MUTATIONAL ANALYSIS OF THE ADENOASSOCIATED VIRUS TYPE-2 REP68 PROTEINHELICASE MOTIFS

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are r equired for viral replication, These proteins are encoded hv unspliced and spliced transcripts, respectively, from the p(5) promoter of AAV and therefore have overlapping amino acid sequences, The Rep78 and Rep 68 proteins share a variety of activities including endonuclease, heli case, and ATPase activities and the ability to hind AAV hairpin DNA. T he part of the amino acid sequence which is identical in Rep78 and Rep 68 contains consensus helicase motifs that are conserved among the par vovirus replication proteins, In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant protein s were synthesized as maltose binding protein-Rep68 fusions in Escheri chia coli cells and affinity purified on amylose resin, The fusion pro teins were assayed in vitro, and their activities were directly compar ed to those of the fusion protein MDP-ReD68 Delta which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstr ated previously to have all of the in vitro activities of wild-type Re p78 and Rep68. Our analysis showed that almost all mutations in the pu tative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.