Quasivirus Infection Model Identifies a Novel Role for Protein Arginine Methyltransferase 1 in the HPV Lifecycle

Abstract

Papillomaviruses (PVs) are a diverse family of small (5.6kb-8.6kb) double stranded DNA viruses. PVs are non-enveloped viruses with histone wrapped genomes. To date over 700 PVs have been identified in a wide range of hosts including mammals, birds, reptiles, and fish. Over 400 HPVs have been identified and these viruses fall into 5 genera termed Alpha-papillomaviruses, Beta-papillomaviruses, Gamma-papillomaviruses, Mu-papillomaviruses, and Nu-papillomaviruses. The Alpha-papillomaviruses genus contains a subset of HPVs adapted to infect the epithelium of the genitalia, oropharynx, and anus with high efficiency. Alpha-papillomaviruses infections are ubiquitous in the global population, and HPV represents the most common sexually transmitted infection. Most Alpha-papillomaviruses cause benign infections. However, 12 Alpha-papillomaviruses (HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59) are recognized as carcinogens by the International Agency for Research on Cancer. A further 13 Alpha-papillomaviruses are suspected carcinogens based on limited data. Oncogenic HPVs are responsible for 4.5% of the global cancer burden and are the etiological agents of cervical, oropharyngeal, anal, vaginal, vulvar, and penile squamous cell carcinomas. Almost all cervical and anal cancers, 15-48% of vulvar cancers, 53% of penile cancers, 78% of vaginal cancers, and 13-60% of oropharyngeal cancers are HPV driven. Interestingly, two HPV types have higher oncogenic potential than all others. Combined HPV16 and HPV18 account for over 72% of HPV driven cervical carcinomas. HPV18 is the main driver of a less common type of cervical cancer, cervical adenocarcinoma. HPV16 accounts for over 90 percent of HPV driven tumors that occur in tissues other than the cervix. Due to the high mortality of oncogenic HPVs, and the high morbidity of genital wart HPVs, preventative HPV vaccines have been developed. These vaccines are made of viral like particles containing only the L1 capsid protein and they induce humoral immunity to HPV virions. The current form of this vaccine covers the seven most common oncogenic HPV types (HPV16, HPV18, HPV31, HPV33, HPV45, HPV52, HPV58) and the two genital wart HPV types (HPV6, HPV11). Vaccination has been highly effective in reducing the rates of oncogenic HPV infection and early preneoplastic cervical dysplasia in countries with high vaccine coverage. However globally HPV vaccines are in limited supply and the current vaccines cannot cure an established infection. Due to these limitations, over 40 million cases of HPV driven cancer are expected over the next 5 decades. The explosion of HPV research following the discovery of HPV16 and HPV18 has taught us a great deal about oncogenic HPV tumor progression. Animal models based on non-human PVs demonstrated the viral lifecycle and role of the adaptive immune response in controlling infections. Population scale epidemiological data identified the near ubiquity of HPV DNA in cervical cancer, the fact HPV is the primary risk factor for cervical cancer development, and important tumor progression cofactors such as dysbiosis. Viral genome transfection and oncogene overexpression directly demonstrated the carcinogenic potential of oncogenic HPV accessory genes. Tissue culture based serial passaging of clonal populations of HPV immortalized keratinocytes identified host and viral genome changes that promote the progression from persistent infection to HPV induced tumor. However, the host pathogen interactions that occur between initial infection and the isolation of an immortalized clone remain largely unstudied. Importantly, the handful of studies that have investigated early timepoints post infection indicate key differences between freshly infected cells and immortalized clones. My thesis focuses on addressing the following gap in our understanding of HPV driven pathogenesis. What are the host factors oncogenic HPVs exploit to establish persistent infections in primary human keratinocytes? In this work I show the combination of 2D monolayer coculture (coculture) and HPV quasivirus infection are an optimal system for investigating HPV host pathogen interactions at early timepoints post infection. HPV viral entry requires heparin sulfate moieties, extracellular proteases, and actively replicating host cells. Cocultures provide a large population of actively replicating primary keratinocytes and the growth inactivated fibroblasts provide the ECM components necessary for viral entry. In addition, keratinocytes from a wide range of HPV relevant cutaneous or mucosal epithelium are easily propagated in coculture. The use of quasivirus to deliver full length viral genomes improves the physiological relevance of this model. Importantly, the full set of viral genes are expressed from the endogenous HPV URR. These genes are transcribed in their natural context of multiple polycistronic mRNA isoforms that encode for a balance of growth promoting and growth inhibiting HPV gene products. In addition, delivery of the viral genome via HPV virion avoids the innate immune activation induced by transfection. By combining keratinocyte coculture and quasivirus infection one can generate pools of HPV infected keratinocytes at any desired timepoint post infection. In this work I leveraged quasivirus infection, coculture, and single cell RNA sequencing to identify Protein Arginine Methyltransferase 1 (PRMT1) as a novel host factor required for persistent HPV18 infection. In addition, through transcriptomic analysis I found HPV18 does not drive its host keratinocyte to adopt the gene expression of an epithelial stem cell. Instead, it upregulates a unique combination of genes in extracellular matrix, cytoskeletal structure, and cholesterol pathways. The aforementioned pathways have been demonstrated to drive keratinocyte self-renewal. Lastly, I found that HPV18 infected keratinocytes at the earliest stage of differentiation upregulate genes in the mitochondrial and ribosomal pathways.