Partial purification of adeno-associated virus Rep78, Rep52, and Rep40 and their biochemical characterization

Abstract

We have used differential cell extraction and conventional chromatography to separate and partially purify the four adeno-associated virus (AAV) nonstructural proteins Rep78, Rep68, Rep52, and Rep4O. In the cytoplasmic extracts Rep52 and Rep4O were present in greater abundance than Rep68 and Rep78, with Rep78 being the least abundant. In nuclear extracts the four Rep proteins were approximately equal in abundance. Regardless of the subcellular fraction examined, three of the Rep proteins (Rep78, Rep68, and Rep4O) consisted of two protein species with slightly different mobilities during polyacrylamide gel electrophoresis. In contrast, Rep52 consisted of only one protein species. Both Rep78 and Rep68 were capable of binding efficiently to AAV terminal hairpin DNA substrates, but we could not detect site-specific DNA binding by Rep52 and Rep4O. Like Rep68, Rep78 had both an ATP-dependent trs endonuclease and a DNA helicase activity. Both Rep78 and Rep68 cut the terminal AAV sequence at the same site (nucleotide 124). The binding, trs endonuclease, and DNA helicase activities comigrated during sucrose density gradient centrifugation with a mobility expected for a monomer of the protein, suggesting that the three biochemical activities were intrinsic properties of the larger Rep proteins. The chromatographic behavior and the DNA-binding properties of the four Rep proteins identified at least two domains within the rep coding region, an exposed hydrophobic domain within the C-terminal end (amino acids 578 to 621) and a region within the N terminus (amino acids 1 to 214) which was necessary for binding to the terminal repeat sequence. No site-specific nuclease activity was seen in the presence of nucleotide analogs ATP-y-S or AMP-PNP, suggesting that ATP hydrolysis was required for the endonuclease reaction. Furthermore, although ATP was the only cofactor which would support the trs endonuclease activity of Rep78, Rep68 nuclease activity was seen in the presence of several other nucleotide cofactors, including CTP, GTP, and UTP.

The Use of Heterologous Promoters for Adeno‐Associated Virus (AAV) Protein Expression in AAV Vector Production

“…AAV protein expression was analyzed by Western blotting. All four types of Rep protein were detected, and Rep78 appeared as a doublet as described previously (12). The additional bands migrating below Rep40 and of about 55 kilodaltons (kD) were detected, but the origins of these products are unknown.…”

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle

“…Completion of the replication scheme requires resolution of the covalently closed hairpin structure by Repdependent nicking, which results in the transfer of this sequence to the progeny DNA. The nicking reaction generates a new 3Ј OH on the parental strand, providing the substrate for copying back the transferred ITR sequences (13,15 for a review, see reference 22).…”

The cellular transcription factor SP1 and an unknown cellular protein are required to mediate Rep protein activation of the adeno-associated virus p19 promoter

“…Genetic analysis of the AAV genome has demonstrated that the p5 Rep proteins (Rep78 and Rep68) are required to reg-ulate transcription from all three promoters and to replicate the viral DNA (20,53). Several biochemical properties have been characterized for the p5 Rep proteins during replication; these include site-specific endonuclease (24,51), DNA helicase (24,58), and DNA binding (24,25,38) activities. In the absence of Ad, Rep has been shown to repress both p5 and p19 transcription (21,30).…”