The high affinity binding site on polyoma virus DNA for the viral large-T protein

Gaudray, Patrick; Tyndall, Chiara; Kamen, Robert; Cuzin, François

doi:10.1093/nar/9.21.5697

Cited by 92 publications

(89 citation statements)

References 25 publications

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“…1B) The present observation that wild-type Py A2 DNA can replicate in EC cells is at variance with two reports (7,27) showing that wild-type Py A3 and large plaque Toronto strains do not replicate in F9 cells. Our data (not shown) with Py A3 strains confirm this result, but it should be pointed out that the A3 strain is lacking, on the early side of the HpaII 3/ 5 junction, a stretch of 11 nucleotides which includes one of the three T-antigen-binding sites which have been identified on the A2 strain (8). This deletion leads to a 10-fold decrease in T antigen affinity for strain A3 DNA as compared with strain A2 DNA.…”

supporting

confidence: 81%

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Dandolo¹,

Aghion²,

Blangy³

1984

Mol. Cell. Biol.

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Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells does replicate in EC cells, although less efficiently than the DNA of PyEC mutants. This replication proceeds via a Tantigen-independent mechanism as confirmed by the behavior of a Py tsa mutant, which produces a thermosensitive large T antigen, of a deletion mutant which lacks part of the large T protein-coding sequence, and of a double mutant carrying both the tsa and the PyEC F9 mutations. We also show that the ability to replicate Py DNA in the absence of T antigen is lost when EC cells are allowed to differentiate. Finally, we discuss the involvement of both viral DNA sequences and cellular proteins in this phenomenon. MATERIALS AND METHODSCell cultures and virus strains. NIH 3T6 mouse cells and the EC cell lines PCC4-aza-1, F9, and LT1 (12,16,21) were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (GIBCO Diagnostics, Madison, Wis.). F9 cells were grown on gelatin-coated (0.1%) dishes. All virus strains were propagated by infecting secondary mouse embryo cells at a multiplicity of 0.01 PFU per cell. The wild-type Py used in these studies was the large plaque strain A2 (10). The nucleotide numbers refer to the Py DNA sequence as reported by Soeda et al. (19). The PyEC mutant PyF9-1 has been previously described (13

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supporting

confidence: 81%

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Dandolo¹,

Aghion²,

Blangy³

1984

Mol. Cell. Biol.

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“…Both the origins of replication and the T-antigen polypeptides of SV40 and Py virus have similar sequences, suggesting that these viruses have similar modes of replication. However, replication of SV40 occurs in primate and human cells, whereas Py virus replicates in mouse cells (15,17,18). Since we had both the SV40 and Py DNA replication systems operational, we tested the specificity of DNA, T antigen, and extract in each system (Table 2).…”

Section: Methodsmentioning

confidence: 99%

“…These findings and previous results (8) Although Py and SV40 DNA replication in vitro follow a similar overall pattern in their requirements for T antigen and the viral replication origin, there are both similarities and differences in the properties of these viral components. SV40 and Py T antigens are fairly homologous proteins (15) that display high-affinity, sequence-specific DNA binding, particularly to sequences containing repeats of the consensus pentanucleotide GRGGC (14,17), and exhibit ATPase activity (18). However, SV40 T antigen, but not Py T antigen, binds to the cellular p53 protein and is capable of inducing the transformed phenotype in both permissive and nonpermissive cells (ref.…”

Section: Methodsmentioning

confidence: 99%

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

Murakami¹,

Eki²,

Yamada³

et al. 1986

Proc. Natl. Acad. Sci. U.S.A.

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In vitro replication of DNA containing the polyoma (Py) virus orign of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase a in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. Cell-free extracts of monkey and human cells that replicate SV40 DNA have been described (4-7). The in vitro replication of SV40 DNA requires, in addition to suitably prepared cell extracts, circular DNA containing the viral replication origin (ori) and purified SV40 large tumor (T) antigen. Since SV40 T antigen and DNA containing the SV40 replication origin are the only viral components required, this system should be useful for identifying and characterizing the eukaryotic proteins involved in DNA replication. In previous reports (7-9), it was shown that DNA polymerase a (pol a) and DNA primase are essential for the replication of SV40 DNA in vitro and that the source of these enzymes is important in determining whether SV40 DNA replication will occur. In contrast to extracts of human cells, mouse cell extracts supplemented with SV40 T antigen did not support replication of DNA containing the SV40 origin unless supplemented with the pol a-primase complex from HeLa cells.In this report we describe the establishment of a system that replicates Py DNA in vitro; it requires mouse cell extract, the Py replication origin, and Py T antigen. In addition, we have confirmed and further defined the important role played by pol a-primase in determining the species specificity of papovavirus DNA replication. MATERIALS AND METHODSPurification of Py T Antigen. Py T antigen was purified from CV-1 cells infected with the helper-dependent recombinant adenovirus vector Ad-SVR587 (10) as follows. Cells were infected with wild-type adenovirus [2-5 plaque-forming units (pfu) per cell] and Ad-SVR587 recombinant virus (a gift from S. Mansour, T. Grodzicker, and R. Tjian) (5-10 pfu per cell). After incubation for 36 hr at 370C, cells were scraped from thirty 150-mm plates into -3 ml of cold Dulbecco's phosphate-buffered saline (PBS: Ca2+-and Mg2+-free), washed twice with cold PBS, suspended in 10 volumes of pH 9.0 buffer [20 mM Tris HCl, pH 9.0/0.2 M NaCl/1 mM EDTA/1 mM dithiothreitol/10% (vol/vol) glycerol/1% (vol/vol) Nonidet P-40 (NP-40)/1 mM phenylmethylsulfonyl fluoride (PMSF)], and lysed on ice for 10: min. The lysate was centrifuged for 10 min at 2000 rpm in a Sorvall H-6000A rotor, and the supernatant was then centrifuged for 10 min at 20,000 rpm in a Sorvall SS34 rotor. The supernatant was mixed with 0.5 volume of pH 6.8 buffer (0.1 M Tris'HCl, pH 6.8/1 mM EDTA/1 mM dithiothre...

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“…Large T antigen acts at the initiation step to trigger DNA replication by an unknown mechanism (8). This likely requires the physical interaction of large T antigen with specific sequences present in the core element of the functional replication origin (11,33 Fig. 7.…”

Section: Resultsmentioning

confidence: 99%

“…These include the capacity to hydrolize ATP (10) and the capacity to bind to specific regions on PyV DNA (11,33). Large T antigen protects three regions near the origin for DNA replication * Corresponding author.…”

mentioning

confidence: 99%

Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.

Massie¹,

Gluzman²,

Hassell³

1986

Mol. Cell. Biol.

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Adenovirus-polyomavirus recombinant viruses were constructed in vitro by inserting a hybrid transcription unit composed of the adenovirus type 2 major late promoter and the early coding region of polyomavirus into the adenovirus type 5 vector Ad5AE1/d1309. The vector lacks the Ela and Elb transcription units and contains a unique restriction endonuclease cleavage site in their place. The polyomavirus genomic insert contained a small deletion which precluded the synthesis of functional small and middle T antigen but allowed for the synthesis of large T antigen. One recombinant virus, Ad5PyR39, which contained the hybrid transcription unit in the opposite transcriptional orientation from the overall direction of late-gene transcription, was studied in detail. Ad5PyR39 replicated efficiently without a helper virus in human 293 cells and expressed hybrid mRNAs of the expected size and composition that were translated to yield large T antigen. The large T antigen synthesized in 293 cells was the same size as that produced in mouse 3T6 cells lytically infected with polyomavirus, and this protein bound efficiently and specifically to the large-T-antigen-binding sites in polyomavirus DNA. Moreover, the large T antigen encoded by the recombinant virus proved capable of catalyzing the replication in mouse 3T6 cells of a plasmid containing the polyomavirus origin for DNA replication. Comparison of the amount of large T antigen produced in 3T6 cells infected with polyomavirus with that in 293 cells infected with Ad5PyR39, under optimal conditions for each system, revealed at least a fivefold greater yield of the protein on a per cell basis in the latter system compared with the former. Ad5PyR39 should prove to be useful to isolate large quantities of functional polyomavirus large T antigen for structural and biochemical studies.

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The high affinity binding site on polyoma virus DNA for the viral large-T protein

Cited by 92 publications

References 25 publications

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

Construction of a helper-free recombinant adenovirus that expresses polyomavirus large T antigen.

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