Moloney murine leukemia virus (MLV) is a prototypical gammaretrovirus that replicates to high titer in neariy all mitotic rodent cells, causes a persistent viremia in infected mice, and induces a T-cell leukemia at a high incidence through insertional activation of host protooncogenes. Much of what we know about retrovirus replication was first learned through the study of the simple viruses such as the MLVs. In the eariy phases of infection, these viruses enter the cell through specific receptors, synthesize a DNA copy of the viral RNA genome by reverse transcription in the cytoplasm, direct the movement ofthe resulting preintegration complex (PIC) into the nucleus, and integrate the viral DNA into the host genome to form the provirus. In the late phases this DNA is expressed to form viral RNAs and proteins, and progeny virions are assembled at the plasma membrane and released to begin a new infectious cycle. In this project we propose to examine the early post-entry events of infection, the most pooriy characterized portion ofthe viral life cycle, focusing on a key host protein, IQGAP, microtubules, and dynein motors. We have previously identified IQGAPI as a major host protein interacting with the Moloney MuLV Gag matrix protein (MA), and have documented the critical importance of that interaction for virus replication. The IQGAPs are large cytoskeletal scaffolding proteins involved in the regulation of cell motility and morphology, and are noteworthy in binding and regulating both actin and microtubule networks. They integrate multiple inputs (especially from small GTPases) and produce multiple outputs, including stabilization of microtubules and capture of microtubule ends. We will determine the role ofthe IQGAPs in MLV infection, and the precise time and step in the life cycle at which they act. Using live-cell imaging of fluorescence-tagged virions, we will examine the trafficking of MLV mutants that do not bind IQGAP, and of wild-type virus blocked by dominant-negative fragments of IQGAP, to determine whether virions fail to be properly delivered to microtubules. We will test for the importance of phosphorylation of IQGAP, thought to be mediated by PKCe, in normal virus trafficking. Finally, we will test the key subunits of the dynein motor and dynactin for their roles in movement of the MLV PICs along both stable and dynamic microtubules. With the help of our cell biologist colleagues in this program, we hope to fill in a majorgap in our current understanding of the MLV life cycle.
Retroviruses are agents of serious human diseases, including leukemias and AIDS, and conversely hold out great promise as tools for gene therapy. Recent work has shown that these viruses are critically dependent on the cytoskeleton and microtubules (MTs) for their intracellular trafficking. In this proposal we aim to define the role of particular MT regulators and motors in eariy steps of MLV infection. Deeper understanding of these processes will provide new insights into retrovirus replication, potentially define new antiviral targets and increase our knowledge of trafficking of cargos on MTs.
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