Helicase, DNA-binding, and immunological properties of replication-defective simian virus 40 mutant T antigens

Abstract

Simian virus 40 T antigen (TAg) exhibits nonspecific and origin-specific DNA binding (ori binding) and ATPase and helicase activities, all of which are related to its roles in viral DNA replication. We have characterized some of the properties of four replication-defective but transformation-competent mutant TAgs, C6-2, T22, Cil, and C8A. C6-2 and T22 TAgs were each previously determined to lack ori-binding properties, while Cll TAg was reported to lack ATPase activity. The C8A TAg did not exhibit defects in either ori-binding or ATPase functions. We have analyzed additional aspects of these mutant TAgs pertaining to their helicase, DNA-binding, and immunological properties. With the exception of the C11 TAg, all the other TAgs exhibited helicase activity. The lack of helicase activity by Cll TAg was consistent with its previously shown inability to hydrolyze ATP or to replicate viral DNA. These results therefore show that on-binding and helicase activities are separate functions of TAg. Wild-type and mutant TAgs bound with similar efficiency to either native or denatured calf thymus DNA-cellulose, indicating no marked differences in their nonspecific DNA-binding properties. We also tested the binding of wild-type and mutant TAgs to a monoclonal antibody, PAb 100, that was previously shown to recognize an extremely small class of TAg that may represent a unique conformational form of the protein. Interestingly, while less than 10% of the wild-type, C6-2, Cll, and T22 mutant TAgs were recognized by PAb 100, more than 60% of the C8A mutant TAg was bound by this antibody. Therefore, although no defect in biochemical function was observed with the C8A TAg, its deficiency in viral DNA replication may be related to an unusual conformation, as detected by its dramatically increased recognition by PAb 100. These results show that the helicase activity of TAg is not required for its transformation function.

A Single Rep Protein Initiates Replication of Multiple Genome Components of Faba Bean Necrotic Yellows Virus, a Single-Stranded DNA Virus of Plants

“…5), similar to the geminivirus Rep proteins (22,35,40,54,65). Replication initiator proteins of some animal viruses, such as Rep proteins of parvoviruses and large T-antigen of simian virus 40 (SV40) and other polyomaviruses, are also helicases that require ATP hydrolysis for the unwinding of dsDNA (45,73), and, consequently, mutants with mutations in their NTPbinding site are defective in viral replication (6,16,21,38,59). Whether in gemini-and nanovirus Rep proteins as well the ATPase is part of a helicase function remains unknown.…”

Purification and characterization of a DNA-binding heterodimer of 52 and 100 kDa from HeLa cells

“…Sequence-specific DNA-binding proteins (Wingender, 1988;Mitchell and Tjian, 1989;Johnson and McKnight, 1989) were shown to play specific roles in cellular gene expression and regulation (Latchman, 1990;Evan, 1990). Sequence-non-specific DNA-binding proteins, such as TFIIC (Slattery et al, 1983;Kurl and Jacob, 1985), topoisomerases (Osheroff, 1989), c-Myc (Eisenman et al, 1985;Watt et al, 1985), Rb protein (Wang et al, 1990), simian-virus-40 T-antigens (Prives et al, 1980;Auborn et al, 1989), Ku antigen (Mimori and Hardin, 1986;Yaneva and Busch, 1986), also play important roles in many cellular processes associated with DNA.…”

Constitutive expression of simian virus 40 large T antigen in monkey cells activates their capacity to support polyomavirus replication

“…3A, compare lanes 5, 6, 9, and 10 with lanes 13 and 14). Mutant C6 large T antigen interacts weakly with the SV40 origin sequences; mutant Cll large T antigen lacks ATPase and helicase activities (1,30,41). DNAs containing the polyomavirus origin replicated very poorly in these cells (less than 1% the level observed in COS-1 cells ; Fig.…”