Submission
| Title: | The Detection and Characterization of R-loops on the HPV Genome |
| Presenter: | Ishanpreet Sran |
| Institution: | IUSM |
| Authors: | Ishanpreet Sran, Leny Jose, Marsha DeSmet, Elliot Androphy |
Abstract
| Background/Significance/Rationale: | Papillomaviruses (PVs) are double-stranded 8 kb DNA viruses that infect mucosal and cutaneous epithelia. The HPV E2 protein is responsible for viral transcription and replication on viral episomes. We hypothesize that viral replication forks collide with nascent viral RNA formed during transcription of viral genes. We predict that these DNA-RNA hybrids (R-loops) composed of three stranded structures occur on replicating and transcriptionally active viral genomes. Our goal is to determine if R-loops occur on viral genomes and to identify the host machinery necessary to resolve these conflicts. |
| Methods: | DNA from CIN612 keratinocyte cell lines (HPV-31) harboring episomal and integrated genomes was isolated and R-loops were immunoprecipitated and detected with the S9.6 antibody. Next we tested if HPV E2 interacts with host R-loop resolving proteins such as ZPR1, SMN, and a fragment of SETX 1-667 amino acids using co-immunoprecipitation. |
| Results/Findings: | We detected R-loops on the viral p97 promoter in CIN612 episomal cells, but not integrated cells. We observed that HPV-16 E2 co-immunoprecipitated with ZPR1 and SMN, but not SETX 1-667. |
| Conclusions/Discussion: | During replication, we observed a significant increase of R-loops in episomal cell lines when compared to integrated HPV cell lines. The viral protein E2 may be recruiting host cell factors such as ZPR1 and SMN to resolve viral R-loops. |
| Translational/Human Health Impact: | These preliminary experiments indicate that HPV could be using cellular machinery to resolve R-loops, aiding viral propagation as observed in other viruses such as Cytomegalovirus and Herpes Simplex Virus. This finding can revolutionize our understanding of viral replication and potentially alter the approach of treatment research. |
