Stability and function of JC virus large T antigen and T' proteins are altered by mutation of their phosphorylated threonine 125 residues.
JC virus (JCV), a human polyomavirus, exhibits oncogenic activity in rodents and primates. The large tumor antigens (TAgs) of the polyomaviruses play key roles in viral replication and oncogenic transformation. Analyses of JCV TAg phosphorylation mutants indicated that the amino-terminal phosphorylation site at threonine 125 (T125) is critical to TAg replication function. This site is also conserved in the TAg splice variants T'(135), T'(136), and T'(165). By constructing stable cell lines expressing JCV T125A and T125D mutants, we show that mutation of this phosphorylation site to alanine generates an unstable TAg; however, the stability of the three T' proteins is unaffected. JCV T125A mutant proteins bind the retinoblastoma protein (RB) family members p107 and p130 with slightly reduced efficiencies and fail to induce the release of transcriptionally active E2F from RB-E2F complexes. On the other hand, cell lines expressing JCV T125D mutant proteins produce stable TAg and T' proteins which bind p107 and p130 more efficiently than do the wild-type proteins. In addition, T125D mutant proteins efficiently induce the release of E2F from RB-E2F complexes. T125D mutant cell lines, unlike the T125A mutant lines, continue to grow under conditions of low serum concentration and anchorage independence. Finally, both T125A and T125D mutant viruses are replication defective. Phosphorylation of the T125 site is likely mediated by a cyclin-cyclin-dependent kinase, suggesting that JCV TAg and T' protein functions that mediate viral replication and oncogenic transformation events are regulated in a cell cycle-dependent manner.
Tyagarajan
Shiva K
SK
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, 16802, USA.
Frisque
Richard J
RJ
D019943:Amino Acid Substitution; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D050684:E2F Transcription Factors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D017930:Genes, Reporter; D007577:JC Virus / Q000235:genetics / Q000502:physiology*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D050720:Retinoblastoma-Like Protein p107 / Q000378:metabolism; D050717:Retinoblastoma-Like Protein p130 / Q000378:metabolism; D013462:Sulfur Radioisotopes / Q000378:metabolism; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D050684:E2F Transcription Factors; D020033:Protein Isoforms; D050720:Retinoblastoma-Like Protein p107; D050717:Retinoblastoma-Like Protein p130; D013462:Sulfur Radioisotopes; D008156:Luciferases