p53-dependent R-loop formation and HPV pathogenesis

R-loops are trimeric RNA: DNA hybrids that are important physiological regulators of transcription; however, their aberrant formation or turnover leads to genomic instability and DNA breaks. High-risk human papillomaviruses are the causative agents of genital as well as oropharyngeal cancers and exhibit enhanced amounts of DNA breaks. The levels of R-loops were found to be increased up to 50-fold in cells that maintain high-risk HPV genomes and were readily detected in squamous cell cervical carcinomas in vivo but not in normal cells. The high levels of R-loops in HPV positive cells were present on both viral and cellular sites together with RNase H1, an enzyme that controls their resolution. Depletion of RNase H1 in HPV positive cells further increased R-loop levels, resulting in impaired viral transcription and replication and reduced expression of DNA repair genes such as FANCD2 and ATR, which are necessary for viral functions. Overexpression of RNase H1 decreased total R-loop levels, reducing DNA breaks by over 50%. Furthermore, increased RNase H1 expression leads to reduced R-loop levels, blocking viral transcription and replication while enhancing the expression of factors in the innate immune regulatory pathway. This suggests that maintaining elevated R-loop levels is important for the HPV life cycle. The E6 viral oncoprotein was found to be responsible for inducing high levels of R-loops by inhibiting p53’s transcriptional activity. Our studies indicate that high R-loop levels are critical for HPV pathogenesis and that this depends on suppressing the p53 pathway. CIN 612 cells (keratinocytes isolated from a cervical biopsy that stably maintain the HPV 31 genome) were transduced with lentivirus to express either a scrambled sequence (Samples 1/2), shRNA targeting Rnase H1 (Samples 3/4), or a cassette expressing a GFP-tagged RNase H1 (Samples 5/6). RNA sequencing was performed on each of these samples and the scramble control CIN 612 cells were compared to CIN 612 cells either depleted of RNase H1 or overexpressing RNase H1.