The adeno-associated virus type 2 (AAV) Rep68 protein produced in Escherichia coli as a fusion protein with maltose-binding protein (MBP-Rep68⌬) has previously been shown to possess DNA-DNA helicase activity, as does the purified wild-type Rep68. In the present study, we demonstrate that MBP-Rep68⌬ also catalyzes the unwinding of a DNA-RNA hybrid. MBP-Rep68⌬-mediated DNA-RNA helicase activity required ATP hydrolysis and the presence of Mg 2؉ ions and was inhibited by high ionic strength. The efficiency of the DNA-RNA helicase activity of MBP-Rep68⌬ was comparable to its DNA-DNA helicase activity. However, MBP-Rep68⌬ lacked the ability to unwind a blunt-ended DNA-RNA substrate and RNA-RNA duplexes. We have also demonstrated that MBP-Rep68⌬ has ATPase activity which is enhanced by the presence of single-stranded DNA but not by RNA. The MBP-Rep68⌬NTP mutant protein, which has a lysine-to-histidine substitution at amino acid 340 in the putative nucleoside triphosphate-binding site of Rep68, not only lacks DNA-RNA helicase and ATPase activities but also inhibits the helicase activity of MBP-Rep68⌬. DNA-RNA helicase activity of Rep proteins might play a pivotal role in the regulation of AAV gene expression by AAV Rep proteins. Adeno-associated virus type 2 (AAV) is a nonpathogenic human parvovirus which requires the coinfection of adenovirus or herpesvirus as a helper to enable its efficient replication (6). AAV has a linear single-stranded DNA genome which has 145-nucleotide-long inverted terminal repeats (42). The AAV genome contains two genes called rep and cap (14). The rep gene codes for four overlapping in-frame polypeptides referred to as Rep78, Rep68, Rep52, and Rep40 (30, 42). The cap gene encodes proteins called VP-1, VP-2, and VP-3 that form the capsid (7). The genome of AAV has three promoters called p 5 , p 19 , and p 40. The Rep78 and Rep68 proteins expressed from p 5 are required for viral DNA replication, while Rep52 and Rep40 expressed from p 19 play a role in the accumulation of single-stranded progeny genomes used for packaging (8, 31). The VP-1, VP-2, and VP-3 capsid proteins are expressed from the p 40 promoter (7). Rep78 and Rep68 proteins have the ability to bind to the AAV inverted terminal repeats (2, 16) by recognition of a GCTC repeating motif (28, 46), cause a site-specific and strand-specific nick at the terminal resolution site (trs) (17, 41), covalently attach to the trs (40), and have a DNA-DNA heli

Transcript levels from the P 5 promoter of adeno-associated virus type 2 (AAV) are negatively regulated by the AAV Rep78 and Rep68 proteins in the absence of helper virus. We have identified a Rep-responsive negative cis element of the P 5 promoter between the P 5 TATA box and transcription start site by using 5 and 3 deletions of the P 5 promoter fused to the chloramphenicol acetyltransferase gene. This element contains four imperfect GAGC repeats similar to the Rep recognition sequences (RRSs) in the AAV inverted terminal repeats and in the AAV preferred integration locus in chromosome 19. Band shift analyses showed that human 293 cell nuclear extracts containing Rep68 or Rep68/K340H, a putative nucleoside triphosphate (NTP)-binding-site mutant of Rep68, formed Rep-specific complexes with this P 5 RRS DNA. Within the P 5 RRS, mutation of a cytosine at position 273 in the AAV sequence to guanine abolished Rep68 binding to the DNA. A mutation in the P 5 RRS within a full-length AAV genome, which abolished Rep binding, resulted in a 40 to 50% reduction in the ability of wild-type Rep68 to inhibit the accumulation of P 5 transcripts in vivo. In contrast, the Rep68/K340H mutant was unable to down-regulate this mutated promoter. These results indicate that there are at least two mechanisms involved in the negative regulation of P 5 transcript levels by Rep68; one involves Rep68 binding to the P 5 RRS, and another requires the region of Rep68 containing the consensus NTP-binding motif. Furthermore, our studies of AAV genomes containing mutated RRS-and/or YY1-binding elements suggest that transcription factor YY1 binding to the transcription start site of P 5 interferes with Rep68 repression of the P 5 promoter.

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p 5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68⌬, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative 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.

The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences. They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins make a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA. The Rep68 and Rep78 proteins also have helicase and DNA-binding activities. The endonuclease activity is believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage. In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector. The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with amylose resin, and assayed in vitro for Rep68-specific activities. Although several of the mutations disrupted the endonuclease activity, only the mutation of tyrosine 152 abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities. Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities.