Regulation of R-Loops in DNA Tumor Viruses

Abstract

R-loops are triple-stranded nucleic acid structures that occur when newly synthesized single-stranded RNA anneals to duplex DNA upon the collision of replication forks with transcription complexes. These RNA-DNA hybrids facilitate several transcriptional processes in the cell and have been described extensively in the literature. Recently, evidence has emerged that R-loops are key regulators of DNA tumor virus transcription and the replication of their lifecycle. Studies have demonstrated that R-loops on the Human Papillomavirus (HPV) genome must be resolved to maintain genome maintenance and avoid viral integration, a hallmark of HPV cancers. For Epstein-Barr virus (EBV), R-loops are formed at the oriLyt to establish lytic replication. Structural maintenance of chromosome proteins 5/6 (SMC5/6) bind to these viral R-loops to repress EBV lytic replication. Most viruses in the herpesvirales order, such as KSHV, contain R-loop-forming sequences. In this perspective, we will describe the current, although limited, literature demonstrating the importance of RNA-DNA hybrids to regulate DNA virus transcription. We will also detail potential new areas of R-loop research and how these viruses can be used as tools to study the growing field of R-loops.

Keywords: DNA tumor viruses; R-loops; SETX; oncogenesis; replication; transcription.

Figure 1

R-loop resolution on the HPV genome is mediated by Senataxin and RNase H1. HPV is a double-stranded 8 kilobase pair DNA genome that replicates as an episome. HPV transcription and replication is mediated through the viral E2 protein. The HPV-E2 protein binds to E2-binding sites throughout the long control region. E2 mediates HPV transcription and replication through host protein–protein interactions. RNA–DNA hybrids form at the p97 promoter on the HPV genome during transcription. RNase H1 and SETX are recruited to the viral genome to resolve these viral R-loops. The depletion of RNase H1 and SETX increases R-loop accumulation, prevents genome maintenance, and increases viral integration. Figure was created with BioRender.com.

Figure 2

BRD4 activates HPV replication through many mechanisms. BRD4 phosphorylates RNAPII [54] and recruits pTEFb [52,55,56] to transcriptional start sites and to the HPV promoter. Recent studies have identified BRD4 as a key regulator for R-loop resolution on the mammalian genome. Future studies are needed to determine if BRD4 mediates HPV R-loop resolution through pTEFb to prevent RNAPII stalling. Figure was created with BioRender.com.