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No description available.

Cells infected with herpes simplex virus show a rapid cessation of protein synthesis and a dramatic decline in the levels of mRNA; a process known as host shutoff. This effect is attributed to a viral tegument protein called the virion host shutoff protein, or vhs. The mechanism by which vhs induces mRNA degradation is not yet understood. It is not known whether vhs possesses RNase activities or if it acts in combination with other cellular factors. To gain a better understanding of the function of vhs, I examined RNA degradation in detail by analyzing the RNA decay products generated in the presence of vhs. 𝘐𝘯 𝘷𝘪𝘷𝘰 experiments, performed by infecting murine erythroid leukemia cells with HSV, revealed that beta-globin mRNA is rapidly degraded, in the presence of vhs, without being converted to detectable decay intermediates. The half-life of this mRNA was 15 and 60 minutes for HSV-2 and HSV-1, respectively. Using vhs translated in a rabbit reticulocyte lysate system, I found that vhs induced rapid decay at the 5' end of a capped RNA molecule. The decay event was endonucleolytic and occurred at preferred sites downstream of the cap, generating capped oligonucleotides. Unlike influenza RNA polymerase, the cleavage event did not occur at a fixed distance from the cap since capped oligos of differing size were generated from different RNA substrates. My data indicate that vhs induced cleavage exhibits a strong, but not absolute preference for RNAs possessing an m⁷G cap, which may account for vhs' specificity for m RNAs 𝘪𝘯 𝘷𝘪𝘷𝘰. / Thesis / Master of Science (MSc)

A HSV 2 immediate early antigen was prepared and used as an immunogen in an attempt to produce monoclonal antibodies to this set to proteins. The results of three screening assays, ELISA, immunodiffusion and radioimmunoprecipitation with cell extracts and Protein A-Sepharose beads indicated that the hybrid cell lines are nonsecretors of antiHSV 2 antibodies. Other monoclonal cell lines from Dr. Bacchetti's laboratory were characterized by radioimmunoprecipitation with Protein A-Sepharose beads and cell extracts. / Thesis / Master of Science (MS)

The role of highly conserved amino acids within the carboxy-terminal hydrophobic domain of herpes simplex virus I (HSV-I) glycoprotein gB was studied by introducing point mutations using the method of site directed mutagenesis. A segment of this hydrophobic domain of glycoprotein gB contains a nuclear envelope (NE) targeting signal and the effect of these point mutations on targeting to the nuclear envelope was determined. A complementation assay was employed to determine the effect these mutations have on HSV-I infectivity .The point mutations created within the transmembrane domain of glycoprotein gB had no effect on nuclear envelope targeting and localization. However, single point mutations introduced into the first and second hydrophobic domains of glycoprotein gB, G₇₄₃R and F₇₇₀S, affected the targeting and localization of full-length glycoprotein gB at the nuclear envelope. When the transmembrane domain ofHSV-I glycoprotein gB containing the following point mutations A₇₉₀Q, A₇₉₁S, A₇₈₆S, A₇₈₆Y and A₇₉₀S, was introduced into a chimeric protein consisting of the cytoplasmic domain and ectodomain of a plasma membrane protein, vesicular stomatitis virus glycoprotein G, NE targeting and localization were affected. These point mutations may affect the targeting of glycoprotein gB by altering the structure of the targeting signal within the protein. It can be hypothesized that the presence of the cytoplasmic domain. ectodomain domain, and the first and second transmembrane domains within full-length glycoprotein gB can compensate for the effect these point mutations have on nuclear envelope targeting. since the same point mutations had no effect on the targeting · and localization of full-length glycoprotein gB. Complementation assays showed that the glycoprotein gB mutants, A₇₈₆S, A₇₈₆Y, A₇₈₆N, A₇₉₀Q, A₇₉₁S, F₇₇₀S, or G₇₄₃R, were unable to complement a gB-null virus even though these mutant proteins are localized at the nuclear envelope. These proteins may not have been incorporated into the viral capsid due to misfolding or due to the fact that sequences required for interaction with other viral proteins were lost. Another possibility is that the mutant proteins were incorporated into the HSV virion but were not biologically active. / Thesis / Master of Science (MS)

The glycoprotein gB of HSV-1 is involved in viral entry and membrane fusion functions. It is glycosylated, forms homodimers, and is transported to both the inner nuclear membrane and plasma membrane in infected cells. The gB glycoprotein contains a potential membrane anchoring hydrophobic sequence of 69 amino acids, located near the carboxy terminus of the glycoprotein. This sequence is predicted to span the membrane bilayer three times, and can thus be divided into three distinct segments, each of which could span the bilayer. To define both the membrane anchoring sequence and the role of this 69 residue hydrophobic domain, a series of deletion mutants were constructed. These mutants have one, two or all three of the predicted membrane spanning segments deleted, in every combination possible, thereby creating a total of 7 deletion mutants. These mutant constructs were expressed in COS-1 cells using transient expression systems. All the mutant constructs were expressed and glycosylated in a manner similar to the wild type gB glycoprotein. Mutant glycoproteins lacking the third (or most carboxy terminal) predicted spanning segment of this 69 residue hydrophobic domain were found to be secreted from the cells, indicating that this segment may specify the membrane anchoring domain of the gB glycoprotein. Further, the mutant glycoproteins containing this third segment were localized in the nuclear envelope, while mutants lacking this segment were not. All the deletion mutants, except for one, were however defective in intracellular transport and processing of the N-linked oligosaccharides. The only mutant that showed any intracellular transport and processing had only the third segment deleted, but even this mutant was transported and processed much slower than the wild type glycoprotein. The mutant glycoproteins also failed to complement a gB-null virus. These results suggest that the carboxy terminus hydrophobic domain contains essential structural determinants of the gB glycoprotein. / Thesis / Master of Science (MS)

Herpes simplex Virus Type 1 expresses three different classes of genes, immediate early, early, and late, during a lytic infection. Immediate early genes are the first class of genes expressed and they are the only genes expressed independently of de novo viral protein synthesis. This unique characteristic is thought to be the result of the activation of immediate early genes by Vmw65, a protein brought into the cell as a component of the infecting virion. Vmw65 transactivates through the target sequence TAATGARAT (R= purine) which is present at least once in all immediate early transcription regulatory regions. By inserting minimal synthetic promoters, containing the TAATGARAT sequence, into the thymidine kinase locus of the herpes simplex virus type 1 genome I determined that transactivation by Vmw65 is not sufficient to confer on the linked sequences the complete immediate early pattern of gene expression. Furthermore through a transient expression assay I determined that the TAATGARAT sequence element, by itself, when linked to a TATA box is sufficient to act as a target for Vmw65 transactivation. / Thesis / Master of Science (MS)