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Rep-dependent initiation of adeno-associated virus type 2 DNA replication by a herpes simplex virus type 1
P Ward1, M Falkenberg, P Elias
1Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, New York 10029, USA. wardp01@doc.mssm.edu
Journal of Virology
|October 3, 2001
Summary
Adeno-associated virus type 2 (AAV) DNA replication can be initiated in vitro using herpes simplex virus type 1 (HSV-1) replication proteins. This suggests AAV utilizes HSV-1 machinery for its DNA replication, paving the way for a reconstituted replication assay.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Adeno-associated virus type 2 (AAV) productive infection requires helper viruses like adenovirus or herpesviruses.
- Adenovirus coinfection utilizes host cell machinery for AAV DNA replication.
- It is hypothesized that herpesvirus replication machinery may also support AAV DNA replication.
Purpose of the Study:
- To investigate if herpes simplex virus type 1 (HSV-1) replication proteins can reconstitute adeno-associated virus type 2 (AAV) DNA replication in vitro.
- To identify the specific HSV-1 proteins involved in initiating AAV DNA replication.
Main Methods:
- Reconstitution of AAV DNA replication in vitro using purified HSV-1 replication proteins and AAV Rep68 protein.
- Utilizing DNA templates containing AAV origins of replication or Rep binding sites and terminal resolution sites.
- Assessing the ability of protein combinations to initiate DNA synthesis.
Main Results:
- A combination of HSV-1 UL5, UL8, UL29, UL30, UL42, UL52 proteins and AAV Rep68 initiated replication on AAV origins, producing DNA fragments.
- Replication initiation was also observed on simpler templates containing only a Rep binding site and a terminal resolution site.
- A subset of factors, including Rep68, HSV-1 UL29, UL30, and UL42, were sufficient for initiating DNA synthesis.
Conclusions:
- The adeno-associated virus type 2 (AAV) Rep protein likely functions as a helicase for DNA synthesis, in addition to primer creation.
- The HSV-1 single-strand DNA binding protein (UL29) likely prevents strand reannealing.
- These findings suggest AAV can utilize the HSV-1 replication apparatus for its DNA replication and represent a step towards a fully reconstituted AAV replication assay.