Date of Award
Doctor of Philosophy (PhD)
Professor James R. Smiley
The herpes simplex virus (HSV) virion contains the virion host shutoff protein (vhs, product of the UL41 gene) that induces inhibition of host cell protein synthesis, disaggregation of pre-existing polyribosomes, and degradation of mRNA during early times of infection. The data presented in this thesis describe the development of a rabbit reticulocyte lysate-based in vitro system for analysis of the vhs protein activity. I show that vhs protein produced, as the sole HSV protein, in rabbit reticulocyte lysates inhibits translation and induces degradation of reporter RNAs. Detailed analysis of the vhs-dependent RNA decay revealed that it proceeds through an endoribonucleolytic cleavage mechanism that is not affected by the presence of a 5' cap structure or a 3' poly(A) tail in the RNA substrate. The vhs-dependent RNA degradation activity requires Mg++ ions and occurs in the absence of ATP and ribosomes. Characterization of the decay profile of two unrelated substrates indicated that they were initially cleaved at preferential sites non-randomly distributed over their 5' quadrants. This activity is greatly altered by placing an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) or poliovirus in the transcript. Transcripts bearing the IRES were preferentially cleaved by the vhs-dependent endoribonuclease at multiple sites clustered in a narrow zone located immediately downstream of the element. "Targeting" was observed when the IRES was located at the 5' end or placed at internal sites in the substrate, indicating that it is independent of position or sequence context. As with cleavage over the 5' quadrant, cleavage downstream of the IRES elements requires Mg ++ ion but not ATP and ribosomes. Transcripts containing internal IRES elements were also independently cleaved over their 5' quadrant. The vhs-induced RNA decay activity is not restricted to HSV-1 vhs, in that the vhs proteins from HSV-2 and pseudorabies virus also induce RNA degradation in vitro . I also show that the RNA degradation activity of the HSV-1 vhs protein is reproducible when vhs is produced in HeLa, but not wheat germ, cell-free extracts. These data indicate that the vhs-dependent nuclease can be selectively targeted by specific cis -acting elements in the RNA substrate, possibly through secondary structure or a component of the translational machinery.
Elgadi, Mabrouk Mohammed, "The in vitro activity of the herpes simplex virus virion host shutoff (vhs) protein" (1999). Open Access Dissertations and Theses. Paper 1735.