Adeno-Associated VirusDNA Replication InVitro: Activation bya Maltose Binding Protein/Rep 68Fusion Protein

Theadeno-associated virus (AAV)nonstructural protein Rep68isrequired forviral DNAreplication. Anin vitro assayhasbeendeveloped inwhichaddition ofRep68toan extract fromuninfected HeLacells supports AAVDNAreplication. Inthis paper,we report characterization ofthereplication processwhenafusion ofthe maltose binding protein andRep68,expressed inEscherichia coli, was usedintheassay.Replication was observed whenthetemplate was either linear double-stranded AAVDNA or a plasmid construct containing intact AAVDNA.Whentherecombinant plasmid construct was usedasthetemplate, there was replication of pBR322DNA aswell as theAAVDNA;however, linear pBR322DNA was notreplicated. Whentheplasmid construct was thetemplate, replication appeared toinitiate on theintact plasmid andledtoseparation ofthe AAVsequencesfromthose ofthevector, a processwhichhasbeentermed rescue.There was no evidence that replication couldinitiate on theproducts ofrescue.Rep68can makea site-specific nick124nucleotides from the3'endofAAVDNA;thesite ofthenickhasbeencalled theterminal resolution site. Ourdataaremost consistent withinitiation occurring attheterminal resolution site andproceeding towardthe3'terminus. Whenthetemplate was theplasmid construct, either elongation continued pastthejunction intopBR322 sequencesorthenewlysynthesized sequencehairpinned, switched template strands, andreplicated theAAV DNA.Replication was linear for4h,during whichtime70%ofthemaximalsynthesis tookplace. Anadditional finding was thattheRepfusion could resolve AAVdimerlength duplex intermediates into monomer duplexes without DNA synthesis. Thehumanparvovirus adeno-associated virus type2(AAV) isclassified asa dependovirus because oftherequirement for coinfection withhelper virus (either adenovirus [Ad]or herpesvirus) foroptimal replication incell culture (1,2,4,21). In theabsence ofhelper virus coinfection, theAAV genome integrates intothecellular genome toestablish a latent infection(1). Inseveral lines ofhumancells, integration hasbeen reported tobewithin adefined locus on chromosome 19q13.3qter(15-17, 27). Helper virus infection ofthelatently infected cell leads torescueandreplication oftheAAV genome,with virion production. We havereported an invitro assaywhich appearstobeamodelfortherescueandreplication ofAAV. Ithasmany oftheparameters determined forinvivoAAV rescueandreplication (10). Indeed, many oftheparameters of invivoreplication were determined after transfection of Ad-infected humancells withplasmid constructs thesame as or similar tothoseusedintheinvitro assay(11,25).In particular, theinvitro assayrequires an extract fromHeLa cells coinfected byAAV andAd;extracts fromuninfected cells orcells infected byAd orAAV alonedonotsupportreplication. Theinvitro assayalsohasspecific template requirements. TheAAV genome isa linear, single-stranded DNA (4,680 bases) with an inverted terminal repeat(ITR)of145bases (20, 28). Theterminal 125bases are an overall palindrome interrupted bytwosmaller, internal 21-base palindromes, one on either sideoftheoverall axisofsymmetry. Whenthepalindromicregion oftheitrisfolded on itself tomaximize