Investigating assembly of the HSV-1 protein Vhs into tegument
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Assembly of Herpes Simplex viruses is a poorly understood process involving multiple redundant interactions between large numbers of tegument and envelope proteins. Previous results from our laboratory have shown that the tegument protein virion host shutoff (Vhs) is largely insoluble in HSV-infected cells and is also stably associated with membranes. This dissertation was aimed at understanding the membrane association properties of Vhs as it is not a typical membrane protein and is not known to undergo any modification that enable membrane binding.;Using an assay where Vhs was expressed in COS cells which were subsequently infected with a Vhs null virus, membrane association properties of Vhs were tested. We demonstrated that both insolubility and stable membrane binding are stimulated during the course of an HSV infection. Furthermore we found that although the majority of the protein is dispensable, the amino terminal 42 residues of Vhs are sufficient to mediate membrane association and tegument incorporation when fused to a GFP reporter. Particle incorporation correlates with sorting to cytoplasmic punctate structures that may correspond to sites of HSV assembly. Immunogold electron microscopy confirmed the incorporation of GFP tagged amino terminal 42 amino acids of Vhs into the viral particles. Further mutagenesis studies have pinpointed the N-terminal 10 residues to be essential and sufficient for membrane binding and tegument incorporation, and that myristoylation is not part of the targeting mechanism.;Studies using mutant viruses have revealed that membrane association of Vhs is partially dependent on the presence of pUL36. This population of Vhs probably represents the capsid bound fraction. A second population of Vhs is assembled via interactions with the envelope glycoprotein gH. Binding of gH to Vhs has been demonstrated by in vitro GST-pull down assays as well as in vivo co-immunoprecipitation assays using infected cells lysates. The critical residues of gH involved in interactions with Vhs have been identified.
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