Use of monoclonal antibodies as probes of simian virus 40 T antigen ATPase activity.

We have investigated the ATPase activity of simian virus 40 (SV40) large T antigen by using monoclonal antibodies as specific probes of enzymatic activity. Three hybridoma cell lines secreting anti-T antigen antibodies were derived from mice that were immunized with D2 T antigen, an SV40 T antigen-related protein. Monoclonal antibodies secreted by these hybridomas bind to three distinct T-antigen determinants. In order to bind to T antigen, the three antibodies required amino acid residues coded by the region of the A gene between 0.37 and 0.29 map unit. Two of these antibodies (DL3C3 and DL3C4) strongly inhibited T antigen ATPase activity. The third antibody (DL3C5) only weakly inhibited the ATPase activity possibly by decreasing the affinity of T antigen for ATP. These results demonstrate that the ATPase activity is intrinsic to T antigen and suggest that the ATPase function of T antigen maps on the SV40 A gene between 0.37 and 0.29 map unit. A T antigen-specific ATPase assay capable of detecting low levels of T antigen in crude extracts of SV40 infected cells was developed by using 3C5 to immobilize an active form of the enzyme. These results indicate that monoclonal antibodies can be used as probes of enzyme structure and function.