Abstract
The JC virus (JCV) regulatory proteins, large T antigen, small t antigen, T′135, T′136, and T′165, are encoded by five transcripts alternatively spliced from the viral early precursor mRNA. T antigen and the T′ proteins share N-terminal amino acid sequences that include the L × C×E and J domains, motifs in SV40 T antigen known to mediate binding to the retinoblastoma (Rb) proteins and Hsc70, respectively. In this study, G418-resistant cell lines were created that express wild-type or mutant JCV T antigen and T′ proteins individually or in combination. These cell lines were used to evaluate the ability of each viral protein to bind p107 and p130 in vivo, and to influence cellular growth characteristics. Differences were observed in the abilities of individual T′ proteins to bind p107 and p130 and to alter their phosphorylation status. The T′ proteins were also found to localize to the cell's nucleus and to be phosphorylated in a cell cycle-dependent manner. JCV T antigen and T′ proteins expressed from a cytomegalovirus promoter failed to induce dense focus formation in Rat2 cells, but they did cooperate with a mutant Ras protein to overcome cellular senescence and immortalize rat embryo fibroblasts. These data indicate that, despite their sequence similarities, JCV early proteins exhibit unique activities that, in combination, effect the inactivation of cell cycle regulators, a requirement for polyomavirus-induced transformation.
The authors thank Dr. Ana Muñoz-Mármol for the gift of the small t-EGFP fusion construct (pEGFP-C2.ST3), and Dr. Satvir Tevethia and members of our laboratories for experimental assistance and critical review of the manuscript. This work was supported by U.S. Public Health Service grants from the National Institute of Neurological Disorders and Stroke (S11 NS41833) and the National Cancer Institute (CA-115771 to RJF; CA-24694 to MJT), and by a grant from the Pennsylvania Department of Health using Tobacco Settlement Funds. The Department specifically disclaims responsibility for any analyses, interpretations or conclusions. Received 21 August 2006; accepted 7 September 2006.