylated, wild-type p53 as a coactivator, with HIF-1+ binding recruiting p53 to Zp. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression. [ABSTRACT FROM AUTHOR]

The life cycle of human papillomaviruses (HPVs) is tightly linked to the differentiation program of the host's stratified epithelia that it infects. E1∧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∧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∧E4 contributes to the replication of the viral plasmid genome as a nuclear plasmid in basal cells, in which we also found E1∧E4 protein to be expressed at low levels. In the suprabasal compartment of organotypic raft cultures harboring E1∧EA 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∧E4 protein-encoding requirements for these various processes differed. These data support the hypothesis that E1∧E4 is a multifunctional protein and that the different properties of E1∧E4 contribute to different processes in both the early and late stages of the virus life cycle. [ABSTRACT FROM AUTHOR]

High-risk human papillomaviruses encode two oncogenes, E6 and E7, expressed in nearly all cervical cancers. Although E7 protein is best known for its ability to inactivate the retinoblastoma tumor suppressor protein, pRb, many other activities for E7 have been proposed in in vitro studies. Herein, we describe studies that allowed us to define unambiguously the pRb-dependent and -independent activities of E7 for the first time in vivo. In these studies, we crossed mice transgenic for human papillomavirus 16 E7 to knock-in mice genetically engineered to express a mutant form of pRb (pRbΔLXCXE) that is selectively defective for binding E7. pRb inactivation was necessary for E7 to induce DNA synthesis and to overcome differentiation-dependent cell cycle withdrawal and DNA damage-induced cell cycle arrest. While most of E7's effects on epidermal differentiation were found to require pRb inactivation, a modest delay in terminal differentiation with resulting hyperplasia was observed in E7 mice on the RbΔLXCXE mutant background. E7-induced p21 upregulation was also pRb dependent, and genetic Rb inactivation was sufficient to reproduce this effect. While E7-mediated p21 induction was partially p53 dependent, neither p53 nor p21 induction by E7 required p19ARF. These data show that E7 upregulates the expression of p53 and p21 via pRb-dependent mechanisms distinct from the proposed plg-Mdm2 pathway. These results extend our appreciation of the importance of pRb as a relevant target for high-risk E7 oncoproteins. [ABSTRACT FROM AUTHOR]

Papillomaviruses contain small double-stranded DNA genomes that are maintained in persistently infected mammalian host epithelia as nuclear plasmids and rely upon the host replication machinery for replication. Papillomaviruses encode a DNA helicase, E1, which can specifically bind to the viral genome and support DNA synthesis. Under some conditions in mammalian cells, E1 is not required for viral DNA synthesis, leading to the hypothesis that papillomavirus DNA can be replicated solely by the host replication machinery. This machinery is highly conserved among eukaryotes. We and others found that papillomavirus DNA could replicate in a simple eukaryote, Saccharomyces cerevisiae. Specifically, papillomavirus DNA could substitute for the function of the autonomously replicating sequence (ARS) and centromere (CEN) elements that are normally both required for the stable replication of extrachromosomal DNAs in yeast. Furthermore, this form of replication in yeast was E1 independent. In this study, we map the elements in the human papillomavirus type 16 (HPV16) genome that can substitute for yeast ARS and CEN elements. A single element, termed rep, was identified that can substitute for ARS, and multiple elements, termed mtc, could substitute for CEN. The location of one of these mtc elements overlaps the location of rep, and this approximately 1,000-bp region of HPV16 was sufficient to support stable replication of a bacterial-yeast shuttle plasmid deleted of both ARS and CEN elements. [ABSTRACT FROM AUTHOR]

Human papillomavirus type 16 (HPV16) can cause cervical cancer. Expression of the viral E1ˆE4 protein is lost during malignant progression, but in premalignant lesions, E1ˆE4 is abundant in cells supporting viral DNA amplification. Expression of 16E1ˆE4 in cell culture causes G2 cell cycle arrest. Here we show that unlike many other G2 arrest mechanisms, 16E1ˆE4 does not inhibit the kinase activity of the Cdk1/cyclin B1 complex. Instead, 16E1ˆE4 uses a novel mechanism in which it sequesters Cdk1/cyclin B1 onto the cytokeratin network. This prevents the accumulation of active Cdk1/cyclin B1 complexes in the nucleus and hence prevents mitosis. A mutant 16E1ˆE4 (T22A, T23A) which does not bind cyclin B1 or alter its intracellular location fails to induce G2 arrest. The significance of these results is highlighted by the observation that in lesions induced by HPV16, there is evidence for Cdk1/cyclin B1 activity on the keratins of 16E1ˆE4-expressing cells. We hypothesize that E1ˆE4-induced G2 arrest may play a role in creating an environment optimal for viral DNA replication and that loss of E1ˆE4 expression may contribute to malignant progression. [ABSTRACT FROM AUTHOR]

Prior studies, which have relied upon the use of pseudovirions generated in heterologous cell types, have led to sometimes conflicting conclusions regarding the role of the minor capsid protein of papillomaviruses, L2, in the viral life cycle. In this study we carry out analyses with true virus particles assembled in the natural host cell to assess L2's role in the viral infectious life cycle. For these studies we used the organotypic (raft) culture system to recapitulate the full viral life cycle of the high-risk human papillomavirus HPV31, which was either wild type or mutant for L2. After transfection, the L2 mutant HPV31 genome was able to establish itself as a nuclear plasmid in proliferating populations of poorly differentiated (basal-like) human keratinocytes and to amplify its genome to high copy number, support late viral gene expression, and cause formation of virus particles in human keratinocytes that had been induced to undergo terminal differentiation. These results indicate that aspects of both the nonproductive and productive phases of the viral life cycle occur normally in the absence of functional L2. However, upon the analysis of the virus particles generated, we found an approximate 10-fold reduction in the amount of viral DNA encapsidated into L2-deficient virions. Furthermore, there was an over-100-fold reduction in the infectivity of L2-deficient virus. Because the latter deficiency cannot be accounted for solely by the 10-fold decrease in encapsidation, we conclude that L2 contributes to at least two steps in the production of infectious virus. [ABSTRACT FROM AUTHOR]

The biological activities of the papillomavirus E2 protein in transcription, replication, and maintenance of the papillomavirus genome rely on the E2 protein's ability to bind that genome specifically. The E2 binding sites (E2BSs), located within the long control region (LCR) of human papillomavirus (HPV) genomes, contain potential sites for 5' methylation at cytosine (CpG) residues. The E2 protein's capacity to bind E2BS in vitro is inhibited by methylation of these cytosines (59). Herein, we describe experiments to assess the influence of methylation on E2 function in cells. E2's ability to activate transcription was inhibited by the global methylation of CpG dinucleotides in E2-responsive transcriptional templates or when only the CpG dinucleotides within the E2BSs of a transcriptional template were methylated. Thus at least one biological activity of E2 that is dependent on its ability to bind DNA in a site-specific manner is influenced by the methylation status of its cognate binding site. The activity of DNA methylases is influenced by the differentiation status of mammalian cells. The life cycle of HPVs is tied to the differentiation of its host cells within stratified squamous epithelia. To investigate whether methylation of the papillomavirus genomes is influenced by the differentiation status of host epithelial cells, we analyzed HPV16 DNA harvested from a cervical epithelial cell line that was isolated from an HPV16-infected patient. We found, using bisulfite treatment to discriminate between methylated and unmethylated cytosines, that the HPV16 LCR was selectively hypomethylated in highly differentiated cell populations. In contrast, the HPV16 LCR from poorly differentiated, basal cell-like cells contained multiple methylated cytosines and were often methylated at E2BSs, particularly E2BS[sub 2]. These experiments indicate that the methylation state of the viral genome, and particular that of E2BSs, may vary during the viral life cycle, providing a novel means for modulating E2 function. These studies also uncovered an extensive pattern of methylation at non-CpG dinucleotides indicative of de novo methylation. The potential implications of this de novo methylation pattern are discussed. [ABSTRACT FROM AUTHOR]

compares precancerous lesions caused by human papillomaviruses (HPV) type 16 with lesions caused by HPV types that are not generally associated with human cancer. Generation and labeling of antibodies; Immunofluorescence and immunoenzymatic staining; Visualization of events during productive papillomavirus infection using antibodies to MCM, E7, E4, and L1.

Human papillomaviruses (HPVs) are the causative agent of warts. Infections with high-risk HPVs are associated with anogenital and head and neck cancers. One of the viral genes responsible for HPV's oncogenic activity is E6. Mice expressing the HPV-16 E6 protein in their epidermis (K14E6[sup WT]) develop epithelial hyperplasia and squamous carcinomas. Numerous cellular proteins interact with E6, some of which can be grouped based on common amino acid motifs in their E6-binding domains. One such group, the PDZ partners, including hDLG, hSCRIBBLE, MUPP1, and MAGI, bind to the carboxy-terminal four amino acids of E6 through their PDZ domains. E6's interaction with the PDZ partners leads to their degradation. Additionally, E6's binding to PDZ proteins has been correlated with its ability to transform baby rat kidney cells in tissue culture and to confer tumorigenicity onto cells in xenograft experiments. To address whether the ability of E6 to bind PDZ domain partners is necessary for E6 to confer epithelial hyperproliferation in vivo, we generated transgenic mice that express in stratified squamous epithelia a mutant of E6 lacking the last six amino acids at its carboxyl terminus, E6[sup Δ146-151], from the human keratin 14 (K14) promoter. The K14E6[sup Δ146-151] mice exhibit a radiation response similar to that of the K14E6[sup WT] mice, demonstrating that this protein, as predicted, retains an ability to inactivate p53. However, the K14E6[sup Δ146-151] mice fail to display epithelial hyperplasia. These results indicate that an interaction of E6 with PDZ partners is necessary for its induction of epithelial hyperplasia. [ABSTRACT FROM AUTHOR]

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... [ABSTRACT FROM AUTHOR]

High-risk human papillomaviruses (HPVs) deregulate epidermal differentiation and cause anogenital and head and neck squamous cell carcinomas (SCCs). The E7 gene is considered the predominant viral oncogene and drives proliferation and genome instability. While the implementation of routine screens has greatly reduced the incidence of cervical cancers which are almost exclusively HPV positive, the proportion of HPV-positive head and neck SCCs is on the rise. High levels of HPV oncogene expression and genome load are linked to disease progression, but genetic risk factors that regulate oncogene abundance and/or genome amplification remain poorly understood. Fanconi anemia (FA) is a genome instability syndrome characterized at least in part by extreme susceptibility to SCCs. FA results from mutations in one of 15 genes in the FA pathway, whose protein products assemble in the nucleus and play important roles in DNA damage repair. We report here that loss of FA pathway components FANCA and FANCD2 stimulates E7 protein accumulation in human keratinocytes and causes increased epithelial proliferation and basal cell layer expansion in the HPV-positive epidermis. Additionally, FANCD2 loss stimulates HPV genome amplification in differentiating cells, demonstrating that the intact FA pathway functions to restrict the HPV life cycle. These findings raise the possibility that FA genes suppress HPV infection and disease and suggest possible mechanism(s) for reported associations of HPV with an FA cohort in Brazil and for allelic variation of FA genes with HPV persistence in the general population. [ABSTRACT FROM AUTHOR]

Fanconi anemia (FA) patients have an increased risk for squamous cell carcinomas (SCCs) at sites of predilection for infection with high-risk human papillomavirus (HPV) types, including the oral cavity and the anogenital tract. We show here that activation of the FA pathway is a frequent event in cervical SCCs. We found that FA pathway activation is triggered mainly by the HPV type 16 (HPV-16) E7 oncoprotein and is associated with an enhanced formation of large FANCD2 foci and recruitment of FANCD2 as well as FANCD1/BRCA2 to chromatin. Episomal expression of HPV-16 oncoproteins was sufficient to activate the FA pathway. Importantly, the expression of HPV-16 E7 in FA-deficient cells led to accelerated chromosomal instability. Taken together, our findings establish the FA pathway as an early host cell response to high-risk HPV infection and may help to explain the greatly enhanced susceptibility of FA patients to squamous cell carcinogenesis at anatomic sites that are frequently infected by high-risk HPVs. [ABSTRACT FROM AUTHOR]

Viral infections in human infants frequently follow a protracted course, with higher viral loads and delayed viral clearance compared to viral infections in older children. To identify the mechanisms responsible for this protracted pattern of infection, we developed an infant infection murine model using the well-characterized lymphocytic choriomeningitis virus (LCMV) WE strain in 2-week-old BALB/c mice. In contrast to adult mice, in which viral clearance occurred as expected 8 days after infection, LCMV titers persisted for several weeks after infection of infant mice. LCMV-specific effector CD8+ T cells were elicited in infant mice and fully functional on day 7 but rapidly waned and could not be recovered from day 12 onwards. We show here that this results from the failure of LCMV-specific CD8+ T cells to expand and the absence of protective LCMV-specific memory CD8+ T cells. Under these early life conditions, viral control and clearance are eventually achieved only through LCMV-specific B cells that contribute to protect infant mice from early death or chronic infection. [ABSTRACT FROM AUTHOR]