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

Abstract

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. Importance Head 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.