scholarly journals Identification of Cellular Proteins That Interact with the Adeno-Associated Virus Rep Protein

2008 ◽  
Vol 83 (1) ◽  
pp. 454-469 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
Max Salganik ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Protein Complexes ◽  
Protein Translation ◽  
Cellular Protein ◽  
Productive Infection ◽  
Cellular Proteins ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins

ABSTRACT Adeno-associated virus (AAV) codes for four related nonstructural Rep proteins. AAV both replicates and assembles in the nucleus and requires coinfection with a helper virus, either adenovirus (Ad) or herpesvirus, for a productive infection. Like other more complex DNA viruses, it is believed that AAV interacts or modifies host cell proteins to carry out its infection cycle. To date, relatively little is known about the host proteins that interact with the viral Rep proteins, which are known to be directly involved in DNA replication, control of viral and cellular transcription, splicing, and protein translation. In this study, we used affinity-tagged Rep protein to purify cellular protein complexes that were associated with Rep in cells that had been infected with Ad and AAV. In all, we identified 188 cellular proteins from 16 functional categories, including 14 transcription factors, 6 translation factors, 15 potential splicing proteins, 5 proteins involved in protein degradation, and 13 proteins involved in DNA replication or repair. This dramatically increases the number of potential interactions over the current number of approximately 26. Twelve of the novel proteins found were further tested by coimmunoprecipitation or colocalization using confocal immunomicroscopy. Of these, 10 were confirmed as proteins that formed complexes with Rep, including proteins of the MCM complex (DNA replication), RCN1 (membrane transport), SMC2 (chromatin dynamics), EDD1 (ubiquitin ligase), IRS4 (signal transduction), and FUS (splicing). Computer analysis suggested that 45 and 28 of the 188 proteins could be placed in a pathway of interacting proteins involved in DNA replication and protein synthesis, respectively. Of the proteins involved in DNA replication, all of the previously identified proteins involved in AAV DNA replication were found, except Ad DBP. The only Ad protein found to interact with Rep was the E1b55K protein. In addition, we confirmed that Rep interacts with Ku70/80 helicase. In vitro DNA synthesis assays demonstrated that although Ku helicase activity could substitute for MCM to promote strand displacement synthesis, its presence was not essential. Our study suggests that the interaction of AAV with cellular proteins is much more complex than previously suspected and provides a resource for further studies of the AAV life cycle.

scholarly journals Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins

2007 ◽  
Vol 82 (3) ◽  
pp. 1458-1464 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Productive Infection ◽  
Nuclear Antigen ◽  
Minichromosome Maintenance ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Mcm Complex ◽  
Factor C

ABSTRACT Adeno-associated virus (AAV) replicates its DNA exclusively by a leading-strand DNA replication mechanism and requires coinfection with a helper virus, such as adenovirus, to achieve a productive infection. In previous work, we described an in vitro AAV replication assay that required the AAV terminal repeats (the origins for DNA replication), the AAV Rep protein (the origin binding protein), and an adenovirus-infected crude extract. Fractionation of these crude extracts identified replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and polymerase δ as cellular enzymes that were essential for AAV DNA replication in vitro. Here we identify the remaining factor that is necessary as the minichromosome maintenance (MCM) complex, a cellular helicase complex that is believed to be the replicative helicase for eukaryotic chromosomes. Thus, polymerase δ, RFC, PCNA, and the MCM complex, along with the virally encoded Rep protein, constitute the minimal protein complexes required to reconstitute efficient AAV DNA replication in vitro. Interfering RNAs targeted to MCM and polymerase δ inhibited AAV DNA replication in vivo, suggesting that one or more components of the MCM complex and polymerase δ play an essential role in AAV DNA replication in vivo as well as in vitro. Our reconstituted in vitro DNA replication system is consistent with the current genetic information about AAV DNA replication. The use of highly conserved cellular replication enzymes may explain why AAV is capable of productive infection in a wide variety of species with several different families of helper viruses.

scholarly journals Adeno-Associated Virus (AAV) Type 5 Rep Protein Cleaves a Unique Terminal Resolution Site Compared with Other AAV Serotypes

1999 ◽  
Vol 73 (5) ◽  
pp. 4293-4298 ◽  
Author(s):  
John A. Chiorini ◽  
Sandra Afione ◽  
Robert M. Kotin
Keyword(s):  
Dna Sequences ◽  
Cleavage Site ◽  
Inverted Terminal Repeat ◽  
Nonstructural Proteins ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins ◽  
A Genome ◽  
Viral Components

ABSTRACT Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) intrans, and inverted terminal repeat (ITR) sequences incis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

scholarly journals Adeno-Associated Virus Rep Protein-Mediated Inhibition of Transcription of the Adenovirus Major Late Promoter In Vitro

2006 ◽  
Vol 80 (13) ◽  
pp. 6207-6217 ◽  
Author(s):  
Patrick G. Needham ◽  
John M. Casper ◽  
Vivian Kalman-Maltese ◽  
Kristin Verrill ◽  
John David Dignam ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Initiation ◽  
Nucleotide Binding ◽  
Dnase I ◽  
Tata Box ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Rep Proteins ◽  
Gel Shift

ABSTRACT Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication. In transient plasmid transfection assays, Rep proteins inhibit gene expression from a variety of transcription promoters. We have examined Rep protein-mediated inhibition of transcription of the Ad major late transcription promoter (AdMLP) in vitro. Rep78/68 are the strongest transcription suppressors and the purine nucleotide binding site in the Rep proteins, and by implication, the ATPase activity or conformational change induced by nucleotide binding is required for full repression. Rep52 has modest effects, and Rep40 exerts no significant effect on transcription. Rep78/68 and their N-terminal 225-residue domain bind to a 55-bp AdMLP DNA fragment in gel shift assays, suggesting that protein-DNA interactions are required for inhibition. This interaction was confirmed in DNase I protection assays and maps to a region extending from the TATA box to the transcription initiation site. Gel shift, DNase I, and chemical cross-linking assays with TATA box-binding protein (TBP) and Rep68 indicate that both proteins interact with each other and with the promoter at adjacent sites. The demonstration of Rep interaction with TBP and the AdMLP suggests that Rep78/68 alter the preinitiation complex of RNA polymerase II transcription. These observations provide new insight into the mechanism of Rep-mediated inhibition of gene expression.

scholarly journals A Role for Single-Stranded Templates in Cell-Free Adeno-Associated Virus DNA Replication

2000 ◽  
Vol 74 (2) ◽  
pp. 744-754 ◽  
Author(s):  
Peter Ward ◽  
R. Michael Linden
Keyword(s):  
Dna Replication ◽  
Infected Cell ◽  
Dna Binding Protein ◽  
Strand Displacement ◽  
Dna Templates ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Cell Extracts ◽  
Infected Cells

ABSTRACT Assays have been described in which duplex adeno-associated virus (AAV) DNA can be replicated in HeLa cell extracts with exogenous AAV Rep protein. These assays appear to mimic the AAV DNA replication that occurs in the cell, including the ability of extracts from adenovirus (Ad)-infected cells to replicate duplex AAV DNA templates more efficiently than extracts from uninfected cells can. We showed previously that the Ad-infected extract was able to support a more processive replication than the uninfected extract. When the Ad single-stranded DNA binding protein (Ad-DBP) was added to an uninfected extract, DNA replication became processive. Based on a strand displacement replication model, we hypothesized that the Ad-DBP was stabilizing the displaced single-stranded DNA during strand displacement replication. In this report, we show that in Ad-infected extracts most of the newly replicated duplex DNA is converted into a single-stranded form shortly after synthesis. Using the results of assays for the replication of single-stranded AAV DNA, we show that these single-stranded molecules serve as templates for additional replication. In addition, we identify a class of molecules which are likely to be intermediates of replication on single-stranded templates. We discuss a possible role for replication of single-stranded molecules in the infected cell.

scholarly journals Efficient Expression of the Adeno-Associated Virus Type 5 P41 Capsid Gene Promoter in 293 Cells Does Not Require Rep

2006 ◽  
Vol 80 (13) ◽  
pp. 6559-6567 ◽  
Author(s):  
Chaoyang Ye ◽  
Jianming Qiu ◽  
David J. Pintel
Keyword(s):  
Virus Type ◽  
Gene Promoter ◽  
Capsid Gene ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Adenovirus E1a ◽  
Amino Terminal ◽  
293 Cells ◽  
Rep Proteins ◽  
Efficient Expression

ABSTRACT Efficient expression of the adeno-associated virus type 5 (AAV5) P41 capsid gene promoter required adenovirus E1A and/or E1B; however, in contrast to what was observed for expression of the AAV2 capsid gene promoter (P40), neither adenovirus infection nor the large Rep protein was required. Although both the AAV2 and the AAV5 large Rep proteins efficiently bound the (GAGY)3 Rep-binding element, the AAV5 large Rep protein transactivated transcription of the inducible AAV2 P40 promoter much less well than AAV2 large Rep. Differences in their activation potentials were mapped to the amino-terminal region of the proteins, and the poorly transactivating AAV5 Rep protein could competitively inhibit AAV2 Rep transactivation.

scholarly journals Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

1998 ◽  
Vol 18 (5) ◽  
pp. 2748-2757 ◽  
Author(s):  
Jacqueline M. Sterner ◽  
Susan Dew-Knight ◽  
Christine Musahl ◽  
Sally Kornbluth ◽  
Jonathan M. Horowitz
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Protein Complexes ◽  
Replication Assay ◽  
Amino Terminal ◽  
Licensing Factor ◽  
Human Proteins ◽  
Carboxy Terminal ◽  
Related Proteins

ABSTRACT A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

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

2001 ◽  
Vol 75 (7) ◽  
pp. 3230-3239 ◽  
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.

scholarly journals Independent regions of adenovirus E1A are required for binding to and dissociation of E2F-protein complexes.

1993 ◽  
Vol 13 (12) ◽  
pp. 7267-7277 ◽  
Author(s):  
A R Fattaey ◽  
E Harlow ◽  
K Helin
Keyword(s):  
Transcription Factor ◽  
Protein Complexes ◽  
Susceptibility Gene ◽  
Cyclin A ◽  
Cellular Proteins ◽  
Adenovirus E1a ◽  
E2f Transcription Factor ◽  
E1a Gene

The transcription factor E2F is present in independent complexes with the product of the retinoblastoma susceptibility gene, pRB, and a related gene product, p107, in association with the cyclin A-cdk2 or the cyclin E-cdk2 kinase complex. pRB and p107 can negatively regulate E2F activity, since overexpression of pRB or p107 in cells lacking a functional pRB leads to the repression of E2F activity. The products of the adenovirus E1A gene can disrupt E2F complexes and result in free and presumably active E2F transcription factor. The regions of E1A required for this function are also essential for binding to a number of cellular proteins, including pRB and p107. Through the use of a number of glutathione S-transferase fusion proteins representing different regions of E1A, as well as in vivo expression of E1A proteins containing deletions of either conserved region 1 (CR1) or CR2, we find that CR2 of E1A can form stable complexes with E2F. E1A proteins containing both CR1 and CR2 also associate with E2F, although the presence of these proteins results in the release of free E2F from its complexes. In vitro reconstitution experiments indicate that E1A-E2F interactions are not direct and that pRB can serve to facilitate these interactions. Complexes containing E1A, p107, cyclin A, and E2F were identified in vivo, which indicates that E1A may associate with E2F through either p107 or pRB. Peptide competition experiments demonstrate that the pRB-binding domain of the human E2F-1 protein can compete with the CR1 but not CR2 domain of E1A for binding to pRB. These results indicate that E1A CR1 and E2F-1 may bind to the same or overlapping sites on pRB and that E1A CR2 binds to an independent region. On the basis of our results, we propose a two-step model for the release of E2F from pRB and p107 cellular proteins.

scholarly journals Distinct Functions of the Two Specificity Determinants in Replication Initiation of Plasmids ColE2-P9 and ColE3-CA38

2007 ◽  
Vol 189 (6) ◽  
pp. 2392-2400 ◽  
Author(s):  
Kazuteru Aoki ◽  
Miki Shinohara ◽  
Tateo Itoh
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Replication Initiation ◽  
Mobility Shift ◽  
Rep Protein ◽  
Rep Proteins ◽  
Mobility Shift Assay ◽  
Hand Region ◽  
A Site ◽  
Specificity Determinants

ABSTRACT The plasmid ColE2-P9 Rep protein specifically binds to the cognate replication origin to initiate DNA replication. The replicons of the plasmids ColE2-P9 and ColE3-CA38 are closely related, although the actions of the Rep proteins on the origins are specific to the plasmids. The previous chimera analysis identified two regions, regions A and B, in the Rep proteins and two sites, α and β, in the origins as specificity determinants and showed that when each component of the region A-site α pair and the region B-site β pair is derived from the same plasmid, plasmid DNA replication is efficient. It is also indicated that the replication specificity is mainly determined by region A and site α. By using an electrophoretic mobility shift assay, we demonstrated that region B and site β play a critical role for stable Rep protein-origin binding and, furthermore, that 284-Thr in this region of the ColE2 Rep protein and the corresponding 293-Trp of the ColE3 Rep protein mainly determine the Rep-origin binding specificity. On the other hand, region A and site α were involved in the efficient unwinding of several nucleotide residues around site α, although they were not involved in the stable binding of the Rep protein to the origin. Finally, we discussed how the action of the Rep protein on the origin involving these specificity determinants leads to the plasmid-specific replication initiation.

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