This new project headed by Dr. DiMaio is based on a discovery made in project 2 during the current funding period. The polyomaviruses, BK virus andJC virus, cause serious diseases in immunosuppressed individuals including cancer patients and, like their close relative SV40, are putative human tumor viruses. With the support of this grant, we discovered that the cellular co-chaperones DNAJ-B12 and DNAJ-B14 are required for efficient infection by these three viruses. When expression of these genes is repressed by shRNAs, there is a substantial reduction in expression of the major early protein, large T antigen. Virus binding to the cell surface appears unimpaired, suggesting that the DNAJ-B12/14 sensitive step(s) is in some aspect of virus entry, intracellular trafficking, or uncoating. We will conduct a series of biochemical, cell biological and genetic studies to elucidate the mechanistic role played by DNAJ-B12/14 in SV40 infection. We will determine how far infection proceeds in cells lacking DNAJ-B12/14 function, and determine what step in the virus entry/trafficking/uncoating process is blocked. We will conduct mutational and biochemical analysis of DNAJ-B12/14 to determine its mode of action at the molecular level. Viral escape mutants that allow infection despite DNAJ-B12/14 repression will be isolated and characterized. Finally, we will use shRNA technology to determine whether other members of the DNAJ gene family are required for infection by the polyomaviruses and other viruses, including Epstein-Barr virus in collaboration with Dr. Miller. These experiments will provide new insights into the process of tumor virus infection and characterize the role of new putative anti-viral targets.