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Journal of Virology, November 1999, p. 9433-9445, Vol. 73, No. 11
Gene Therapy Center,1
Curriculum in Genetics and Molecular
Biology,3 Structural BioInformatics Core
Facility,4 and Department of
Pharmacology,2 University of North Carolina at
Chapel Hill, Chapel Hill, North Carolina 27599, and Gene
Therapy Center,5 and Department of
Molecular Genetics and Microbiology,6
University of Florida, Gainesville, Florida 32610
Received 26 April 1999/Accepted 23 July 1999
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 a
ts 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 ts
mutants 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.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Charge-to-Alanine Mutagenesis of the
Adeno-Associated Virus Type 2 Rep78/68 Proteins Yields
Temperature-Sensitive and Magnesium-Dependent Variants
*
Corresponding author. Mailing address: Gene Therapy
Center, 7119 Thurston Bowles CB 7352, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599. Phone: (919) 962-3285. Fax: (919)
966-0907. E-mail: rjs{at}med.unc.edu.
Present address: Institute for Gene Therapy, University of
Pennsylvania, and The Wistar Institute, Philadelphia, PA 19104.
Journal of Virology, November 1999, p. 9433-9445, Vol. 73, No. 11
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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