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Human papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States and are a major etiological agent of cancers in the anogenital tract and oral cavity. Growing evidence suggests changes in the host microbiome are associated with the natural history and ultimate outcome of HPV infection. We sought to define changes in the host cervicovaginal microbiome during papillomavirus infection, persistence, and pathogenesis using the murine papillomavirus (MmuPV1) cervicovaginal infection model. Cervicovaginal lavages were performed over a time course of MmuPV1 infection in immunocompetent female FVB/N mice and extracted DNA was analyzed by qPCR to track MmuPV1 viral copy number. 16S ribosomal RNA (rRNA) gene sequencing was used to determine the composition and diversity of microbial communities throughout this time course. We also sought to determine whether specific microbial communities exist across the spectrum of MmuPV1-induced neoplastic disease. We, therefore, performed laser-capture microdissection to isolate regions of disease representing all stages of neoplastic disease progression (normal, low- and high-grade dysplasia, and cancer) from female reproductive tract tissue sections from MmuPV1-infected mice and performed 16S rRNA sequencing. Consistent with other studies, we found that the natural murine cervicovaginal microbiome is highly variable across different experiments. Despite these differences in initial microbiome composition between experiments, we observed that MmuPV1 persistence, viral load, and severity of disease influenced the composition of the cervicovaginal microbiome. These studies demonstrate that papillomavirus infection can alter the cervicovaginal microbiome.IMPORTANCEHuman papillomaviruses (HPVs) are the most common sexually transmitted infection in the United States. A subset of HPVs that infect the anogenital tract (cervix, vagina, anus) and oral cavity cause at least 5% of cancers worldwide. Recent evidence indicates that the community of microbial organisms present in the human cervix and vagina, known as the cervicovaginal microbiome, plays a role in HPV-induced cervical cancer. However, the mechanisms underlying this interplay are not well-defined. In this study, we infected the female reproductive tract of mice with a murine papillomavirus (MmuPV1) and found that key aspects of papillomavirus infection and disease influence the host cervicovaginal microbiome. This is the first study to define changes in the host microbiome associated with MmuPV1 infection in a preclinical animal model of HPV-induced cervical cancer. These results pave the way for using MmuPV1 infection models to further investigate the interactions between papillomaviruses and the host microbiome., Competing Interests: The authors declare no conflict of interest.

Objective: Mouse papillomavirus MmuPV1 causes both primary and secondary infections of the larynx in immunocompromised mice. Understanding lateral and vertical transmission of papillomavirus to the larynx would benefit patients with recurrent respiratory papillomatosis (RRP). To test the hypothesis that the larynx is uniquely vulnerable to papillomavirus infection, and to further develop a mouse model of RRP, we assessed whether immunocompetent mice were vulnerable to secondary or vertical laryngeal infection with MmuPV1., Methods: Larynges were collected from 405 immunocompetent adult mice that were infected with MmuPV1 in the oropharynx, oral cavity, or anus, and 31 mouse pups born to immunocompetent females infected in the cervicovaginal tract. Larynges were analyzed via polymerase chain reaction (PCR) of lavage fluid or whole tissues for viral DNA, histopathology, and/or in situ hybridization for MmuPV1 transcripts., Results: Despite some positive laryngeal lavage PCR screens, all laryngeal tissue PCR and histopathology results were negative for MmuPV1 DNA, transcripts, and disease. There was no evidence for lateral spread of MmuPV1 to the larynges of immunocompetent mice that were infected in the oral cavity, oropharynx, or anus. Pups born to infected mothers were negative for laryngeal MmuPV1 infection from birth through weaning age., Conclusion: Secondary and vertical laryngeal MmuPV1 infections were not found in immunocompetent mice. Further work is necessary to explore immunologic control of laryngeal papillomavirus infection in a mouse model and to improve preclinical models of RRP., Level of Evidence: NA Laryngoscope, 134:2322-2330, 2024., (© 2023 The Authors. The Laryngoscope published by Wiley Periodicals LLC on behalf of The American Laryngological, Rhinological and Otological Society, Inc.)

A murine papillomavirus, MmuPV1, infects both cutaneous and mucosal epithelia of laboratory mice and can be used to model high-risk human papillomavirus (HPV) infection and HPV-associated disease. We have shown that estrogen exacerbates papillomavirus-induced cervical disease in HPV-transgenic mice. We have also previously identified stress keratin 17 (K17) as a host factor that supports MmuPV1-induced cutaneous disease. Here, we sought to test the role of estrogen and K17 in MmuPV1 infection and associated disease in the female reproductive tract. We experimentally infected wild-type and K17 knockout (K17KO) mice with MmuPV1 in the female reproductive tract in the presence or absence of exogenous estrogen for 6 mon. We observed that a significantly higher percentage of K17KO mice cleared the virus as opposed to wild-type mice. In estrogen-treated wild-type mice, the MmuPV1 viral copy number was significantly higher compared to untreated mice by as early as 2 wk postinfection, suggesting that estrogen may help facilitate MmuPV1 infection and/or establishment. Consistent with this, viral clearance was not observed in either wild-type or K17KO mice when treated with estrogen. Furthermore, neoplastic disease progression and cervical carcinogenesis were supported by the presence of K17 and exacerbated by estrogen treatment. Subsequent analyses indicated that estrogen treatment induces a systemic immunosuppressive state in MmuPV1-infected animals and that both estrogen and K17 modulate the local intratumoral immune microenvironment within MmuPV1-induced neoplastic lesions. Collectively, these findings suggest that estrogen and K17 act at multiple stages of papillomavirus-induced disease at least in part via immunomodulatory mechanisms.

Human tumor viruses cause various human cancers that account for at least 15% of the global cancer burden. Among the currently identified human tumor viruses, two are small DNA tumor viruses: human papillomaviruses (HPVs) and Merkel cell polyomavirus (MCPyV). The study of small DNA tumor viruses (adenoviruses, polyomaviruses, and papillomaviruses) has facilitated several significant biological discoveries and established some of the first animal models of virus-associated cancers. The development and use of preclinical in vivo models to study HPVs and MCPyV and their role in human cancer is the focus of this review. Important considerations in the design of animal models of small DNA tumor virus infection and disease, including host range, cell tropism, choice of virus isolates, and the ability to recapitulate human disease, are presented. The types of infection-based and transgenic model strategies that are used to study HPVs and MCPyV, including their strengths and limitations, are also discussed. An overview of the current models that exist to study HPV and MCPyV infection and neoplastic disease are highlighted. These comparative models provide valuable platforms to study various aspects of virus-associated human disease and will continue to expand knowledge of human tumor viruses and their relationship with their hosts., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author. Published by Elsevier B.V. All rights reserved.)

Merkel cell polyomavirus (MCPyV) is the only human polyomavirus currently known to cause human cancer. MCPyV is believed to be an etiological factor in at least 80% of cases of the rare but aggressive skin malignancy Merkel cell carcinoma (MCC). In these MCPyV+ MCC tumors, clonal integration of the viral genome results in the continued expression of two viral proteins: the viral small T antigen (ST) and a truncated form of the viral large T antigen. The oncogenic potential of MCPyV and the functional properties of the viral T antigens that contribute to neoplasia are becoming increasingly well-characterized with the recent development of model systems that recapitulate the biology of MCPyV+ MCC. In this review, we summarize our understanding of MCPyV and its role in MCC, followed by the current state of both in vitro and in vivo model systems used to study MCPyV and its contribution to carcinogenesis. We also highlight the remaining challenges within the field and the major considerations related to the ongoing development of in vitro and in vivo models of MCPyV+ MCC.

Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT's LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT's LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: J.A.D. received research funding from Rain Therapeutics. The remaining authors have declared that no competing interests exist.

Merkel cell polyomavirus (MCPyV) is a human polyomavirus causally linked to the development of Merkel cell carcinoma (MCC), an aggressive malignancy that largely arises within the dermis of the skin. In this study, we recapitulate the histopathology of human MCC tumors in vitro using an organotypic (raft) culture system that is traditionally used to recapitulate the dermal and epidermal equivalents of skin in three dimensions (3D). In the optimal culture condition, MCPyV+ MCC cells were embedded in collagen between the epidermal equivalent comprising human keratinocytes and a dermal equivalent containing fibroblasts, resulting in MCC-like lesions arising within the dermal equivalent. The presence and organization of MCC cells within these dermal lesions were characterized through biomarker analyses. Interestingly, co-culture of MCPyV+ MCC together with keratinocytes specifically within the epidermal equivalent of the raft did not reproduce human MCC morphology, nor were any keratinocytes necessary for MCC-like lesions to develop in the dermal equivalent. This 3D tissue culture system provides a novel in vitro platform for studying the role of MCPyV T antigens in MCC oncogenesis, identifying additional factors involved in this process, and for screening potential MCPyV+ MCC therapeutic strategies.

Merkel cell polyomavirus (MCPyV) causes the majority of human Merkel cell carcinomas (MCC), a rare but highly aggressive form of skin cancer. We recently reported that constitutive expression of MCC tumor-derived MCPyV tumor (T) antigens in the skin of transgenic mice leads to hyperplasia, increased proliferation, and spontaneous epithelial tumor development. We sought to evaluate how the MCPyV T antigens contribute to tumor formation in vivo using a classical, multi-stage model for squamous cell carcinoma development. In this model, two chemical carcinogens, DMBA and TPA, contribute to two distinct phases of carcinogenesis-initiation and promotion, respectively-that are required for tumors to develop. By treating the MCPyV transgenic mice with each chemical carcinogen, we determined how the viral oncogenes contributed to carcinogenesis. We observed that the MCPyV T antigens synergized with the tumor initiator DMBA, but not with the tumor promoter TPA, cause tumors. Therefore, the MCPyV tumor antigens function primarily as tumor promoters, similar to that seen with human papillomavirus (HPV) oncoproteins. These studies provide insight into the role of MCPyV T antigen expression in tumor formation in vivo and contribute to our understanding of how MCPyV may function as a human DNA tumor virus.

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, we discuss the discovery of a murine papillomavirus, Mus musculus papillomavirus 1 (MmuPV1), and how its experimental use represents a major advancement in models of papillomavirus-induced pathogenesis/carcinogenesis, and their transmission., (Copyright © 2020 American Society for Microbiology.)

The papillomavirus E5 gene contributes to transformation and tumorigenesis; however, its exact function in these processes and viral pathogenesis is unclear. While E5 is present in high-risk mucosotropic HPVs that cause anogenital and head and neck cancers, it is absent in cutaneous HPVs and the recently discovered mouse papillomavirus (MmuPV1), which causes papillomas and squamous cell carcinomas of the skin and mucosal epithelia in laboratory mice. We infected K14E5 transgenic mice, which express the high-risk mucosotropic HPV16 E5 gene in stratified epithelia, with MmuPV1 to investigate the effects of E5 on papillomavirus-induced pathogenesis. Skin lesions in MmuPV1-infected K14E5 mice had earlier onset, higher incidence, and reduced frequency of spontaneous regression compared to those in non-transgenic mice. K14E5 mice were also more susceptible to cervicovaginal cancers when infected with MmuPV1 and treated with estrogen compared to non-transgenic mice. Our studies support the hypothesis that E5 contributes to papillomavirus-induced pathogenesis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Human papillomaviruses (HPVs) are the most common sexually transmitted infectious agents. Because of the species specificity of HPVs, study of their natural transmission in laboratory animals is not possible. The papillomavirus, MmuPV1, which infects laboratory mice ( Mus musculus ), can cause infections in the female cervicovaginal epithelium of immunocompetent mice that progress to cancer. Here, we provide evidence that MmuPV1 is sexually transmitted in unmanipulated, immunocompetent male and female mice. Female 'donor' mice experimentally infected with MmuPV1 in their lower reproductive tract were housed with unmanipulated male mice. The male mice were then transferred to cages holding 'recipient' female mice. One third of the female recipient mice acquired cervicovaginal infections. Prolonged infections were verified by histopathology and in situ hybridization analyses of both male and recipient female mice at the study endpoint. These findings indicate that MmuPV1 is a new model animal papillomavirus with which to study sexually transmission of papillomaviruses., Competing Interests: MS, PL No competing interests declared, (© 2019, Spurgeon and Lambert.)

Megan E. Spurgeon works in the field of viral oncology. In this mSphere of Influence article, she reflects on how the paper "Rescuing US biomedical research from its systemic flaws" by Bruce Alberts, Marc W. Kirschner, Shirley Tilghman, and Harold Varmus (Proc Natl Acad Sci U S A 111:5773-5777, 2014, https://doi.org/10.1073/pnas.1404402111) made an impact on her by influencing her research and career outlook. The way researchers think about their science is shaped by a variety of factors, and these often include research articles that influence their technical or experimental approaches and interpretations. However, it is rare that an article influences the broader career and research perspectives of scientists by compelling them to look beyond the bench. The article highlighted in this commentary prompted the realization that a sphere of influence beyond our routine scientific focus can create a scientific community that is better equipped to address the systemic threats facing the larger research ecosystem to which we all belong., (Copyright © 2019 Spurgeon.)

Papillomaviruses exhibit species-specific tropism, thereby limiting understanding and research of several aspects of HPV infection and carcinogenesis. The discovery of a murine papillomavirus (MmuPV1) provides the opportunity to study papillomavirus infections in a tractable, in vivo laboratory model. MmuPV1 infects and causes disease in the cutaneous epithelium, as well as the mucosal epithelia of the oral cavity and anogenital tract. In this report, we describe a murine model of MmuPV1 infection and neoplastic disease in the female reproductive tracts of wild-type immunocompetent FVB mice. Low-grade dysplastic lesions developed in reproductive tracts of FVB mice infected with MmuPV1 for 4 months, and mice infected for 6 months developed significantly worse disease, including squamous cell carcinoma (SCC). We also tested the contribution of estrogen and/or UV radiation (UVR), two cofactors we previously identified as being involved in papillomavirus-mediated disease, to cervicovaginal disease. Similar to HPV16 transgenic mice, exogenous estrogen treatment induced high-grade precancerous lesions in the reproductive tracts of MmuPV1-infected mice by 4 months and together with MmuPV1 efficiently induced SCC by 6 months. UV radiation and exogenous estrogen cooperated to promote carcinogenesis in MmuPV1-infected mice. This murine infection model represents the first instance of de novo papillomavirus-mediated carcinogenesis in the female reproductive tract of wild-type mice resulting from active virus infection and is also the first report of the female hormone estrogen contributing to this process. This model will provide an additional platform for fundamental studies on papillomavirus infection, cervicovaginal disease, and the role of cellular cofactors during papillomavirus-induced carcinogenesis. IMPORTANCE Tractable and efficient models of papillomavirus-induced pathogenesis are limited due to the strict species-specific and tissue-specific tropism of these viruses. Here, we report a novel preclinical murine model of papillomavirus-induced cervicovaginal disease in wild-type, immunocompetent mice using the recently discovered murine papillomavirus, MmuPV1. In this model, MmuPV1 establishes persistent viral infections in the mucosal epithelia of the female reproductive tract, a necessary component needed to accurately mimic HPV-mediated neoplastic disease in humans. Persistent MmuPV1 infections were able to induce progressive neoplastic disease and carcinogenesis, either alone or in combination with previously identified cofactors of papillomavirus-induced disease. This new model will provide a much-needed platform for basic and translational studies on both papillomavirus infection and associated disease in immunocompetent mice., (Copyright © 2019 Spurgeon et al.)

High-risk human papillomaviruses (HPVs) infect epithelial cells and are causally associated with cervical cancer, but HPV infection is not sufficient for carcinogenesis. Previously, we reported that estrogen signaling in the stromal tumor microenvironment is associated with cervical cancer maintenance and progression. We have now determined how HPV oncogenes and estrogen treatment affect genome-wide host gene expression in laser-captured regions of the cervical epithelium and stroma of untreated or estrogen-treated nontransgenic and HPV-transgenic mice. HPV oncogene expression in the cervical epithelium elicited significant gene-expression changes in the proximal stromal compartment, and estrogen treatment uniquely affected gene expression in the cervical microenvironment of HPV-transgenic mice compared with nontransgenic mice. Several potential estrogen-induced paracrine-acting factors were identified in the expression profile of the cervical tumor microenvironment. The microenvironment of estrogen-treated HPV-transgenic mice was significantly enriched for chemokine/cytokine activity and inflammatory and immune functions associated with carcinogenesis. This inflammatory signature included several proangiogenic CXCR2 receptor ligands. A subset of the same CXCR2 ligands was likewise increased in cocultures of early-passage cells from human cervical samples, with levels highest in cocultures of cervical fibroblasts and cancer-derived epithelial cells. Our studies demonstrate that high-risk HPV oncogenes profoundly reprogram the tumor microenvironment independently of and synergistically with estrogen. These observations illuminate important means by which HPVs can cause cancer through alterations in the tumor microenvironment., Competing Interests: Conflict of interest statement: D.J.B. holds equity in Bellbrook Labs LLC, Tasso Inc., Stacks to the Future LLC, Lynx Biosciences LLC, Onexio Biosystems LLC, and Salus Discovery LLC. D.D. is a member of the external advisory board for the University of Wisconsin Cancer Center, for which he receives an honorarium. He is not an active collaborator with any of the authors.

Human papillomaviruses (HPVs) are double-stranded DNA (dsDNA) tumor viruses that are causally associated with human cancers of the anogenital tract, skin, and oral cavity. Despite the availability of prophylactic vaccines, HPVs remain a major global health issue due to inadequate vaccine availability and vaccination coverage. The HPV life cycle is established and completed in the terminally differentiating stratified epithelia, and decades of research using in vitro organotypic raft cultures and in vivo genetically engineered mouse models have contributed to our understanding of the interactions between HPVs and the epithelium. More recently, important and emerging roles for the underlying stroma, or microenvironment, during the HPV life cycle and HPV-induced disease have become clear. This review discusses the current understanding of the bidirectional communication and relationship between HPV-infected epithelia and the surrounding microenvironment. As is the case with other human cancers, evidence suggests that the stroma functions as a significant partner in tumorigenesis and helps facilitate the oncogenic potential of HPVs in the stratified epithelium., Competing Interests: The authors declare no conflict of interest.

Infection with Merkel cell polyomavirus (MCPyV) can lead to Merkel cell carcinoma (MCC), a lethal form of skin cancer. However, the skin cell type productively infected by MCPyV remains a central question. We combined cell culture and ex vivo approaches to identify human dermal fibroblasts as natural host cells that support productive MCPyV infection. Based on this, we established a cell culture model for MCPyV infection, which will facilitate investigation of the oncogenic mechanisms for this DNA virus. Using this model, we discovered that induction of matrix metalloproteinase (MMP) genes by the WNT/β-catenin signaling pathway and other growth factors stimulates MCPyV infection. This suggests that MCC risk factors such as UV radiation and aging, which are known to stimulate WNT signaling and MMP expression, may promote viral infection and thus drive MCC. Our study also introduces the FDA-approved MEK antagonist trametinib as an effective inhibitor for controlling MCPyV infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Merkel cell polyomavirus (MCPyV) is frequently associated with Merkel cell carcinoma (MCC), a highly aggressive neuroendocrine skin cancer. Most MCC tumors contain integrated copies of the viral genome with persistent expression of the MCPyV large T (LT) and small T (ST) antigen. MCPyV isolated from MCC typically contains wild-type ST but truncated forms of LT that retain the N-terminus but delete the C-terminus and render LT incapable of supporting virus replication. To determine the oncogenic activity of MCC tumor-derived T antigens in vivo, a conditional, tissue-specific mouse model was developed. Keratin 14-mediated Cre recombinase expression induced expression of MCPyV T antigens in stratified squamous epithelial cells and Merkel cells of the skin epidermis. Mice expressing MCPyV T antigens developed hyperplasia, hyperkeratosis, and acanthosis of the skin with additional abnormalities in whisker pads, footpads, and eyes. Nearly half of the mice also developed cutaneous papillomas. Evidence for neoplastic progression within stratified epithelia included increased cellular proliferation, unscheduled DNA synthesis, increased E2F-responsive genes levels, disrupted differentiation, and presence of a DNA damage response. These results indicate that MCPyV T antigens are tumorigenic in vivo, consistent with their suspected etiologic role in human cancer., (©2015 American Association for Cancer Research.)

Estrogen and its nuclear receptor, estrogen receptor α, are necessary cofactors in the initiation and multistage progression of carcinogenesis in the K14E6/E7 transgenic mouse model of human papillomavirus-associated cervical cancer. Recently, our laboratory reported that raloxifene, a selective estrogen receptor modulator, promoted regression of high-grade dysplasia and cancer that arose in the cervix of K14E6/E7 transgenic mice treated long-term with estrogen. Herein, we evaluated the recurrence of cervical cancer after raloxifene therapy in our preclinical model of human papillomavirus-associated cervical carcinogenesis. We observed recurrence of cervical cancer in mice re-exposed to estrogen after raloxifene treatment, despite evidence suggesting the antagonistic effects of raloxifene persisted in the reproductive tract after treatment had ceased. We also observed recurrence of neoplastic disease in mice that were not retreated with exogenous estrogen, although the severity of disease was less. Recurrent neoplastic disease and cancers retained functional estrogen receptor α and responded to retreatment with raloxifene. Moreover, continuous treatment of mice with raloxifene prevented the emergence of recurrent disease seen in mice in which raloxifene was discontinued. These data suggest that cervical cancer cells are not completely eradicated by raloxifene and rapidly expand if raloxifene treatment is ceased. These findings indicate that a prolonged treatment period with raloxifene might be required to prevent recurrence of neoplastic disease and lower reproductive tract cancers., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

A marked escalation in the rate of discovery of new types of human polyomavirus has occurred over the last five years largely owing to recent technological advances in their detection. Among the newly discovered viruses, Merkel Cell Polyomavirus (MCPyV or MCV) has gained the most attention due to its link with a rare human cancer. Infection with MCPyV is common in the human population, and the virus is detected in several anatomical locations, but most frequently in skin. Study of MCPyV molecular virology has been complicated by the lack of straightforward cell culture models, but recent in vitro studies are making strides towards understanding the virus life cycle, its cellular tropism, and mode of transmission. While MCPyV shares several traditional traits with other human polyomaviruses, the burst of research since its discovery reveals insight into a virus with many unique genetic and mechanistic features. The evidence for a causal link between MCPyV and the rare neuroendocrine cancer, Merkel Cell Carcinoma (MCC), is compelling. A majority of MCCs contain clonally integrated viral DNA, express viral T antigen transcripts and protein, and exhibit an addiction to the viral large T and small t antigen oncoproteins. The MCPyV large T antigen contains MCC tumor-specific mutations that ablate its replication capacity but preserve its oncogenic functions, and the small t antigen promotes an environment favorable for cap-dependent translation. The mechanisms of MCPyV-induced transformation have not been fully elucidated, but the likely etiological role of this new polyomavirus in human cancer provides a strong opportunity to expand knowledge of virus-host interactions and viral oncology., (Copyright © 2012 Elsevier B.V. All rights reserved.)

During a productive infection, species C adenovirus reprograms the host cell to promote viral translation at the expense of cellular translation. The E1B 55-kilodalton (E1B-55K) and E4 open reading frame 6 (E4orf6) proteins are important in this control of gene expression. As part of a ubiquitin-protein ligase, these viral proteins stimulate viral mRNA export, inhibit cellular mRNA export, promote viral gene expression, and direct the degradation of certain host proteins. We report here that the E1B-55K and E4orf6 proteins limited phosphorylation of eIF2alpha and the activation of the eIF2alpha kinase PKR. Phospho-eIF2alpha levels were observed to rise and fall at least twice during infection. The E1B-55K and E4orf6 proteins prevented a third increase at late times of infection. PKR appeared to phosphorylate eIF2alpha only in the absence of E1B-55K/E4orf6 function. PKR activation and eIF2alpha phosphorylation was unrelated to the cytoplasmic levels of the adenovirus inhibitor of PKR, VA-I RNA. Nonetheless, expression of a PKR inhibitor, the reovirus double-stranded RNA-binding protein sigma 3, prevented PKR activation and eIF2alpha phosphorylation. The sigma 3 protein largely corrected the defect in viral late protein synthesis associated with the E1B-55K and E4orf6 mutant viruses without affecting cytoplasmic levels of the late viral mRNA. The ubiquitin-protein ligase activity associated with the E1B-55K/E4orf6 complex was necessary to prevent activation of PKR and phosphorylation of eIF2alpha. These findings reveal a new contribution of the E1B-55K/E4orf6 complex to viral late protein synthesis and the existence of multiple layers of regulation imposed on eIF2alpha phosphorylation and PKR activation in adenovirus-infected cells.