# APOBEC3B regulates HPV replication by inducing R-loop formation and DNA damage

## TL;DR

APOBEC3B promotes HPV replication by forming R-loops and causing DNA damage, which affects both viral and cellular gene expression.

## Contribution

APOBEC3B is shown to regulate R-loop formation and DNA damage in HPV-positive cells, acting as a proviral factor.

## Key findings

- APOBEC3B binds to HPV and cellular chromatin, inducing R-loop formation and DNA damage.
- Depletion of APOBEC3B reduces R-loops and DNA breaks while altering immune gene expression.
- APOBEC3B regulates viral replication and gene expression in HPV-positive cells.

## Abstract

APOBECs are cytidine deaminases whose levels are increased in cells with high-risk HPV genomes and are responsible for most mutations in HPV associated cancers. APOBEC3B is a nuclear member of this family and is shown to be a positive regulator of HPV replication as well as expression. The proviral effects of A3B found in HPV positive cells contrast with its role as a restriction factor for many other viruses. Studies demonstrated that A3B can bind and regulate the formation of R-loops, which are trimeric nucleic acid structures consisting of an RNA paired with its complementary DNA strand, displacing one of the DNA strands. The present study demonstrates that A3B binds stably to both cellular and viral chromatin at sequences containing high R-loop levels, including the HPV URR and early polyadenylation sites. Importantly, A3B was found to play a critical role in the replication of HPV genomes and in regulating viral expression. Reduction of R-loop levels through overexpression of the R-loop specific RNase, RNase H1, impaired A3B binding to viral genomes as well as at multiple cellular sites. When A3B was depleted, total R-loop levels decreased by ~50%, leading to impaired viral transcription and an increase in the expression of immune genes, such as OASL, IL6, and IRF1. Mapping R-loop formation in A3B depleted cells revealed that A3B regulated a subset of R-loops that form on the transcriptional start (TSS) and termination sites (TTS) of cellular genes, including at the HPV URR. Furthermore, A3B depletion resulted in over a 50% reduction of DNA breaks along with altered expression of DNA damage repair proteins. This study demonstrates that A3B is an inducer of R-loop formation and DNA damage in HPV positive cells, thereby regulating cellular and viral gene expression along with HPV replication.

The APOBEC3 (A3) family of proteins plays a significant role in the generation of mutations in the development of HPV positive cancers. A3 levels are increased in HPV positive cancers that often contain only integrated copies of the HPV genome, as well as in precancerous lesions that contain viral episomes, but little is known about their role in viral pathogenesis. In this study, A3B, one of the cytosine deaminases within the A3 family, was found to be present at high levels in cells that maintain viral episomes, and these elevated levels were shown to be crucial for viral DNA maintenance and gene expression. This is in contrast to A3B’s role as a restriction factor during the life cycle of other DNA viruses. Increased A3B expression in HPV positive cells induced R-loop formation as well as DNA damage, which was important for regulating the expression of a subset of cellular genes. In addition, many, but not all, of the R-loops within HPV positive cells are bound by A3B, including those on the viral URR and early polyA site. Knockdown of A3B reduced R-loop formation at the URR, and similar effects were observed on cellular genes, altering their expression. These data indicate that A3B acts as a proviral factor in HPV replication by regulating R-loop formation and cellular and viral gene expression.