Analysis of G418-selected Rat2 cells containing prototype, variant, mutant, and chimeric JC virus and SV40 genomes

Abstract

The human polyomavirus JC virus (JCV) is highly tumorigenic in rodents, but transforms cells in culture inefficiently. To explore the basis for JCV's restricted transforming behavior, nonpermissive Rat2 cells were contransfected with pSV2-neo (encodes G418 resistance) and viral DNAs including prototype, variant, and mutant JCV genomes and two JCV-SV40 chimeras. By selecting cells displaying G418 resistance, lines were established that contain viral DNA and exhibit a wide range of transformed phenotypes. The G418-resistant lines were tested for their ability to grow under anchorage-independent conditions, to overgrow a monolayer of untransformed cells, and to form dense colonies on plastic. Expression of the viral T and t proteins and interaction of T protein with the cellular anti-oncoprotein p53 were measured. Also determined was the number of intact viral early coding regions integrated within the cellular DNA. The results of these studies suggested that most of the G418-resistant lines failed to express JCV T protein above a minimum threshold level required for their conversion to a fully transformed phenotype. In anchorage-independent growth assays, higher levels of a 17-kDa T-related peptide in JCV transformants appeared to compensate for decreased T antigen levels. Comparison of the T to p53 ratios in the cell lysates suggested that the quaternary structure of the JCV protein differed from that of its SV40 counterpart in the T-p53 complex. The presence of multiple vs single integrated copies of the viral genome in the cells did not correlate with elevated T antigen expression or an enhanced transformation status.