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 Human herpesvirus-6 rep/U94 gene product has single-stranded DNA-binding activity

2002 ◽  
Vol 83 (4) ◽  
pp. 847-854 ◽  
Author(s):  
Panadda Dhepakson ◽  
Yasuko Mori ◽  
Yun Bao Jiang ◽  
Hong Lan Huang ◽  
Pilailuk Akkapaiboon ◽  
...  
Keyword(s):  
Human Herpesvirus ◽  
Binding Activity ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Rep Protein ◽  
Dna Binding Activity ◽  
Late Protein ◽  
Glycoprotein H ◽  
Infected Cells

The characterization is reported of the human herpesvirus-6B (HHV-6B) rep/U94 gene, which is a homologue of the adeno-associated virus type 2 rep. In this study, a monoclonal antibody was produced against HHV-6B REP (anti-REP mAb). Immunofluorescence staining using the anti-REP mAb showed that REP was localized to the nucleus in HHV-6-infected MT4 cells. It was first detected at 24 h post-infection (p.i.) and accumulated to higher levels by 72 h p.i. REP may be expressed only at very low levels in HHV-6-infected cells: even when the late protein glycoprotein H was detected in nearly 90% of HHV-6-infected cells, REP was detected in only a small percentage of them. Western blot analysis showed that the anti-REP mAb recognized a 56-kDa polypeptide in HHV-6B-infected MT4 cells. Furthermore, the REP protein was shown to bind single-stranded DNA.

scholarly journals In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

PLoS Genetics ◽  
2014 ◽  
Vol 10 (12) ◽  
pp. e1004832 ◽  
Author(s):  
Javier Miralles Fusté ◽  
Yonghong Shi ◽  
Sjoerd Wanrooij ◽  
Xuefeng Zhu ◽  
Elisabeth Jemt ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Strand Displacement ◽  
Single Stranded Dna ◽  
Displacement Mode

scholarly journals Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication

2007 ◽  
Vol 81 (11) ◽  
pp. 5777-5787 ◽  
Author(s):  
Kevin Nash ◽  
Weijun Chen ◽  
William F. McDonald ◽  
Xiaohuai Zhou ◽  
Nicholas Muzyczka
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Binding Protein ◽  
Dna Binding Protein ◽  
Dna Helicase ◽  
Strand Displacement ◽  
Adeno Associated Virus ◽  
Replication System ◽  
Factor C

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.

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

1998 ◽  
Vol 72 (1) ◽  
pp. 420-427 ◽  
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.

scholarly journals Cellular Proteins Required for Adeno-Associated Virus DNA Replication in the Absence of Adenovirus Coinfection

1998 ◽  
Vol 72 (4) ◽  
pp. 2777-2787 ◽  
Author(s):  
Tie-Hua Ni ◽  
William F. McDonald ◽  
Irene Zolotukhin ◽  
Thomas Melendy ◽  
Shou Waga ◽  
...  
Keyword(s):  
Dna Replication ◽  
Hela Cell ◽  
Dna Polymerase ◽  
Single Stranded Dna ◽  
Adeno Associated Virus ◽  
Cell Extracts ◽  
Terminal Repeats ◽  
Factor C

ABSTRACT We previously reported the development of an in vitro adeno-associated virus (AAV) DNA replication system. The system required one of the p5 Rep proteins encoded by AAV (either Rep78 or Rep68) and a crude adenovirus (Ad)-infected HeLa cell cytoplasmic extract to catalyze origin of replication-dependent AAV DNA replication. However, in addition to fully permissive DNA replication, which occurs in the presence of Ad, AAV is also capable of partially permissive DNA replication in the absence of the helper virus in cells that have been treated with genotoxic agents. Limited DNA replication also occurs in the absence of Ad during the process of establishing a latent infection. In an attempt to isolate uninfected extracts that would support AAV DNA replication, we discovered that HeLa cell extracts grown to high density can occasionally display as much in vitro replication activity as Ad-infected extracts. This finding confirmed previous genetic analyses which suggested that no Ad-encoded proteins were absolutely essential for AAV DNA replication and that the uninfected extracts should be useful for studying the differences between helper-dependent and helper-independent AAV DNA replication. Using specific chemical inhibitors and monoclonal antibodies, as well as the fractionation of uninfected HeLa extracts, we identified several of the cellular enzymes involved in AAV DNA replication. They were the single-stranded DNA binding protein, replication protein A (RFA), the 3′ primer binding complex, replication factor C (RFC), and proliferating cell nuclear antigen (PCNA). Consistent with the current model for AAV DNA replication, which requires only leading-strand DNA synthesis, we found no requirement for DNA polymerase α-primase. AAV DNA replication could be reconstituted with purified Rep78, RPA, RFC, and PCNA and a phosphocellulose chromatography fraction (IIA) that contained DNA polymerase activity. As both RFC and PCNA are known to be accessory proteins for polymerase δ and ɛ, we attempted to reconstitute AAV DNA replication by substituting either purified polymerase δ or polymerase ɛ for fraction IIA. These attempts were unsuccessful and suggested that some novel cellular protein or modification was required for AAV DNA replication that had not been previously identified. Finally, we also further characterized the in vitro DNA replication assay and demonstrated by two-dimensional (2D) gel electrophoresis that all of the intermediates commonly seen in vivo are generated in the in vitro system. The only difference was an accumulation of single-stranded DNA in vivo that was not seen in vitro. The 2D data also suggested that although both Rep78 and Rep68 can generate dimeric intermediates in vitro, Rep68 is more efficient in processing dimers to monomer duplex DNA. Regardless of the Rep that was used in vitro, we found evidence of an interaction between the elongation complex and the terminal repeats. Nicking at the terminal repeats of a replicating molecule appeared to be inhibited until after elongation was complete.

An RNA polymerase II transcription factor inactivated in poliovirus-infected cells copurifies with transcription factor TFIID

1988 ◽  
Vol 8 (8) ◽  
pp. 3175-3182
Author(s):  
S Kliewer ◽  
A Dasgupta
Keyword(s):  
Transcription Factor ◽  
Infected Cell ◽  
Rna Polymerase ◽  
Host Cell ◽  
Rna Polymerase Ii ◽  
Heat Inactivation ◽  
Early Event ◽  
Cell Extracts ◽  
Infected Cells ◽  
Rna Polymerase Ii Transcription

Inhibition of host cell RNA polymerase II-mediated transcription by poliovirus infection was studied in vitro. Whole-cell extracts prepared from poliovirus-infected HeLa cells at 3 h postinfection were shown to be deficient in a factor required for specific transcription from the adenovirus major late promoter. Three lines of evidence suggest that transcription factor TFIID is deficient in poliovirus-infected cells. First, the activity required to specifically restore transcription in poliovirus-infected cell extracts was shown to copurify with TFIID through three chromatographic steps. Second, transcription reactions reconstituted with phosphocellulose-derived chromatographic fractions revealed a fourfold decrease in the specific activity of the TFIID-containing fraction prepared from poliovirus-infected cells compared with that of the same fraction prepared from mock-infected cells. Finally, TFIID and the activity required to specifically restore transcription in virus-infected cell extracts were shown to have the same kinetics of heat inactivation. Together, these results suggest that inactivation of TFIID is an early event in the inhibition of host cell RNA polymerase II transcription by poliovirus.

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