Bs. Collins et Jm. Pipas, T ANTIGENS ENCODED BY REPLICATION-DEFECTIVE SIMIAN-VIRUS-40 MUTANT-DL1135 AND MUTANT-5080, The Journal of biological chemistry, 270(25), 1995, pp. 15377-15384
We present a preliminary biochemical characterization of two simian vi
rus 40 mutants that affect different T antigen replication functions,
SV40 T antigen mutants dl1135 (Delta 17-27 amino acids) and 5080 (P-L)
have been studied extensively with regard to their ability to transfo
rm cells in culture and induce tumors in transgenic mice. Both mutants
are defective for viral DNA replication in vivo. In order to assess i
n more detail the molecular basis for the in vivo replication defects
of 5080 and dl1135, we expressed the mutant proteins using the baculov
irus system and purified them by immunoaffinity chromatography. With e
ach of the purified proteins, we examined some of the biochemical acti
vities of T antigen required for replication, viz, ATPase, binding to
the origin of replication (ori) and assembly on ori, DNA helicase and
unwinding, and replication in in vitro assays, Consistent with previou
s studies, we found that the 5080 protein is defective for multiple bi
ochemical activities including ATPase, helicase, ori-specific unwindin
g, and ATP-induced hexamerization. However, this mutant retains some s
equence-specific DNA binding activity. In contrast, the dl1135 protein
exhibited significant levels of activity in all assays, including the
ability to drive SV40 DNA replication in vitro. Thus, dl1135 is one o
f several mutants with an altered aminoterminal domain which can repli
cate DNA in vitro, but not in vivo. Thus, while the 5080 mutation affe
cts a T antigen enzymatic function directly required far viral DNA syn
thesis, dl1135 may alter an activity required to prepare the cell for
viral replication.