Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

ABSTRACT Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase δ, but not polymerase ε or α, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase δ complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase δ, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase δ, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

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Cloning, Expression and Purification of Full-Length Rep78 of Adeno-associated Virus as a Fusion Protein with Maltose Binding Protein in Escherichia coli

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1995.1396 ◽

1995 ◽

Vol 208 (2) ◽

pp. 714-720 ◽

Cited By ~ 7

Author(s):

R.B. Batchu ◽

D.A. Miles ◽

T.M. Rechtin ◽

R.R. Drake ◽

P.L. Hermonat

Keyword(s):

Escherichia Coli ◽

Fusion Protein ◽

Binding Protein ◽

Maltose Binding Protein ◽

Full Length ◽

Expression And Purification ◽

Adeno Associated Virus

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Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

Journal of Virology ◽

10.1128/jvi.72.1.420-427.1998 ◽

1998 ◽

Vol 72 (1) ◽

pp. 420-427 ◽

Cited By ~ 41

Author(s):

Peter Ward ◽

Frank B. Dean ◽

Michael E. O’Donnell ◽

Kenneth I. Berns

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Basic Question ◽

Adeno Associated Virus ◽

Vitro Assay ◽

Cell Extracts ◽

Vector Sequences

ABSTRACT A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences.

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A maltose-binding protein/adeno-associated virus Rep68 fusion protein has DNA-RNA helicase and ATPase activities.

Journal of Virology ◽

10.1128/jvi.69.6.3542-3548.1995 ◽

1995 ◽

Vol 69 (6) ◽

pp. 3542-3548 ◽

Cited By ~ 41

Author(s):

R S Wonderling ◽

S R Kyöstiö ◽

R A Owens

Keyword(s):

Fusion Protein ◽

Binding Protein ◽

Rna Helicase ◽

Maltose Binding Protein ◽

Adeno Associated Virus

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Production of MBP(maltose binding protein)-GroES fusion protein and utilization to stimulate GroEL-mediated protein refolding

Journal of Fermentation and Bioengineering ◽

10.1016/s0922-338x(97)80356-x ◽

1998 ◽

Vol 85 (1) ◽

pp. 69-73 ◽

Cited By ~ 2

Author(s):

Yoshiyuki Ishii ◽

Kaoru Murakami ◽

Hiroaki I.-Ogawa ◽

Akihiko Kondo ◽

Yasuhiko Kato

Keyword(s):

Fusion Protein ◽

Binding Protein ◽

Protein Refolding ◽

Maltose Binding Protein

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Effect of immunization of hamsters against recombinant P26h on fertility rates

Reproduction ◽

10.1530/rep.0.1230307 ◽

2002 ◽

pp. 307-313 ◽

Cited By ~ 18

Author(s):

C Gaudreault ◽

L Montfort ◽

R Sullivan

Keyword(s):

Zona Pellucida ◽

Binding Protein ◽

Maltose Binding Protein ◽

Fertilization Rate ◽

Active Immunization ◽

Antigenic Determinants ◽

Binding Assays ◽

Contraceptive Vaccine

Despite the various contraceptive methods available, an effective and inexpensive method remains to be established. Immunocontraception may help to achieve this goal. P26h has been proposed as a candidate for the development of a male contraceptive vaccine. P26h, a hamster sperm protein, interacts with the zona pellucida. Furthermore, in vivo fertilization can be blocked completely by active immunization of male hamsters against P26h. Maltose binding protein (MBP)-P26 shares antigenic determinants with the native P26h present on cauda epididymal spermatozoa. The aim of the present study was to reproduce the immunocontraceptive properties of native P26h by immunizing male hamsters against a recombinant P26h fused with a maltose binding protein (MBP). Active immunization of male hamsters with the MBP-P26h showed that specific anti-P26h circulating IgGs could be generated. Mating of immunized male hamsters with superovulated females resulted in a significant decrease, 20-25%, in the fertilization rate. This result is in agreement with results from in vitro sperm-zona pellucida binding assays. Indeed, the anti-recombinant P26h IgGs showed lower inhibitory properties when compared with anti-native P26h IgG. Despite the high anti-P26h IgG titres generated in hamsters, histological studies showed that active immunization has no pathological sequelae to the reproductive tissues. The potential of P26h as a candidate for a contraceptive vaccine is discussed.

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Amino-Terminal Domain Exchange Redirects Origin-Specific Interactions of Adeno-Associated Virus Rep78 In Vitro

Journal of Virology ◽

10.1128/jvi.75.7.3230-3239.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3230-3239 ◽

Cited By ~ 21

Author(s):

Miran Yoon ◽

Deborah H. Smith ◽

Peter Ward ◽

Francisco J. Medrano ◽

Aneel K. Aggarwal ◽

...

Keyword(s):

Dna Replication ◽

Catalytic Domain ◽

Adeno Associated Virus ◽

Free System ◽

Site Specific ◽

Amino Terminal ◽

Terminal Domain ◽

Site Specific Integration ◽

Amino Terminal Domain

ABSTRACT The unique ability of adeno-associated virus type 2 (AAV) to site-specifically integrate its genome into a defined sequence on human chromosome 19 (AAVS1) makes it of particular interest for use in targeted gene delivery. The objective underlying this study is to provide evidence for the feasibility of retargeting site-specific integration into selected loci within the human genome. Current models postulate that AAV DNA integration is initiated through the interactions of the products of a single viral open reading frame,REP, with sequences present in AAVS1 that resemble the minimal origin for AAV DNA replication. Here, we present a cell-free system designed to dissect the Rep functions required to target site-specific integration using functional chimeric Rep proteins derived from AAV Rep78 and Rep1 of the closely related goose parvovirus. We show that amino-terminal domain exchange efficiently redirects the specificity of Rep to the minimal origin of DNA replication. Furthermore, we establish that the amino-terminal 208 amino acids of Rep78/68 constitute a catalytic domain of Rep sufficient to mediate site-specific endonuclease activity.

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