Journal article
Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication
mBio, Vol.14(1), e0352822
02/2023
DOI: 10.1128/mbio.03528-22
PMCID: PMC9973366
PMID: 36719192
Abstract
Adeno-associated virus (AAV) belongs to the
genus of the
family. AAV replication relies on a helper virus, such as adenovirus (Ad). Co-infection of AAV and Ad induces a DNA damage response (DDR), although its function in AAV DNA replication remains unknown. In this study, monoinfection of AAV2 in HEK293T cells expressing a minimal set of Ad helper genes was used to investigate the role of the DDR solely induced by AAV. We found that AAV2 DNA replication, but not single stranded (ss)DNA genome accumulation and Rep expression only, induced a robust DDR in HEK293T cells. The induced DDR featured the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all 3 phosphatidylinositol 3-kinase-related kinases (PIKKs). We also found that the kinase ataxia telangiectasia and Rad3-related protein (ATR) plays a major role in AAV2 DNA replication and that Y family DNA repair DNA polymerases η (Pol η) and Pol κ contribute to AAV2 DNA replication both
and in HEK293T cells. Knockout of
and
in HEK293T cells significantly decreased wild-type AAV2 replication and recombinant AAV2 production. Thus, our study has proven that AAV2 DNA replication induces a DDR, which in turn initiates a DNA repairing process that partially contributes to the viral genome amplification in HEK293T cells.
Recombinant AAV (rAAV) has emerged as one of the preferred delivery vectors for clinical gene therapy. rAAV production in HEK293 cells by transfection of a rAAV transgene plasmid, an AAV Rep and Cap expression packaging plasmid, and an Ad helper plasmid remains the popular method. Here, we demonstrated that the high fidelity Y family DNA repair DNA polymerase, Pol η, and Pol κ, plays a significant role in AAV DNA replication and rAAV production in HEK293T cells. Understanding the AAV DNA replication mechanism in HEK293T cells could provide clues to increase rAAV vector yield produced from the transfection method. We also provide evidence that the ATR-mediated DNA repair process through Pol η and Pol κ is one of the mechanisms to amplify AAV genome, which could explain AAV replication and rAAV ssDNA genome conversion in mitotic quiescent cells.
Details
- Title: Subtitle
- Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication
- Creators
- Kang Ning - University of Kansas Medical CenterCagla Aksu Kuz - University of Kansas Medical CenterFang Cheng - University of Kansas Medical CenterZehua Feng - University of IowaZiying Yan - University of IowaJianming Qiu - University of Kansas Medical Center
- Resource Type
- Journal article
- Publication Details
- mBio, Vol.14(1), e0352822
- DOI
- 10.1128/mbio.03528-22
- PMID
- 36719192
- PMCID
- PMC9973366
- NLM abbreviation
- mBio
- eISSN
- 2150-7511
- Grant note
- DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: AI150877, AI156448; DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: AI156448
- Language
- English
- Electronic publication date
- 01/31/2023
- Date published
- 02/2023
- Academic Unit
- Anatomy and Cell Biology
- Record Identifier
- 9984363682302771
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