Role of the Herpes Simplex Virus Helicase-Primase Complex during Adeno-Associated Virus DNA Replication
- 1 June 2006
- journal article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 80 (11) , 5241-5250
- https://doi.org/10.1128/jvi.02718-05
Abstract
A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.Keywords
This publication has 24 references indexed in Scilit:
- Mutations in the Putative Zinc-Binding Motif of UL52 Demonstrate a Complex Interdependence between the UL5 and UL52 Subunits of the Human Herpes Simplex Virus Type 1 Helicase/Primase ComplexJournal of Virology, 2005
- The Rep Protein of Adeno-Associated Virus Type 2 Interacts with Single-Stranded DNA-Binding Proteins That Enhance Viral ReplicationJournal of Virology, 2004
- Recruitment of Polymerase to Herpes Simplex Virus Type 1 Replication Foci in Cells Expressing Mutant Primase (UL52) ProteinsJournal of Virology, 2003
- Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCRJournal of Virology, 2002
- The UL5 and UL52 Subunits of the Herpes Simplex Virus Type 1 Helicase-Primase Subcomplex Exhibit a Complex Interdependence for DNA BindingJournal of Biological Chemistry, 2001
- High-titer recombinant adeno-associated virus production utilizing a recombinant herpes simplex virus type I vector expressing AAV-2 Rep and CapGene Therapy, 1999
- A Mutation in the C-terminal Putative Zn2+ Finger Motif of UL52 Severely Affects the Biochemical Activities of the HSV-1 Helicase-Primase SubcomplexJournal of Biological Chemistry, 1999
- The UL8 Subunit of the Herpes Simplex Virus Type-1 DNA Helicase-Primase Optimizes Utilization of DNA Templates Covered by the Homologous Single-strand DNA-binding Protein ICP8Journal of Biological Chemistry, 1996
- The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 proteinJournal of General Virology, 1994
- Herpes simplex virus type 1 origin-dependent DNA replication in insect cells using recombinant baculovirusesJournal of General Virology, 1992