Different genotypes of JC virus (JCV) will be examined to determine if these differences in the viral genome affect viral infectivity and/or DNA replication. Specifically, the different variants of the large T- antigen will be studied. The large T-antigen of different JCV genotypes are highly homologous to each other but contain a few significant differences. One area of interest is the single zinc finger domain. The prototype JCV strain (Mad1) has a glutamine residue near the N-terminal end of the zinc finger domain, while in other genotypes, there is a leucine substitution at the same position. This nonconservation variation may affect the structure of the zinc finger and thus may affect function. This variation will be tested in two ways: (1) The mutant strains of the prototype strain, Mad1, will be constructed using site-directed mutagenesis. The glutamine residue in the large T-antigen of Mad1 has been mutated to leucine. Other mutations, such as alanine or proline may be done. These viral strains will then be used to transfect primary human fetal glial (PHFG) cells. Various timepoints will be taken to taken to analyze JCV infectivity and DNA replication activity to demonstrate if there are any differences in activities between the different genotypes. (2) The large T-antigen from different genotypes of JCV will be cloned into a baculovirus expression vector and used to infect and overexpress in insect cells. Several of these constructs have already been made. The overexpressed protein will be purified using a monoclonal antibody to the large T-antigen by affinity chromatography. The pure large T-antigen proteins can then be used in various biochemical assays such as DNA binding and protein-to-protein binding. If differences in the in vitro or the tissue culture activities of the different variants in the zinc finger domain of the large T-antigen are found, the structure of the zinc finger domain may be determined using NMR techniques. Perhaps differences in activity can be correlated with differences in the structure of the zinc finger.
