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

Area of Science:

• Molecular Biology
• Virology
• Genetics

Background:

• APOBEC3B (A3B) levels are elevated in high-risk HPV genomes, contributing to mutations in HPV-associated cancers.
• A3B acts as a positive regulator of HPV replication and expression, contrasting with its viral restriction roles in other contexts.
• A3B is known to bind and regulate R-loop formation, which are RNA:DNA hybrids.

Purpose of the Study:

• To investigate the role of A3B in R-loop formation and its impact on HPV replication and gene expression.
• To determine how A3B interacts with cellular and viral chromatin.
• To elucidate the consequences of A3B depletion on R-loop levels, viral transcription, and DNA damage.

Main Methods:

• Chromatin binding assays to assess A3B interaction with HPV and cellular DNA.
• Overexpression of RNase H1 to reduce R-loop levels.
• Depletion of A3B via genetic manipulation.
• RNA sequencing to analyze gene expression changes.
• DNA break quantification and analysis of DNA damage repair proteins.

Main Results:

• A3B stably binds to chromatin with high R-loop levels, including the HPV URR and polyadenylation sites.
• Reducing R-loops with RNase H1 impaired A3B binding and HPV replication.
• A3B depletion decreased R-loops by ~50%, impaired viral transcription, and increased immune gene expression (OASL, IL6, IRF1).
• A3B regulates R-loops at transcriptional start and termination sites of cellular genes and the HPV URR.
• A3B depletion reduced DNA breaks by over 50% and altered DNA damage repair protein expression.

Conclusions:

• A3B is a key inducer of R-loop formation and DNA damage in HPV-positive cells.
• A3B regulates both cellular and viral gene expression and is critical for HPV replication.
• Targeting A3B or R-loop formation may offer therapeutic strategies for HPV-associated cancers.