Transcriptional activation of growth regulated genes by polymavirus small tumor antigen

Polyomavirus, like the other DNA tumorviruses (adenovirus, human papillomavirus and simian virus 40) often infect differentiated cells which are in the quiescent Go state of the cell cycle. As for their duplication these viruses depend to a large extent on the replication machinery of the host cell which is expressed in sufficient quantity only during the S phase, these viruses had to evolve in a way which copes with this situation. Thus every one of these viruses codes for one or more proteins which interfere with cellular growth control. These same proteins are responsible for the immortalising and transforming activities of DNA tumorviruses. Polyomavirus has a circular genome of about 5000 base pairs which can be devided into two halves, one of which carries the information for three capsid proteins, the other one codes for the three tumor antigens (large middle and small T antigen). In each case all three reading frames are used. While middle T counteracts apoptosis and thus has transforming activity, large and small T are involved in S phase induction. To study the activity of large and small T individually and in combination, we created cell lines which carry the information for one or both of these proteins in hormone- inducible form and thus could grow these cells in the absence of viral protein and hence in the absence of selection pressure. The cells also could easily be growth arrested by serum withdrawal. Upon addition of the glucocorticoid, dexamethasone, the viral protein is induced allowing to study its effect on particular reactions in the cell. With this system it was possible to examine in some detail the transactivation of genes coding for DNA synthesis- and precursor-producing enzymes by large T antigen. This requires the capacity of large T to bind the socalled pocket proteins, retinoblastoma protein and its relatives, p107 and p130. It turned out, however, that despite of very active induction of such enzymes, large T does not by itself drive a sizable fraction of cells into S phase. This requires small T antigen in addition. It turned out the the major limiting proteins for S phase induction by large T alone are the cyclins E and A and in particular their respective kinase activities. It is well known that cyclin E/cdk2 and cyclin A/cdk2 are necessary for an initiation of S phase. Our previous experiments showed that while both T antigens can transactivate the cyclin A promoter, small T is absolutely necessary for development of the kinase activities. It does so by causing the destruction of the kinase inhibitor, p27. For this the capacity of small T to bind the protein phosphatase PP2A is essential. More recently we found that both viaral proteins exert their transactivating activity by acting on protein complexes building up on various transcription factor binding sites on the cyclin A promoter. A protein sequence present in both proteins which allows them to bind the chaperone HSC70 is essential for this activity in addition to the interaction of large T with pocket proteins and of small T with PP2A. The surprinsing capacity of small T protein to transactivate the cyclin A promoter led to the present research proposal in which we plan 1. To learn in more detail as to how small T transactivates the cyclin A gene, in this context it has to be clarified with which cellular proteins (aside of PP2A and chaperones) the viral protein can interact, and 2. to search for further genes which are transactivated by small T using DNA chip technology.

In this project it was shown that one of the tumorantigens of the DNA tumorvirus polyoma, the small tumorantigen, has a profound effect on the expression of cellular genes which can be up- or downregulated. It is suggested that these effects contribute significantly to the capacity of polyomavirus to form tumors in mice and to transform cells in culture. The replication of small DNA tumorvirus affords the deregulation of cellular growth regulatory pathways in order to drive arrested cells into S phase when virus replication can take place. They therefore code for proteins, the tumor antigens, which deregulate gene expression. The major protein of polyomavirus for this aim is the large T antigen which activates the transcription factor family E2F. For S phase induction, however, it requires the additional activity of the small tumorantigen which interfers with and inhibits an enzyme involved in dephosphorylation of proteins, including those implicated in cell cycle and growth regulation. This interaction with the phosphorylation status of cellular proteins, however, allows this viral protein to influence a great variety of cellular processes. In order to understand the potential of this tumorantigen, we have carried out a search for genes deregulated by the small tumorantigen of polyomavirus. Our data show that this protein deregulates the expression of more than 500 genes about half of them being upregulated and the other half downregulated. Particularly important among the upregulated genes are those which are expressed in cells when they are driven into the S phase of the cell cycle. Small T antigen thus duplicates in part the activities of the large tumor antigen. Among the genes downregulated are some which normally block the cell cycle. Their downregulation again aids the growth induction of cells and thus the provision of optimal conditions for virus replication but also for tumorformation.These data help to understand the role played by this protein in tumorigenesis and in the transformation of cells in culture.