Our investigations primarily center on the viral protein SV40 large T antigen (LT), a highly multifunctional protein critical for viral replication and oncogenic transformation. LT has been studied extensively for many years, and it has been instrumental for identification and characterization of key cellular tumor suppressors, most famously p53 and the retinoblastoma family. Mutant analysis indicates that additional proteins bound to LT are likely to be relevant for its biological activities. 

Figure 1 

Schematic depiction of SV40 large T antigen (LT). LT is a highly multifunctional protein directing both initiation of viral replication and oncogenic transformation. LT binds and alters cellular signaling proteins, some of which are indicated (Hsc70, Cul7, Bub1, pRB, p107, p130, p53, p300, CBP, Fbw7). In addition, the DNA binding domain (DNA), ATPase domain (ATP), Zn binding element (Zn), nuclear localization signal (NLS) and DnaJ domain (DnaJ) are diagrammed. Two clusters of phosphorylation sites are also indicated.

In collaboration with Parmjit Jat at the Ludwig Institute we first discovered and characterized a novel interaction of LT with the Bub1 mitotic kinase. Bub1 is critical for the spindle checkpoint, a quality control mechanism that ensures high fidelity mitotic segregation and genome stability. Since Bub1 is mutated in human cancer and reduced Bub1 expression can lead to increased tumor susceptibility in mouse models, we believe that studies of LT binding to Bub1 are going to inform us further regarding contributions of genomic instability to tumorigenesis.

Our studies have led to a fascinating insight to functions of LT that were poorly understood: The ability of LT to cause structural and numerical chromosome instability. Our LT mutant analysis first linked Bub1 binding to oncogenic transformation and viral replication. Our recent analysis has also connected it with induction of both tetraploidy and a DNA damage response (DDR). Moreover, we find that LT-expressing cells have much higher frequencies of anaphase bridges and micronuclei. Experiments are under way to investigate by time-lapse imaging how LT affects mitotic progression and fidelity via Bub1 binding. It is our goal to understand the molecular mechanisms, whereby LT binding to Bub1 facilitates generation of tetraploidy and induction of the DDR.   

Figure 2 

LT induces multiple cellular phenotypic alterations. LT causes genomic instability manifested in micronuclei, anaphase bridges and tetraploidy. LT also induces DNA damage responses and affects homology-directed repair. Moreover, LT causes oncogenic transformation, demonstrated by a focus formation assay, and drives viral replication, shown by Southern blotting of DpnI digested low molecular weight DNA.

            Our study of the DDR induced by LT via Bub1 binding resulted in greater appreciation of the general interplay between LT and DDR signaling pathways. Comet assays demonstrate that LT generates DNA damage. A major conclusion is that different domains of LT are capable of inducing distinct sub-components of the DDR and repair machinery. The LT N-terminus, via Bub1 binding, induces nuclear g-H2AX/ 53BP1 foci that are the hallmarks of a DDR. LT also activates the Fanconi anemia pathway, which is normally linked to a replication stress response. LT furthermore, in unknown ways, induces foci of the homologous recombination protein Rad51. LT itself is localized on chromatin near Rad51 and PML (promyelocytic leukemia), the nucleating component of ND10 bodies. Interestingly, we and others have shown that SV40 has usurped the DDR to enhance its own replication. We have demonstrated that the homologous recombination machinery (Rad51) and FancD2 are required for high-level extra-chromosomal viral replication with high fidelity.

Another major research emphasis centers on other polyomaviruses. Drs Patrick Moore and Yuan Chang discovered Merkel cell polyomavirus (MCPyV) in 2008 and found it to be integrated in about 80% of Merkel cell carcinomas, a highly aggressive skin cancer with poor prognosis. Accumulating evidence strongly supports a causal role of MCPyV in Merkel cell carcinoma, making it the first human polyomavirus with a persuasive direct link to cancer etiology. We have collaborated extensively with the Chang/ Moore laboratory. Using tandem-affinity purification, we have identified and characterized a new binding protein for MCPyV LT, which is believed to participate in vesicular trafficking and lysosomal fusion.   

Figure 3

Identification of novel Merkel cell polyomavirus large T antigen binding proteins by tandem affinity purification. SV40 LT 1-136 or MCV LT 350 were tagged with Flag and strep tags. Stable cell lines were derived in 293H. Tandem affinity purification (TAP) was performed, products separated by SDS-PAGE and proteins visualized by silver staining. A new binding protein hVam6p was identified by mass spectrometry.

Taken together, our interests span a wide range but our emphasis is on cell cycle, checkpoints, genomic instability, DDR signaling and oncogenic transformation mechanisms. We are committed to cross-disciplinary approaches combining molecular biology techniques with both biological and biochemical assays. We strongly believe, with the advent of a new polyomavirus directly linked to human cancer, that the viral model systems will continue to reveal important new facets of cellular signaling, and how it goes awry in cancer.