Properties of the simian virus 40 (SV40) large T antigens encoded by SV40 mutants with deletions in gene A

Simian virus 40 large T antigen (T) can transform cultured cells, but the mechanisms by which it functions are not entirely understood. Several lines of evidence have suggested that the amino-terminal 130 residues of T may be sufficient to confer the transforming capability. Oligonucleotide-directed mutagenesis was used to generate a series of deletion and substitution mutants within the amino-terminal 82 residues of T, the segment which is shared with simian virus 40 small t antigen (t). Results of stability and transformation assays of these mutants strongly suggest that the 1-to-82 region of T contains sequences which govern T transforming activity and affect in vivo stability. Instability and a defect in transforming activity could be separated from one another genetically. Thus, the 1-to-82 region appears to contain a specific region that contributes to the transforming function of the protein. This segment operates by means other than the simple binding of pRb and/or p107.

mentioning

confidence: 99%

The T/t common region of simian virus 40 large T antigen contains a distinct transformation-governing sequence

Marsilio

Cheng

Schaffhausen

et al. 1991

“…The host range mutants, which map to the C terminus of T antigen (16,17,50,51,64), produce stable, truncated T antigens and replicate to near wild-type levels (12,50 gradient fractionation. In these SDS gels, every other gradient fraction was precipitated with anti-SV40 antibody recognizing VPI and T antigen.…”

Section: Discussionmentioning

confidence: 99%

“…All T antigen functions required for viral DNA replication, transcriptional control, and neoplastic transformation are localized to the N terminus and middle portion of the protein (12,17,60,67). The role of the C terminus of T antigen (amino acids 660 to 708) in lytic growth of SV40 has been less well understood.…”

mentioning

confidence: 99%

“…Compared with wild-type virus, mutant stocks give 10-fold-lower yields on BSC cells and 10,000-fold-lower yields on CV1 cells (50). Mutant viruses replicate DNA at nearly wild-type levels under nonpermissive conditions, suggesting a postreplicative block to lytic growth similar to the adenovirus helper function block (50,64 ( 1992) Virol 189 762 4d12465 -truncated at 626 -E for 627 Cole et al ( 1986) J. Virol. 57 539 C8B -aa 1-659. terminating in a nonsense mutation Manos and Gluzrran ( 1985) J Virol 53 120 Both of these lack the host range domain and would be expected to lack both host range and adenovirus helper activity.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Simian virus 40 large T antigen host range domain functions in virion assembly

Spence

Pipas

1994

The simian virus 40 (SV40) T antigen host range mutants d11066 and d11140 display a postreplicative block to plaque formation which suggests a novel role for T antigen late in the viral life cycle. The host range mutants d11066 and d11140 are able to grow in and plaque on BSC but not on CV1 monkey kidney cells, a normally permissive host. Previous work showed that in CV1 cells infected with d11066 and d11140, levels of viral DNA replication and of late capsid protein accumulation were only slightly reduced and the failure to accumulate agnoprotein was not likely to be the major factor responsible for the mutants' growth defect. Here we show that the host range mutants are defective in the assembly of viral particles. SV40 assembly proceeds as the progressive conversion of 75S viral chromatin complexes to 200S-240S assembled virions. When virus-infected cell extracts are separated on 5 to 40%0 sucrose gradients, wild-type extracts show the greatest accumulation of viral late protein in the 200S-240S fractions corresponding to the assembled virus peak and lesser amounts in the 75S-150S fractions corresponding to immature assembly intermediates. The host range mutants d11066 and d11140 grown in nonpermissive CV1 cells, however, failed to assemble any appreciable amounts of mature 200S-240S virions and accumulate 75S intermediates, whereas in permissive BSC cells, levels of assembly were more slightly reduced than those of the wild type. Analysis of the protein composition of gradient fractions suggests that SV40 assembly proceeds by a mechanism similar to that proposed for polyomavirus and suggests

“…T antigen is a 708-amino-acid multifunctional protein that possesses several biochemical activities, some of which map to discrete domains that can act independently of the rest of the polypeptide (18,45). For example, the binding of T antigen to specific sequences within the viral origin of DNA replication is mediated through its DNA binding domain (amino acids 135 to 249), while residues 371 to 625 are sufficient for ATP binding and hydrolysis (1,9,10,18,52). In some cases, proper T-antigen function requires the coordinated action of multiple domains.…”

mentioning

confidence: 99%

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

Castellino

Cantalupo

Marks

et al. 1997

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.