Interaction of the single-stranded DNA-binding protein Puralpha with the human polyomavirus JC virus early protein T-antigen.
Large T-antigen, the major regulatory protein encoded by polyomaviruses, including Simian Virus 40 (SV40) and JC virus (JCV), is a multifunctional phosphoprotein that is involved in many viral and cellular events. In addition to its integral role in viral replication and cellular transformation, T-antigen also regulates transcription of both viral and cellular genes. In particular, the viral late promoter has been used as a model for the analysis of T-antigen-mediated transcriptional activation. Earlier studies have demonstrated that the cellular protein Puralpha is able to attenuate the transcriptional activity of JCV T-antigen. We investigated the mechanism whereby Puralpha affects T-antigen function. Co-immunoprecipitation studies demonstrated that Puralpha and JCV T-antigen associate in vivo, and glutathione S-transferase affinity binding assays revealed that these two proteins interact in vitro. Moreover, we localized the sequences of Puralpha that are important for the interaction between Puralpha and JCV T-antigen. In addition, we demonstrated that Puralpha interacts with the SV40 T-antigen. Transient transfection studies demonstrated that Puralpha and JCV T-antigen interact functionally as well. More specifically, Puralpha and a deletion mutant that interacts with T-antigen attenuated T-antigen-mediated transcriptional activation. A Puralpha deletion mutant that is unable to interact with JCV T-antigen, however, was found to be incapable of abrogating JCV T-antigen transactivation. Taken together, these data demonstrate that Puralpha and T-antigen interact both physically and functionally and that this interaction modulates T-antigen-mediated transcriptional activation. The implication of these findings with respect to the cellular role of Puralpha is discussed.
Gallia
G L
GL
Center for NeuroVirology and NeuroOncology, Allegheny University of the Health Sciences, Philadelphia, Pennsylvania 19102, USA.
Safak
M
M
Khalili
K
K
D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D002461:Cell Line, Transformed; D017362:Cyclic AMP Response Element-Binding Protein; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007577:JC Virus / Q000235:genetics / Q000276:immunology*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017384:Sequence Deletion; D014157:Transcription Factors; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D000952:Antigens, Polyomavirus Transforming; D017362:Cyclic AMP Response Element-Binding Protein; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C501130:PURA protein, human; D014157:Transcription Factors