Thesis (M.S.)--University of Wisconsin--Madison, 1981. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 60-69).

Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1962. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1968. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Membrane fusion plays a crucial role in many biological processes from virus infection to release of neurotransmitters (Hughson 1999). Membrane -bound surface glycoproteins are involved in the fusion process. The enveloped animal virus infection is initiated by interactions between the virus and the cell membrane through the surface glycoproteins called fusion glycoproteins (Eckert and Kim 2001). The fusion glycoproteins are responsible for both receptor binding and membrane fusion activity. The fusion proteins are characterized by a large ectodomain containing fusion peptides, a transmembrane (TM) domain, and a cytoplasimic domain. The viruses can enter cells either at neutral pH or at acidic pH. When exposed to appropriate conditions, the fusion protein undergoes conformational changes, which in turn drives the fusion process. The fusion glycoproteins can be classified as Class I and Class II fusion proteins (Lescar eta/. 2001 ). The Class I fusion proteins are synthesized as a precursor molecule, which then undergoes proteolytic cleavage to generate a mature molecule containing the hydrophobic fusion peptide at the N -terminal. The class II fusion glycoproteins are not synthesized as precursor molecules, and they have internal fusion peptides. The vesicular stomatitis virus (VSV) glycoprotein G is a class Ill fusion protein. It has a neutral internal fusion peptide and upon exposure to low pH, the protein undergoes reversible conformational change (Gaudin 2000, Yao eta/. 2003). A 62kDa soluble ectodomain of VSV G (Gs) has been generated by limited trypsin digestion. The SDS PAGE gel electrophoresis indicates that the trypsin has possibly cleaved near the transmembrane (TM) domain. Liposome binding experiment suggests that Gs can bind to liposomes in a pH dependent manner. Liposome fusion studied by RET assay suggests that the Gs can induce significant amount of hemifusion. However, it failed to induce any content mixing mainly due to considerable amount of membrane leakage activity. This indicates that the binding to the membrane through the TM domain is required for complete membrane fusion. Unlike TBE E soluble ectodomain, Gs can form dimers and trimers at neutral and fusion active pH. Light scattering experiment shows that the aggregation of Gs increases with a decrease in pH. The conformational change with changes in pH was evident from the trypsin sensitivity assay and CD spectroscopy. It was observed that Gs became resistant to trypsin digestion at low pH and a-helicity content of the molecule increased upon lowering the pH. However, the maximum amount of a-helicity was observed at pH 6. The removal of the TM domain also shifts the optimum fusion pH towards more acidic pH in comparison to VSV G. These results indicate that the TM domain is not required for the oligomerization of G protein, but some role has been reserved for the TM domain during membrane fusion. The CD spectroscopic data also indicated that the G protein undergoes structural rearrangement between pH 7.4-6, which could be responsible for the exposure of fusion peptide and subsequent target membrane binding. / Thesis / Master of Science (MSc)

Typescript (photocopy). / Digitized by Kansas Correctional Industries

Call number: LD2668 .T4 1979 T516 / Master of Science

Vesicular stomatitis virus (VSV) is considered a prototype for studying non-segmented negative-stranded ribonucleic acid (RNA) viruses. Livestock are naturally infected by VSV, causing severe economic impact due to lack of any effective treatment. RNA interference (RNAi)-based therapeutics are promising alternatives to control viral infections. Lentiviral vector systems deliver artificial short hairpin RNA (shRNA) into the genome of cells to activate the RNAi pathway. In this study, an RNAi-based approach to generate cell lines with reduced susceptibility to VSV (Indiana) infection was tested. First, eight shRNAs targeting either the nucleocapsid (N), phosphoprotein (P), or the polymerase (L) viral genes were designed and introduced into cell systems. To test the potency of the shRNAs for silencing the target viral transcripts, semi-quantitative polymerase chain reaction (PCR) analysis of viral N, P, and L transcripts was performed. Then, supernatants from infected groups were evaluated by microtitration and immunoblot. Finally, the effect of VSV genomic variability in the target region of shRNAs was predicted by partial sequencing field and laboratory-adapted strains. Viral transcripts were significantly reduced in cells stably expressing shRNAs targeting the N viral gene (nucleotides 67-97 or 1312-1332; p<0.05) or P gene (nucleotides 1772-1792; p&lt;0.05). Reduction in viral transcripts was not observed by other VSV-shRNAs tested. Reduction of viral transcripts by the N-shRNA (sh-1312) was accompanied by a decrease in viral protein. Also, a reduction in the viral particles shed from cells expressing N-shRNAs (nucleotides 67-97, p&lt;0.05) was noted. The results also showed complementarity of target gene sequences for shRNAs in the sequence from the laboratory-adapted strain and single base substitutions in the corresponding regions from VSV field isolates. However, these mismatches did not occur within the seed region of the shRNAs. In conclusion, partial silencing of viral transcripts by a single shRNA does not block VSIV replication; however, partial impairment of VSIV replication was observed in N-shRNAs expressing cells. During infection, the naturally high level of N gene transcription may have modulated the sh-RNA effect. The combination of the most potent shRNAs identified here into a multiple shRNA vector may result in further reduction of viral replication. These data contribute to ongoing development of effective RNAi-based technologies to combat viral diseases.

Thesis (M.S.)--Northern Michigan University, 2008. / Includes bibliographical references (leaves 75-81).

Thesis (M.S.)--University of Wisconsin--Madison, 1970. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.