Charge-to-alanine mutagenesis of the adeno-associated virus type 2 rep78/68 proteins yields temperature-sensitive and magnesium-dependent variants

The adeno-associated virus type 2 (AAV) replication (Rep) proteins Rep78 an d 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. Individ ual and clusters of charged amino acids were converted to alanines in an ef fort to generate a collection of conditionally defective Rep78/68 proteins. Rep78 variants were expressed in human 293 cells and analyzed for their ab ility to mediate replication of recombinant AAV vectors at various temperat ures. The biochemical activities of Rep variants were further characterized in vitro by using Rep68 His-tagged proteins purified from bacteria. The re sults of these analyses identified a temperature-sensitive (ts) Rep protein (D40,42,44A-78) that exhibited a delayed replication phenotype at 32 degre es C, which exceeded wild-type activity by 48 h. Replication activity was r educed by more than threefold at 37 degrees C and was undetectable at 39 de grees C. Stability of the Rep78 protein paralleled replication levels at ea ch 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 x 10(6) and 3 x 10(3), respectively), d emonstrating 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 mut ant demonstrated an Mg2+-dependent phenotype that was specific to Rep endon uclease activity and did not affect helicase activity. The two mutants desc ribed here are unique, in that Rep ts mutants have not previously been desc ribed and the D412A Rep mutant represents the first mutant in which the hel icase and endonuclease functions can be distinguished biochemically. Furthe r understanding of these mutants should facilitate our understanding of AAV replication and integration, as well as provide novel strategies for produ ction of viral vectors.