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A: Vaccinia Virus: Vaccinia virus is the prototypic poxvirus and was the virus used in the vaccination campaign that led to the global eradication of smallpox. Vaccinia virus replicates solely within the cytoplasm of infected cells, and the 192 kb DNA genome encodes most, if not all, of the functions required for the progression of the viral life cycle. We have focused our attention primarily on viral DNA replication, the role of virally encoded kinases and phosphatases within the infectious cycle, and virion morphogenesis. We are also exploring the interplay between the viral life cycle and cellular bioenergetics. Our work integrates diverse approaches drawn from the disciplines of virology, molecular genetics, cell biology, and biochemistry.
With regard to DNA replication, we are interested in understanding how replication is organized within dedicated cytoplasmic domains, in deciphering the mechanism of replication, and in pursuing a biochemical and genetic investigation of the proteins involved. We are interested in how the core polymerase, processivity factor (A20+UDG), single-strand DNA binding protein (I3), DNA ligase (A50), and FEN-1 like nuclease work together to accomplish faithful DNA replication and repair. Additionally, we are pursuing the hypothesis that the abundant H5 protein serves as a scaffold to support replication within the membrane-delimited cytoplasmic replication domains.
With regard to virion morphogenesis, our current interest is focused on the biogenesis of the poxvirus membrane, which is quite unique and involves the enlargement of planar lipid bilayers within the cytoplasm. We are using a genetic, biochemical, cell biological and ultrastructural approaches to understand how the F10 kinase, a group of regulatory proteins (A6, A11, A30.5, H7, L2), and the two major structural proteins within the membrane (A14 and A17) mediate diversion of membranes from the endoplasmic reticulum (ER) and their remodeling and enlargement. The overall process of virion assembly involves a cascade of protein/protein, protein/DNA, and protein/lipid interactions that serve as an excellent model for the process of cellular organelle biogenesis.
B: VRK1: A cellular protein kinase involved in nuclear architecture, mitotic and meiotic progression, cell proliferation, and oncogenesis.
We became interested in the VRK family of cellular protein kinases because of the sequence similarity between their catalytic domains and that of the vaccinia-encoded B1 kinases. We performed the first thorough characterization of the VRK family (VRK1, nuclear; VRK2, nuclear envelope and ER; VRK3, nuclear pseudokinase) and purified and characterized their biochemical properties. We identified and validated the cellular BANF1 (BAF) protein as a highly efficient substrate for both VRK1. Within the interphase nucleus, BANF1 binds to chromatin and to proteins in the inner nuclear membrane (INM). We have shown that VRK1-mediated phosphorylation of BANF1, which peaks at the onset of mitosis, abrogates BANF’s DNA binding activity and reduces it’s interactions with proteins at the INM. VRK1 depletion leads to aberrant nuclear envelopes in interphase nuclei, and to the abnormal retention of BANF1 on chromosomes during early stages of mitosis (prophase, metaphase and anaphase). These effects have impacts on mitotic fidelity as well as cell proliferation.
Because overexpression of VRK1 has shown to correlate with poor clinical outcome in a subset of breast cancer patients, we have focused much of our work on mammary epithelial cells (normal and malignant). Using a mouse xenograft model, we showed that malignant cells depleted of VRK1 formed smaller tumors than control cells, and that mice receiving this cells did not develop distal metastases. We have also shown that VRK1 overexpression accelerates acinus growth in a 3D culture model, but reduces cell migration in a 2D wound-healing model. We are using a variety of “omic” strategies, as well as cell biological and biochemical approaches, to understand the roles played by VRK1 in regulating cell structure and function in normal and cancerous cells.
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