Studies of protein interactions with SV40 large T antigen in mammals and yeast: T antigen interacts with cell cycle proteins and transcription factors

SV40 large T antigen is a viral oncoprotein which interacts with numerous host proteins and is capable of many biochemical functions including transformation and transcriptional activation. Using Saccharomyces cerevisiae, we have identified an interaction between T antigen and a cell cycle regulatory protein, suggesting a mechanism by which T antigen can alter cell growth. T antigen expression in Saccharomyces cerevisiae causes dramatic morphological changes in the cells and a lag in progression from G$\sb1$ to S. One gene which regulates this transition is p34$\sp{CDC28}$. GST-fusion assays showed that T antigen binds both p34$\sp{CDC28}$ and p34 $\sp{Cdc2Hs}$ in vitro. Also, p34$\sp{CDC28}$ coprecipitates with T antigen from yeast cell extracts. Overexpression of p34$\sp{CDC28}$ does not rescue the T antigen phenotypes suggesting that the T antigen-p34$\sp{CDC28}$ complex has altered function. Our data shows that this complex has kinase activity which differs from the endogenous p34$\sp{CDC28}$ kinase activity. These data, along with the previous finding that phosphorylation by human p34$\sp{CDC2}$ kinase can regulate certain T antigen functions, suggests that T antigen forms a complex with mammalian p34$\sp{CDC2}$, or another Cdk, altering its kinase activity. T antigen transcriptionally activates many viral and cellular promoters. The amino-terminal 172 amino acids of T antigen bind to the TATA-binding protein (TBP) and contain the 82 amino acids which are shared by small t antigen (the common region). Small t also activates some genes and GST-fusion assays showed that the common region is also able to bind TBP. In mapping the regions of TBP which bind to both large T and to the common region, we found that while both bind to the conserved carboxy-terminus of TBP, they utilize distinct binding domains within this region. Large T antigen requires TBP amino acids 164-214 to bind, while the common region requires TBP amino acids 214-252. Adenovirus E1A also binds to amino acids 214-252, suggesting that large T antigen utilizes basal transcription components differently than other viral transactivating proteins