James M. Pipas, PhD

Molecular Biology of SV40. 

Simian virus 40 (SV40) belongs to a small collection of viruses that induce tumors. We utilize SV40 as a model system for understanding the molecular events that drive tumorigenesis. Our studies focus on the virus-encoded master regulatory protein, large T antigen.  Large T antigen controls several aspects of viral infection including DNA replication, transcription and virion assembly.  In addition, T antigen is necessary and, in most cases, sufficient for SV40-mediated tumorigenesis. T antigen induces tumors in rodents and the neoplastic transformation of cells in culture (Figure 1) by binding to key cellular proteins that regulate proliferation and survival, and altering their activities. Our basic strategy is to use a combination of genetics and proteomics to identify cellular targets of T antigen and then to use molecular biology and mouse model approaches to understand how these actions contribute to tumorigenesis.

Currently we are studying the biochemical mechanisms by which T antigen acts on complexes containing p130 or pRb with the E2F family of transcription factors, and how these actions contribute to neoplasia. Our studies are focused on understanding how the molecular chaperone function of T antigen through its J domain disrupts the p130/E2F4 repressive complex, and on how T antigen blocks the ability of pRb to repress E2F-dependent transcription.

Figure 1 (right). Transformation potential of TAg on an established rodent cell line, REF52. A) Dense focus assay. B) Anchorage-independent assay.

We employ mouse model systems to study the effects of T antigen actions on Rb-E2F complexes in tumorigenesis. We have generated a series of transgenic mouse lines that express wild-type or various T antigen mutants in the intestinal epithelium (Figure 2). We have also generated gene knockout mice in which pRb or various E2Fs have been ablated (Chong et al. Nature 2009). We plan to use this combination of transgenic and gene knockout technologies coupled with molecular studies such as gene microarrays and ChIP experiments to explore differences in cell-cycle control exhibited by progenitor and differentiated cells.

Figure 2. The SV40 TAg induces ectopic proliferation in villus enterocytes of transgenic mice, leading to hyperplasia and dysplasia.

Virus Discovery and Functional Viral Metagenomics

All viruses encode proteins that are involved in replicating the viral nucleic acid or in the assembly of progeny virions. In addition, viruses must encode activities that block or diminish host defenses against infection. We term this latter group of proteins as HIPs (Host Interactive Proteins). We have developed computational methods for identifying HIPs and are now in the process of applying these methods to all known viruses that infect multicellular organisms. Our strategy is to develop a retrovirus library that expresses viral HIPs and to use the library in high throughput screens to identify viral proteins that modulate cell proliferation, death or the innate immune response. We then use a combination of proteomics and genetics to identify the cellular target and pathway that is altered. Given that there are over 50,000 virus-encoded proteins listed in Genbank and that fewer than 5% are of known function, we expect these HIP screens will identify novel cellular pathways and regulatory proteins.

Our computational studies have revealed evidence for the exchange of genes within and across virus families. Based on these observations we hypothesize that new viruses are being constantly created by recombination. To test this hypothesis we are collecting samples from selected biomes around the world and identifying viruses present in the samples by deep sequencing (Loh et al. J. Virol 2009; Holtz et al. Virology 2009). We are also developing the computational tools needed to identify known and novel viruses from these metagenomic data sets and to search for recombination among viruses in nature. Our preliminary studies suggest a remarkable amount of viral diversity and suggest that the 2,200 or so currently recognized viruses likely represent a very small fraction of the planet’s virome.

Lab Personnel

Ping An, Research Assistant Professor
Maria Teresa Sáenz Robles, Research Assistant Professor
Ashok Srinivasan
Shelley Cockrell, Post Doc
Tushar Gupta, Graduate Student
Nicole Seneca, Graduate Student
Paul Cantalupo, Staff
Nicholas Giacobbi, Staff
Han Na Choi, Staff
Joshua Katz
Jenny Koffman
Alfred Lentzsch
Christina Comunale
Chris Owens
Danielle Walheim
Zoe Cesarz
Will Okoniewski