Properties of a simian virus 40 mutant T antigen substituted in the hydrophobic region: defective ATPase and oligomerization activities and altered phosphorylation accompany an inability to complex with cellular p53

We generated a series of COOH-terminal truncated simian virus 40 large tumor (T) antigens by using oligonucleotide-directed site-specific mutagenesis. The mutant proteins [T(1-650) to T(1-516)] were expressed in insect cells infected with recombinant baculoviruses. T(1-623) and shorter proteins [T(1-621) to T(1-516)1 appeared to be structurally changed in a region between residues 269 and 522, as determined by increased sensitivities to trypsin digestion and by altered reactivities to several monoclonal antibodies. These same mutant proteins bound significantly less nonorigin plasmid DNA (15%) and calf thymus DNA (25%) than longer proteins [T(1-625) to T(1-708)]. However, all mutant T antigens exhibited a nearly wild-type level of viral origin-specific DNA binding and binding to a helicase substrate DNA. This indicated that binding to origin and helicase substrate DNAs is separable from about 85% of nonspecific binding to double-stranded DNA. As an independent confirmation that a region distinct from the origin-binding domain (amino acids 147 to 247) is * Corresponding author. origin-binding domain (residues 147 to about 247) (2, 49-52, 61) has been the best characterized. The maximal p53binding and helicase substrate-binding regions map to residues 272 to 517 (46) and 131 to 517 (75), respectively, and the ATP-binding region maps to amino acids (aa) 418 to 528 (4, 5). The first N-terminal 121 amino acids have been implicated in transactivation (57, 79), and a small region (residues 105 to 114) is responsible for binding to the Rb protein (13).One activity of T antigen that is less well characterized is its ability to bind nonspecifically to double-stranded and single-stranded DNAs (7,38,56). The exact function of this activity is not yet clear, but it has been implicated in origin-specific DNA binding (51). It may also be involved in subsequent steps during viral DNA replication, in the induction of cellular DNA synthesis, or in the activation of rRNA genes. We have mapped nonspecific DNA binding to a region between aa residues 131 and 517 (75). Recently, we showed that at least 5 residues (mapping between positions 149 and 203) within the DNA binding domain were important in binding nonspecifically to doubleand single-stranded DNAs (51,52). All mutations in the domain which significantly reduced nonspecific binding also prevented originspecific binding (although the converse was not true). This suggested that T antigen first binds to virus DNA nonspecifically and then locates the origin sequences.Several years ago, Prives et al. (44) reported that a 56-kDa adeno-SV40 hybrid protein (containing T-antigen sequences from about residues 252 to 708) bound calf thymus DNA but could not bind specifically to the SV40 origin. A smaller, 45-kDa protein, with T-antigen sequences from about residues 336 to 708 was unable to bind to any DNA. These 5443 Vol. 66,No. 9

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Nonspecific DNA binding activity of simian virus 40 large T antigen: evidence for the cooperation of two regions for full activity

Lin

Upson

Simmons

1992

“…Tevethia et al (62) have characterized a deletion mutant missing amino acids 587 to 589 which does not bind p53. Tack et al (61) have shown that, in fact, a base substitution mutation at amino acid 584 affects a number of functions of SV40 large T antigen, including the ability to hydrolyze ATP and to oligomerize. This suggests that the region of amino acids 570 to 588 may play a general structural role and is not necessarily contained within the domain of SV40 large T antigen which contacts p53.…”

Section: Discussionmentioning

confidence: 99%

Binding of p53 and p105-RB is not sufficient for oncogenic transformation by a hybrid polyomavirus-simian virus 40 large T antigen

Manfredi

Prives

1990

To identify regions on the large T antigens of simian virus 40 (SV40) and polyomavirus which are involved in oncogenic transformation, we constructed plasmids encoding hybrid polyomavirus-SV40 large T antigens. The hybrid T antigens were expressed in G418 sulfate-resistant pools of rat F2408 cells, and extracts of such pools were immunoprecipitated with an antibody against p53. Two hybrid T antigens containing SV40 amino acids 337 to 708 bound to p53, whereas another hybrid T antigen containing SV40 amino acids 412 to 708 did not. This suggests that a binding domain on SV40 large T antigen for p53 is contained within amino acids 337 to 708, with amino acids 337 to 411 playing an important role. One of the two hybrids that bound to p53 was chosen for further study. This T antigen contained SV40 large T antigen amino acids 336 to 708 joined to polyomavirus large T antigen amino acids 1 to 521 (PyT1 521-SVT336-708). Immunoprecipitation with antibodies directed against the product of the retinoblastoma susceptibility gene, p105-RB, showed that this hybrid bound p105-RB as well as p53. Pools expressing the hybrid PyTl-521-SVT336-708 did not grow in soft agar, nor did they form foci on confluent monolayers of nontransformed F2408 cells. The hybrid T antigen was expressed at levels comparable to those seen in retrovirus-infected F2408 cells expressing only SV40 large T antigen, which do show a transformed phenotype. Thus, this level of expression was sufficient for transformation by SV40 large T antigen but not for the hybrid large T antigen. These data, combined with genetic studies from other laboratories, suggest that complex formation with p53 and p105-RB is necessary but not sufficient for the oncogenic potential of papovavirus large T antigens. * Corresponding author. MATERIALS AND METHODS Plasmids, cells, and antibodies. pSVBam is plasmid pBR322 with the entire SV40 genome inserted into its BamHI site. pSVLT is the plasmid pMK 16 with the entire 5250

“…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%

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

Castellino

Cantalupo

Marks

et al. 1997

Self Cite

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.