Differential Roles of Type I Topoisomerases in Regulating HPV Pathogenesis

High-risk human papillomaviruses (HPVs) activate the ATM and ATR pathways by inducing DNA breaks through the action of viral oncoproteins E6 and E7, which target factors such as topoisomerases. Type I topoisomerases cleave and relegate a single strand of DNA, and little is known about how they regulate HPV pathogenesis. The levels of type I topoisomerases, TOP1A, TOP3A, and TOP3B, were all elevated in cells maintaining high-risk HPV genomes, as well as in squamous cell carcinomas. Only TOP1A and TOP3B, but not TOP3A, bound to HPV genomes and were critical for regulating viral gene transcription and replication with little effect on cell growth. Furthermore, the knockdown of TOP1A or TOP3B reduced levels of DNA breaks and differentially altered the expression of genes in key pathways. TOP1A knockdown reduced the expression of IL6 and activation of the pro-cytokine signaling pathway. In contrast, TOP3B targeted EGR3, which regulates growth and differentiation. Finally, TOP1A and TOP3B differentially regulate the formation of R-loops, which are critical for viral replication. These findings demonstrate the differential roles of type I topoisomerases in HPV pathogenesis. Overall design: 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 TOP1A (Samples 3/4), or TOP3B (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 depleted with TOP1A or TOP3B