Infection of a host cell by the Herpes Simplex Virus Type 1 leads to the efficient reprogramming of the cells' synthetic machinery to replicate the viral genome ultimately producing progeny virions. Two proteins introduced upon viral fusion are thought to initiate this effect. The potent transactivator of immediate early genes (VP16) and the mRNA destabilizing virion host shutoff protein (vhs), work in concert with one another to invoke the cascade of viral gene expression, and to destroy pre-existing cellular mRNA. Due to the non-specific nature of vhs induced mRNA degradation, its activity is downregulated at later times during infection to spare virally encoded mRNA. Recent evidence has shown that VP16 is responsible for this vhs downregulation, a process thought to occur by mutual interactions between the two proteins and a potential compartmentalization of vhs within the nucleus (Lam 𝘦𝘵 𝘢𝘭., 1996; Smibert 𝘦𝘵 𝘢𝘭., 1994). Furthermore, such an event is also thought to position vhs so it can be efficiently packaged, a supposition supported by the observation that vhs lacking the ability to bind VP16 is not incorporated into new virions (Read 𝘦𝘵 𝘢𝘭. , 1993). To ascertain if VP16 was indeed capable of relocalizing vhs to the nucleus of a cell in the absence of any other viral factors, we created multiple constructs consisting of various portions of vhs fused in frame to the fluorescent marker protein EGFP. In addition, various truncated forms of VP16 were also fused to EGFP for the purpose of delineating the region of VP16 that is responsible for VP16 and possibly vhs nuclear localization. Co-transfection experiments utilizing EGFP-vhs fusions demonstrated that vhs relocalizes to perinuclear regions in the presence of VP16, an effect absolutely dependent upon its ability to interact with VP16. In addition, deletion mapping of VP16 implicated the region spanning amino acids 335 to 355 as being necessary for this localization, with a stretch of 15 amino acids (330 to 344) appearing to constitute a putative bipartite nuclear localization signal. Interestingly, our observation that the vhs/VP16 complex localizes to a region of the cell thought to ultimately encompass the tegument of new virions gives credence to the notion that this interaction and subsequent localization may indeed function to package vhs into new virions. Furthermore, it is also suggested that vhs may in fact be downregulated at intermediate times during infection through VP16 mediated compartmentalization within the nucleus. For these reasons we propose that the disruption of the vhs/VP16 interaction could severely abrogate the infectivity of HSV and as such could present a novel target for antiviral intervention. / Thesis / Master of Science (MS)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/23155 |
| Date | 08 1900 |
| Creators | Inglis, Jamie |
| Contributors | Capone, John, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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