Adeno-Associated Virus 2 (AAV2) - Induced RPA exhaustion generates cellular DNA damage and restricts viral gene...

Area of Science:

• Virology
• Molecular Biology
• Genetics

Background:

• Parvoviruses, including Adeno-Associated Virus (AAV), are single-stranded DNA viruses.
• Recombinant AAV (rAAV2) vectors are used for gene therapy, while wild-type AAV2 (wtAAV2) can replicate.
• Viral infection often triggers a host DNA Damage Response (DDR) to support replication.

Purpose of the Study:

• To investigate whether wtAAV2 and rAAV2 genomes induce cellular DDR signals.
• To determine the impact of these viral genomes on host chromosome integrity.
• To elucidate the mechanism by which viral genomes interact with host replication machinery.

Main Methods:

• BrdU analog pulse-labeling of infected cells.
• Single-molecule imaging of cellular replisomes.
• Proteomic analysis of host replication forks.
• Investigating the role of RPA and viral Inverted Terminal Repeats (ITRs).

Main Results:

• Non-replicative wtAAV2/rAAV2 genomes induce replication stress and DDR signals in a dose-dependent manner.
• wtAAV2 infection enriches replication stress proteins, DNA repair factors, and RNA processing machinery at replication forks.
• Incoming wtAAV2 genomes bind RPA, shortening replication forks and leading to nuclear DDR accumulation.
• Viral ITRs or empty capsids alone do not induce replication stress.
• RPA depletion by viral genomes restricts viral gene expression; RPA restoration rescues replication stress.

Conclusions:

• Non-replicative wtAAV2/rAAV2 genomes induce significant replication stress and DNA damage in host cells.
• The interaction with RPA is crucial for this phenomenon, impacting viral gene expression and host genome integrity.
• Understanding this mechanism is vital for the safe and effective use of AAV-based gene therapies.