Evans, M. R. | Search bioRxiv

bioRxiv2019

SAMHD1 regulates human papillomavirus 16 induced cell proliferation and viral replication during differentiation of oral keratinocytes.

Human papillomaviruses induce a host of anogenital cancers, and also oropharyngeal cancer (HPV+OPC); HPV16 is causative in around 90% of HPV+OPC. Using TERT immortalized \"normal\" oral keratinocytes (NOKs) we have identified significant host gene reprogramming by HPV16 (NOKs+HPV16), and demonstrated that NOKs+HPV16 support late stages of the viral life cycle. Expression of the cellular dNTPase and homologous recombination factor SAMHD1 is transcriptionally regulated by HPV16 in NOKs, and here we demonstrate that E6 and E7 regulate expression of SAMHD1 at the transcriptional and post-transcriptional levels. CRISPR/Cas9 removal of SAMHD1 from NOKs and NOKs+HPV16 demonstrate that SAMHD1 controls cell proliferation of NOKs only in the presence of HPV16; deletion of SAMHD1 promotes hyper-proliferation of NOKs+HPV16 cells in organotypic raft cultures but has no effect on NOKs. Viral replication is also elevated in the absence of SAMHD1. This new system has allowed us to identify a specific interaction between SAMHD1 and HPV16 that regulates host cell proliferation and viral replication; such studies are problematic in non-immortalized primary oral keratinocytes due to their limited lifespan. To confirm the relevance of our results we repeated the analysis with human tonsil keratinocytes immortalized by HPV16 (HTK16) and observe the same hyper-proliferative phenotype following CRISPR/Cas9 editing of SAMHD1. Identical results were obtained with three independent CRISPR/Cas9 guide RNAs. The isogenic pairing of NOKs with NOKs+HPV16, combined with HTK16, presents a unique system to identify host genes whose products functionally interact with HPV16 to regulate host cellular growth in oral keratinocytes.\n\nImportanceHead and neck cancer is the sixth most common cancer worldwide. The incidence of HPV+OPC has been rising steadily since the 1970s and has recently reached epidemic proportions, according to the WHO. Upwards of 70% of the 600,000 new OPC cases per year are HPV positive, with high-risk type 16 present in 90% of those incidences. A better understanding of the viral life cycle will facilitate the development of novel therapeutics to combat this ongoing epidemic, as well as other HPV positive cancers. Here we present a unique oral keratinocyte model to identify host proteins that specifically interact with HPV16. Using this system, we report that a cellular gene, SAMHD1, is regulated by HPV16 at the RNA and protein level in oral keratinocytes. Elimination of SAMHD1 from these cells using CRISPR/Cas9 editing promotes enhanced cellular proliferation by HPV16 in oral keratinocytes and elevated viral replication, but not in keratinocytes that do not have HPV16. Our study demonstrates a specific intricate interplay between HPV16 and SAMHD1 during the viral life cycle and establishes a unique model system to assist exploring host factors critical for HPV pathogenesis.

cancer biology↗

bioRxiv2018

Human papillomavirus 16 E2 regulates keratinocyte gene expression relevant to cancer and the viral life cycle

Human papillomaviruses (HPV) are causative agents in ano-genital and oropharyngeal cancers. The virus must reprogram host gene expression to promote infection, and E6 and E7 contribute to this via targeting of cellular transcription factors including p53 and pRb, respectively. The HPV16 E2 protein regulates host gene expression in U2OS cells and in this study we extend these observations into TERT immortalized oral keratinocytes (NOKs) that are capable of supporting late stages of the HPV16 life cycle. We observed repression of innate immune genes by E2 that are also repressed by the intact HPV16 genome in NOKs. RNA-seq data identified 167 up and 395 downregulated genes by E2; there was a highly significant overlap of the E2 regulated genes with those regulated by the intact HPV16 genome in the same cell type. siRNA targeting of E2 reversed repression of E2 targeted genes. The ability of E2 to repress innate immune genes was confirmed in an ano-genital immortalized keratinocyte cell line, N/Tert-1. We present analysis of data from The Cancer Genome Atlas (TCGA) for HPV16 positive and negative head and neck cancers (HNC) suggesting that E2 plays a role in regulation of the host genome in cancers. Patients with HPV16 positive HNC with a loss of E2 expression exhibit a worse clinical outcome and we discuss how this could, at least partially, be related to the loss of E2 host gene regulation.\n\nImportanceHPV16 positive tumors that retain expression of E2 have a better clinical outcome than those that have lost E2 expression. It has been suggested that this is due to a loss of E2 repression of E6 and E7 expression but this is not supported by data from tumors where there is not more E6 and E7 expression in the absence of E2. Here we report that E2 regulates host gene expression and place this regulation in context of the HPV16 life cycle and HPV16 positive head and neck cancers (the majority of which retain E2 expression). We propose that this E2 function may play an important part in the increased response of HPV16 positive cancers to radiation therapy. Therefore, host gene regulation by E2 may be important for promotion of the HPV16 life cycle, and also for the response of HPV16 positive tumors to radiation therapy.

molecular biology↗