Microtubules and their nnotors mediate the intracellular movement of cargos and control of cell structure that underlie processes such as cell polarization and motility. Consisting of a/|3-tubulin heteropolymers, MT minus-ends are anchored in the perinuclear MT Organizing Center (MTOC) while their plus-ends radiate toward the cell periphery. Most MTs dynamically grow and shrink rapidly. However, a subset (<10%) become stabilized in response to various physiological signals, acquiring post-translational modifications such as de- tyrosination or acetylation, and are thought to act as specialized tracks for vesicle transport. The end-binding protein, EBl tracks MT plus-ends and regulates both MT dynamics and stabilization by recruiting a variety of plus-end binding regulatory proteins (+TIPs). +TIP binding responds to signal pathways such as Rho-mDia and Akt-GSK-3p. MTs also play a critical yet poorly understood role in the trafficking of pathogens. Herpes Simplex Virus type 1 (HSV-1) infects ~60-90% of the world population, establishing life-long infections that result in periodic disease recurrence, ranging from cold sores to fatal encephalitis. While it is established that HSV-1 induces a-tubulin rearrangements and exploits MT motors for intracellular movement, the precise nature of MT modifications and their role in infection, as well as potential roles for specialized MT regulatory factors remain unknown. Our preliminary data shows that HSV-1 causes centrosomal MTOC dispersal and MT disorganization early in infection of primary normal human dermal fibroblasts. This occurred concomitant with the loss of EB1 comet staining, indicative of MT plus-end tracking, and was dependent on viral gene expression. Co-immunoprecipitation identified a candidate EBI-binding protein synthesized in infected cells while RNAi-mediated depletion of EBl prevented MT reorganization and potently inhibited infection. As infection progressed, EBl comet staining returned as extensive MT re-growth and stabilization occurred. Depletion of EBl or CLASP2, a GSK-3P-regulated +TIP that controls MT growth from the Trans Golgi Network (TGN), a known alternate MTOC, significantly impaired HSV-1 infection. In cells infected with HSV 1 lacking the viral kinase, Us3, which stimulates GSK-3p phosphorylation, MTs remained bundled around the TGN, the site of secondary virus envelopment.
We aim to determine the role of specialized host MT end- binding proteins and viral factors in regulating MT dynamics and stabilization at discrete phases of HSV-1 infection in different natural target cell types, defining their role in mediating virion trafficking and spread. This will provide important insight into not only the lifecycle of this ubiquitous human pathogen but also more general mechanisms of MT regulation and macromolecular transport.
Microtubules regulate intracellular cargo movement and changes in cell structure that play critical roles in various biological processes, yet their regulation remains poorly understood in many contexts including viral infection. In this proposal we aim to determine the role of specialized MT regulatory proteins and MT subsets in HSV-1 infection of a variety of natural target cell types. This will not only provide important insight into the replication of this ubiquitous human pathogen, with the potential to uncover novel antiviral targets, but will also add to our broader understanding of fundamental mechanisms of MT regulation and cargo transport.
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