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J Virol. Feb 1992; 66(2): 1119–1128.
PMCID: PMC240816

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 Rep40. In the cytoplasmic extracts Rep52 and Rep40 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 Rep40) 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 Rep40. 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-gamma-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.

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  • Astell CR, Chow MB, Ward DC. Sequence analysis of the termini of virion and replicative forms of minute virus of mice DNA suggests a modified rolling hairpin model for autonomous parvovirus DNA replication. J Virol. 1985 Apr;54(1):171–177. [PMC free article] [PubMed]
  • Becerra SP, Koczot F, Fabisch P, Rose JA. Synthesis of adeno-associated virus structural proteins requires both alternative mRNA splicing and alternative initiations from a single transcript. J Virol. 1988 Aug;62(8):2745–2754. [PMC free article] [PubMed]
  • Berns KI, Bohenzky RA. Adeno-associated viruses: an update. Adv Virus Res. 1987;32:243–306. [PubMed]
  • Berns KI, Hauswirth WW. Adeno-associated viruses. Adv Virus Res. 1979;25:407–449. [PubMed]
  • Buller RM, Janik JE, Sebring ED, Rose JA. Herpes simplex virus types 1 and 2 completely help adenovirus-associated virus replication. J Virol. 1981 Oct;40(1):241–247. [PMC free article] [PubMed]
  • Cassinotti P, Weitz M, Tratschin JD. Organization of the adeno-associated virus (AAV) capsid gene: mapping of a minor spliced mRNA coding for virus capsid protein 1. Virology. 1988 Nov;167(1):176–184. [PubMed]
  • Cavalier-Smith T. Palindromic base sequences and replication of eukaryote chromosome ends. Nature. 1974 Aug 9;250(5466):467–470. [PubMed]
  • Challberg MD, Kelly TJ., Jr Adenovirus DNA replication in vitro. Proc Natl Acad Sci U S A. 1979 Feb;76(2):655–659. [PMC free article] [PubMed]
  • Chejanovsky N, Carter BJ. Mutagenesis of an AUG codon in the adeno-associated virus rep gene: effects on viral DNA replication. Virology. 1989 Nov;173(1):120–128. [PubMed]
  • Fife KH, Berns KI, Murray K. Structure and nucleotide sequence of the terminal regions of adeno-associated virus DNA. Virology. 1977 May 15;78(2):475–477. [PubMed]
  • Green MR, Roeder RG. Transcripts of the adeno-associated virus genome: mapping of the major RNAs. J Virol. 1980 Oct;36(1):79–92. [PMC free article] [PubMed]
  • Green MR, Straus SE, Roeder RG. Transcripts of the adenovirus-associated virus genome: multiple polyadenylated RNAs including a potential primary transcript. J Virol. 1980 Aug;35(2):560–565. [PMC free article] [PubMed]
  • Hermonat PL, Labow MA, Wright R, Berns KI, Muzyczka N. Genetics of adeno-associated virus: isolation and preliminary characterization of adeno-associated virus type 2 mutants. J Virol. 1984 Aug;51(2):329–339. [PMC free article] [PubMed]
  • Im DS, Muzyczka N. Factors that bind to adeno-associated virus terminal repeats. J Virol. 1989 Jul;63(7):3095–3104. [PMC free article] [PubMed]
  • Im DS, Muzyczka N. The AAV origin binding protein Rep68 is an ATP-dependent site-specific endonuclease with DNA helicase activity. Cell. 1990 May 4;61(3):447–457. [PubMed]
  • Janik JE, Huston MM, Rose JA. Locations of adenovirus genes required for the replication of adenovirus-associated virus. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1925–1929. [PMC free article] [PubMed]
  • Labow MA, Graf LH, Jr, Berns KI. Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes. Mol Cell Biol. 1987 Apr;7(4):1320–1325. [PMC free article] [PubMed]
  • Labow MA, Hermonat PL, Berns KI. Positive and negative autoregulation of the adeno-associated virus type 2 genome. J Virol. 1986 Oct;60(1):251–258. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Laughlin CA, Westphal H, Carter BJ. Spliced adenovirus-associated virus RNA. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5567–5571. [PMC free article] [PubMed]
  • Lusby E, Bohenzky R, Berns KI. Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome. J Virol. 1981 Mar;37(3):1083–1086. [PMC free article] [PubMed]
  • Lusby E, Fife KH, Berns KI. Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA. J Virol. 1980 May;34(2):402–409. [PMC free article] [PubMed]
  • Lusby EW, Berns KI. Mapping of the 5' termini of two adeno-associated virus 2 RNAs in the left half of the genome. J Virol. 1982 Feb;41(2):518–526. [PMC free article] [PubMed]
  • Maxam AM, Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. [PubMed]
  • Mendelson E, Trempe JP, Carter BJ. Identification of the trans-acting Rep proteins of adeno-associated virus by antibodies to a synthetic oligopeptide. J Virol. 1986 Dec;60(3):823–832. [PMC free article] [PubMed]
  • Metcalf JB, Bates RC, Lederman M. Interaction of virally coded protein and a cell cycle-regulated cellular protein with the bovine parvovirus left terminus ori. J Virol. 1990 Nov;64(11):5485–5490. [PMC free article] [PubMed]
  • McPherson RA, Rosenthal LJ, Rose JA. Human cytomegalovirus completely helps adeno-associated virus replication. Virology. 1985 Nov;147(1):217–222. [PubMed]
  • Redemann BE, Mendelson E, Carter BJ. Adeno-associated virus rep protein synthesis during productive infection. J Virol. 1989 Feb;63(2):873–882. [PMC free article] [PubMed]
  • Samulski RJ, Chang LS, Shenk T. A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication. J Virol. 1987 Oct;61(10):3096–3101. [PMC free article] [PubMed]
  • Samulski RJ, Srivastava A, Berns KI, Muzyczka N. Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV. Cell. 1983 May;33(1):135–143. [PubMed]
  • Schlehofer JR, Ehrbar M, zur Hausen H. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus. Virology. 1986 Jul 15;152(1):110–117. [PubMed]
  • Senapathy P, Tratschin JD, Carter BJ. Replication of adeno-associated virus DNA. Complementation of naturally occurring rep- mutants by a wild-type genome or an ori- mutant and correction of terminal palindrome deletions. J Mol Biol. 1984 Oct 15;179(1):1–20. [PubMed]
  • Snyder RO, Im DS, Muzyczka N. Evidence for covalent attachment of the adeno-associated virus (AAV) rep protein to the ends of the AAV genome. J Virol. 1990 Dec;64(12):6204–6213. [PMC free article] [PubMed]
  • Snyder RO, Samulski RJ, Muzyczka N. In vitro resolution of covalently joined AAV chromosome ends. Cell. 1990 Jan 12;60(1):105–113. [PubMed]
  • Srivastava A, Lusby EW, Berns KI. Nucleotide sequence and organization of the adeno-associated virus 2 genome. J Virol. 1983 Feb;45(2):555–564. [PMC free article] [PubMed]
  • Straus SE, Ginsberg HS, Rose JA. DNA-minus temperature-sensitive mutants of adenovirus type 5 help adenovirus-associated virus replication. J Virol. 1975 Jan;17(1):140–148. [PMC free article] [PubMed]
  • Straus SE, Sebring ED, Rose JA. Concatemers of alternating plus and minus strands are intermediates in adenovirus-associated virus DNA synthesis. Proc Natl Acad Sci U S A. 1976 Mar;73(3):742–746. [PMC free article] [PubMed]
  • Teraoka H, Sumikawa T, Tsukada K. Purification of DNA ligase II from calf thymus and preparation of rabbit antibody against calf thymus DNA ligase II. J Biol Chem. 1986 May 25;261(15):6888–6892. [PubMed]
  • Tratschin JD, Miller IL, Carter BJ. Genetic analysis of adeno-associated virus: properties of deletion mutants constructed in vitro and evidence for an adeno-associated virus replication function. J Virol. 1984 Sep;51(3):611–619. [PMC free article] [PubMed]
  • Tratschin JD, Tal J, Carter BJ. Negative and positive regulation in trans of gene expression from adeno-associated virus vectors in mammalian cells by a viral rep gene product. Mol Cell Biol. 1986 Aug;6(8):2884–2894. [PMC free article] [PubMed]
  • Trempe JP, Carter BJ. Alternate mRNA splicing is required for synthesis of adeno-associated virus VP1 capsid protein. J Virol. 1988 Sep;62(9):3356–3363. [PMC free article] [PubMed]
  • Trempe JP, Mendelson E, Carter BJ. Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli. Virology. 1987 Nov;161(1):18–28. [PubMed]
  • Yakobson B, Koch T, Winocour E. Replication of adeno-associated virus in synchronized cells without the addition of a helper virus. J Virol. 1987 Apr;61(4):972–981. [PMC free article] [PubMed]

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