Detection of Simian Virus 40 T-Antigen-related Antigens by a 125I-Protein A-binding Assay and by Immunofluorescence Microscopy on the Surface of SV40-transformed Monolayer Cells

ABSTRACT Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase α-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase α-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia colias a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.

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Amino Acids 257 to 288 of Mouse p48 Control the Cooperation of Polyomavirus Large T Antigen, Replication Protein A, and DNA Polymerase α-Primase To Synthesize DNA In Vitro

Journal of Virology ◽

10.1128/jvi.75.18.8569-8578.2001 ◽

2001 ◽

Vol 75 (18) ◽

pp. 8569-8578 ◽

Cited By ~ 6

Author(s):

Armin R. Kautz ◽

Klaus Weisshart ◽

Annerose Schneider ◽

Frank Grosse ◽

Heinz-Peter Nasheuer

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Protein Complexes ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Specific Factor ◽

Dna Polymerase Α

ABSTRACT Although p48 is the most conserved subunit of mammalian DNA polymerase α-primase (pol-prim), the polypeptide is the major species-specific factor for mouse polyomavirus (PyV) DNA replication. Human and murine p48 contain two regions (A and B) that show significantly lower homology than the rest of the protein. Chimerical human-murine p48 was prepared and coexpressed with three wild-type subunits of pol-prim, and four subunit protein complexes were purified. All enzyme complexes synthesized DNA on single-stranded (ss) DNA and replicated simian virus 40 DNA. Although the recombinant protein complexes physically interacted with PyV T antigen (Tag), we determined that the murine region A mediates the species specificity of PyV DNA replication in vitro. More precisely, the nonconserved phenylalanine 262 of mouse p48 is crucial for this activity, and pol-prim with mutant p48, h-S262F, supports PyV DNA replication in vitro. DNA synthesis on RPA-bound ssDNA revealed that amino acid (aa) 262, aa 266, and aa 273 to 288 are involved in the functional cooperation of RPA, pol-prim, and PyV Tag.

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Identification of the human papovavirus T antigen and comparison with the simian virus 40 protein A

Virology ◽

10.1016/0042-6822(77)90043-5 ◽

1977 ◽

Vol 82 (1) ◽

pp. 206-213 ◽

Cited By ~ 6

Author(s):

Kathleen Rundell ◽

Peter Tegtmeyer ◽

Peter J. Wright ◽

Giampiero Di Mayorca

Keyword(s):

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Human Papovavirus

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The Replication Protein A Binding Site in Simian Virus 40 (SV40) T Antigen and Its Role in the Initial Steps of SV40 DNA Replication

Journal of Virology ◽

10.1128/jvi.72.12.9771-9781.1998 ◽

1998 ◽

Vol 72 (12) ◽

pp. 9771-9781 ◽

Cited By ~ 42

Author(s):

Klaus Weisshart ◽

Poonam Taneja ◽

Ellen Fanning

Keyword(s):

Dna Replication ◽

Binding Site ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Second Step ◽

Dna Unwinding ◽

Viral Dna ◽

Viral Dna Replication

ABSTRACT Physical interactions of simian virus 40 (SV40) large tumor (T) antigen with cellular DNA polymerase α-primase (Pol/Prim) and replication protein A (RPA) appear to be responsible for multiple functional interactions among these proteins that are required for initiation of viral DNA replication at the origin, as well as during lagging-strand synthesis. In this study, we mapped an RPA binding site in T antigen (residues 164 to 249) that is embedded within the DNA binding domain of T antigen. Two monoclonal antibodies whose epitopes map within this region specifically interfered with RPA binding to T antigen but did not affect T-antigen binding to origin DNA or Pol/Prim, ATPase, or DNA helicase activity and had only a modest effect on origin DNA unwinding, suggesting that they could be used to test the functional importance of this RPA binding site in the initiation of viral DNA replication. To rule out a possible effect of these antibodies on origin DNA unwinding, we used a two-step initiation reaction in which an underwound template was first generated in the absence of primer synthesis. In the second step, primer synthesis was monitored with or without the antibodies. Alternatively, an underwound primed template was formed in the first step, and primer elongation was tested with or without antibodies in the second step. The results show that the antibodies specifically inhibited both primer synthesis and primer elongation, demonstrating that this RPA binding site in T antigen plays an essential role in both events.

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Denaturation of the simian virus 40 origin of replication mediated by human replication protein A.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3876 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3876-3883 ◽

Cited By ~ 16

Author(s):

C Iftode ◽

J A Borowiec

Keyword(s):

Protein A ◽

Simian Virus 40 ◽

Replication Protein A ◽

Simian Virus ◽

T Antigen ◽

Replication Protein ◽

Origin Of Replication ◽

Sv40 T Antigen ◽

Viral Origin ◽

Single Stranded Dna

The initiation of simian virus 40 (SV40) replication requires recognition of the viral origin of replication (ori) by SV40 T antigen, followed by denaturation of ori in a reaction dependent upon human replication protein A (hRPA). To understand how origin denaturation is achieved, we constructed a 48-bp SV40 "pseudo-origin" with a central 8-nucleotide (nt) bubble flanked by viral sequences, mimicking a DNA structure found within the SV40 T antigen-ori complex. hRPA bound the pseudo-origin with similar stoichiometry and an approximately fivefold reduced affinity compared to the binding of a 48-nt single-stranded DNA molecule. The presence of hRPA not only distorted the duplex DNA flanking the bubble but also resulted in denaturation of the pseudo-origin substrate in an ATP-independent reaction. Pseudo-origin denaturation occurred in 7 mM MgCl2, distinguishing this reaction from Mg2+-independent DNA-unwinding activities previously reported for hRPA. Tests of other single-stranded DNA-binding proteins (SSBs) revealed that pseudo-origin binding correlates with the known ability of these SSBs to support the T-antigen-dependent origin unwinding activity. Our results suggest that hRPA binding to the T antigen-ori complex induces the denaturation of ori including T-antigen recognition sequences, thus releasing T antigen from ori to unwind the viral DNA. The denaturation activity of hRPA has the potential to play a significant role in other aspects of DNA metabolism, including DNA repair.

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Use of purified antipeptide sera of selected specificity for the detection of the simian virus 40 large T antigen by western blotting

Bioscience Reports ◽

10.1007/bf01117060 ◽

1985 ◽

Vol 5 (2) ◽

pp. 137-142 ◽

Cited By ~ 4

Author(s):

Jean Bernard Dietrich

Keyword(s):

Western Blotting ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

Kidney Cells ◽

Large Tumor ◽

Infected Monkey ◽

Time Specific ◽

First Time

Western blotting was used as a powerful alternative to immunoprecipitation for the detection of the simian virus 40 (SV40) large tumor (T) antigen. After resolution by electrophoresis on a SDS-polyacrylamide gel of a [15S]methionine labeled crude extract from SV40 infected monkey kidney cells, the separated proteins were transferred electrophoretically on nitrocellulose paper. T antigen was detected on nitrocellulose strips by using for the first time, specific, purified antipeptide monoclonal antibodies directed against the N- and C-terminal portions of the molecule, and125I-labeled Protein A.

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Role of the p68 Subunit of Human DNA Polymerase α-Primase in Simian Virus 40 DNA Replication

Molecular and Cellular Biology ◽

10.1128/mcb.22.16.5669-5678.2002 ◽

2002 ◽

Vol 22 (16) ◽

pp. 5669-5678 ◽

Cited By ~ 20

Author(s):

Robert D. Ott ◽

Christoph Rehfuess ◽

Vladimir N. Podust ◽

Jill E. Clark ◽

Ellen Fanning

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Essential Role ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

T Antigen ◽

S Phase ◽

Dna Polymerase Α

ABSTRACT DNA polymerase α-primase (pol-prim) is a heterotetramer with DNA polymerase and primase activities. The polymerase (p180) and primase (p48 and p58) subunits synthesize primers and extend them, but the function of the remaining subunit (p68) is poorly understood. Genetic studies in yeast suggested an essential role for the p68 ortholog in early S phase prior to the hydroxyurea-sensitive step, possibly a regulatory role in initiation of DNA replication, but found no evidence for an essential function of p68 later in S phase. To investigate whether the human p68 subunit has an essential role in DNA replication, we examined the ability of a purified trimeric human pol-prim lacking p68 to initiate simian virus 40 DNA replication in vitro and to synthesize and elongate primers on single-stranded DNA in the presence of T antigen and replication protein A (RPA). Both activities of trimeric pol-prim were defective, but activity was recovered upon addition of separately purified p68. Phosphorylation of p68 by cyclin A-dependent protein kinase also inhibited both activities of pol-prim. The data strongly suggest that the p68 subunit is required for priming activity of pol-prim in the presence of RPA and T antigen, both during initiation at the origin and during lagging strand replication.

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Timed interactions between viral and cellular replication factors during the initiation of SV40 in vitro DNA replication

Biochemical Journal ◽

10.1042/bj20070794 ◽

2007 ◽

Vol 407 (2) ◽

pp. 313-320 ◽

Cited By ~ 4

Author(s):

Poonam Taneja ◽

Heinz-Peter Nasheuer ◽

Hella Hartmann ◽

Frank Grosse ◽

Ellen Fanning ◽

...

Keyword(s):

Dna Replication ◽

Protein A ◽

Simian Virus 40 ◽

Simian Virus ◽

Specific Protein ◽

T Antigen ◽

Initiation Phase ◽

Native Proteins ◽

Replication Factors

The initiation of SV40 (simian virus 40) DNA replication requires the co-operative interactions between the viral Tag (large T-antigen), RPA (replication protein A) and Pol (DNA polymerase α-primase) on the template DNA. Binding interfaces mapped on these enzymes and expressed as peptides competed with the mutual interactions of the native proteins. Prevention of the genuine interactions was accomplished only prior to the primer synthesis step and blocked the assembly of a productive initiation complex. Once the complex was engaged in the synthesis of an RNA primer and its extension, the interfering effects of the peptides ceased, suggesting a stable association of the replication factors during the initiation phase. Specific antibodies were still able to disrupt preformed interactions and inhibited primer synthesis and extension activities, underlining the crucial role of specific protein–protein contacts during the entire initiation process.

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