trans-dominant defective mutants of simian virus 40 T antigen

Farber, Joshua M.; Peden, Keith; Nathans, Daniel

doi:10.1128/jvi.61.2.436-445.1987

Cited by 25 publications

(18 citation statements)

References 95 publications

(99 reference statements)

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“…To construct plasmid pKHF, we subcloned the 13-kb HpaI F fragment of HSV-1 strain KOS (coordinates 0.535 to 0.620) from pSG124 into the vector pUC19 by digestion of pSG124 with HpaI and blunt-end ligation with T4 DNA ligase into the SmaI site of pUC19. To construct plasmid pKpX2, we subcloned the 2.3-kb XhoI fragment of pKHF (coordinates 0.564 to 0.580) into plasmid vector pKP54 (14) by ligating the fragment directly into the unique SalI site of the vector. pKX2-,G3 was constructed by partial digestion of pKpX2 with BamHI and ligation of a BamHI fragment containing the lacZ gene from plasmid pDP503 (37) (generously provided by D. Panicali) into the BamHI site indicated in Fig.…”

Section: Methodsmentioning

confidence: 99%

Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant

Goldstein

Weller

1988

J Virol

359

117

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Herpes simplex virus (HSV) encodes a ribonucleotide reductase consisting of two subunits (140 and 38 kilodaltons) whose genes map to coordinates 0.56 to 0.60 on the viral genome. Host cell lines containing the HpaI F fragment which includes the reductase subunit genes of HSV type 1 strain KOS (coordinates 0.535 to 0.620) were generated. Transfection of these cells with a plasmid containing the immediate-early ICPO gene resulted in the expression of ICP6; interestingly, ICP4 plasmids failed to induce expression, indicating an unusual pattern of ICP6 regulation. One such cell line (D14) was used to isolate a mutant with the structural gene of lacZ inserted into the ICP6 gene such that the lacZ gene is read in frame with the N-terminal region of ICP6. This mutant generated a protein containing 434 amino acids (38%) of the N terminus of ICP6 fused to I8-galactosidase under control of the endogenous ICP6 promoter. Screening for virus recombinants was greatly facilitated by staining virus plaques with 5-bromo-4-chloro-3-indoyl-j(-D-galactoside (X-gal). Enzyme assays of infected BHK cells indicated that the mutant is incapable of inducing viral ribonucleotide reductase activity. Surprisingly, although plaque size was greatly reduced, mutant virus yield was reduced only fourto fivefold compared with that of the wild type grown in exponentially growing Vero cells. Mutant virus plaque size, yields, and ability to synthesize viral DNA were more severely compromised in serum-starved cells as compared with the wild type grown under the same condition. Although our evidence suggests that the HSV type 1 ribonucleotide reductase is not required for virus growth and DNA replication in dividing cells, it may be required for growth in nondividing cells.

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Section: Methodsmentioning

confidence: 99%

Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant

Goldstein

Weller

1988

J Virol

359

117

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“…We have been characterizing a collection of T-antigen mutants that carry amino acid substitutions, deletions, or insertions within the ATPase domain (19,44,54). This study reports the initial characterization of three mutant T antigens that FIG.…”

Section: Discussionmentioning

confidence: 99%

“…Mutant 5030 carries a single amino acid substitution (P417S), while 5031 has three amino acid replacements (D402N, V404M, and V413M). The 5061 mutation results in the replacement of G(431) with three amino acids (A L E) and has been described previously (19).…”

Section: Baculovirus Vectors Expressing Mutant T Antigensmentioning

confidence: 97%

“…In fact, both 5030 and 5031 are readily complemented in trans by wild-type T antigen (44a). On the other hand, the 5061 T antigen is strongly trans dominant negative in vivo, inhibiting the replication activity of wild-type T antigen by about 100-fold (19). Therefore, we examined the influence of each mutant on the ability of wildtype T antigen to stimulate SV40 replication in vitro.…”

Section: Purification Of 5030 5031 and 5061 Mutant T Antigensmentioning

confidence: 99%

“…In the majority of cases, the replication defects of these mutants can be complemented by supplying a wild-type T antigen in trans. However, some of the mutant T antigens cannot be complemented and exhibit a trans-dominant-negative effect on the replication activity of wild-type T antigen (3,19). The biochemical basis for this trans dominance is not known.…”

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confidence: 99%

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trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP

Castellino

Cantalupo

Marks

et al. 1997

J Virol

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Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.

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SV40 DNA Replication

Melendy¹,

Stillman

1992

Nucleic Acids and Molecular Biology

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trans-dominant defective mutants of simian virus 40 T antigen

Cited by 25 publications

References 95 publications

Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant

Herpes simplex virus type 1-induced ribonucleotide reductase activity is dispensable for virus growth and DNA synthesis: isolation and characterization of an ICP6 lacZ insertion mutant

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP

SV40 DNA Replication

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