Journal article
Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants
Journal of virology, Vol.73(11), pp.9433-9445
11/01/1999
DOI: 10.1128/JVI.73.11.9433-9445.1999
PMCID: PMC112978
PMID: 10516052
Abstract
ABSTRACT The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 and 68 (Rep78/68) exhibit a number of biochemical activities required for AAV replication, including specific binding to a 22-bp region of the terminal repeat, site-specific endonuclease activity, and helicase activity. Individual and clusters of charged amino acids were converted to alanines in an effort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ability to mediate replication of recombinant AAV vectors at various temperatures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The results of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32°C, which exceeded wild-type activity by 48 h. Replication activity was reduced by more than threefold at 37°C and was undetectable at 39°C. Stability of the Rep78 protein paralleled replication levels at each temperature, further supporting ats phenotype. Replication differences resulted in a 3-log-unit difference in virus yields between the permissive and nonpermissive temperatures (2.2 × 106 and 3 × 103, respectively), demonstrating that this is a relatively tight mutant. In addition to the ts Rep mutant, we identified a nonconditional mutant with a reduced ability to support viral replication in vivo. Additional characterization of this mutant demonstrated an Mg2+-dependent phenotype that was specific to Rep endonuclease activity and did not affect helicase activity. The two mutants described here are unique, in that Rep tsmutants have not previously been described and the D412A Rep mutant represents the first mutant in which the helicase and endonuclease functions can be distinguished biochemically. Further understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for production of viral vectors.
Details
- Title: Subtitle
- Charge-to-Alanine Mutagenesis of the Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields Temperature-Sensitive and Magnesium-Dependent Variants
- Creators
- Denise K Gavin - Gene Therapy Center,1, Department of Pharmacology,2 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, andSamuel M Young - Gene Therapy Center,1, Curriculum in Genetics and Molecular Biology,3Weidong Xiao - Gene Therapy Center,1, Curriculum in Genetics and Molecular Biology,3Brenda Temple - Structural BioInformatics Core Facility,4 andCorinne R Abernathy - Gene Therapy Center,5 and, Department of Molecular Genetics and Microbiology,6University of Florida, Gainesville, Florida 32610Daniel J Pereira - Gene Therapy Center,5 and, Department of Molecular Genetics and Microbiology,6University of Florida, Gainesville, Florida 32610Nicholas Muzyczka - Gene Therapy Center,5 and, Department of Molecular Genetics and Microbiology,6University of Florida, Gainesville, Florida 32610Richard Jude Samulski - Gene Therapy Center,1, Department of Pharmacology,2 University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, and
- Resource Type
- Journal article
- Publication Details
- Journal of virology, Vol.73(11), pp.9433-9445
- DOI
- 10.1128/JVI.73.11.9433-9445.1999
- PMID
- 10516052
- PMCID
- PMC112978
- ISSN
- 0022-538X
- eISSN
- 1098-5514
- Language
- English
- Date published
- 11/01/1999
- Academic Unit
- Anatomy and Cell Biology; Iowa Neuroscience Institute; Otolaryngology
- Record Identifier
- 9984025575602771
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