Thesis (M. Phil.)--University of Hong Kong, 2002. / Includes bibliographical references (leaves 121-137).

published_or_final_version / Microbiology / Master / Master of Medical Sciences

Thesis (Ph. D.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 85-92). Also available on the World Wide Web.

Thesis (Ph.D.) - University of St Andrews, May 2008.

Thesis (Ph. D. in Molecular Physiology and Biophysics)--Vanderbilt University, Aug. 2009. / Title from title screen. Includes bibliographical references.

Rubella virus (RV) is a small, enveloped, positive-stranded RNA virus in the family Togaviridae, and bears striking similarities to the prototype alphaviruses Semliki Forest virus (SFV) and Sindbis virus (SV) in terms of genome organization and structural protein expression strategy. However unlike alphaviruses, RV infection of cultured cells is characterized by relatively long latency periods, slow replication kinetics, limited cytopathology, and the ability to establish a persistent infection in virtually every cell line capable of supporting its growth. RV virions contain three structural proteins C, E2, and El which are derived by post-translational processing of a precursor polyprotein pllO (NF₂-C-E2-El-COOH). Processing and intracellular transport of RV structural proteins has been studied by jn vitro and jn vivo expression of RV cDNAs. It was found that targeting of El and E2 into the endoplasmic reticulum was mediated by two independently functioning signal peptides. Coincident with translocation into the ER, both proteins underwent addition of N-linked glycans and proteolytic processing. C protein did not appear to play a role in the processing of pllO. Expression of the RV structural proteins in COS cells revealed that E2 exited the ER, and was transported through the Golgi to the cell surface in an El-independent manner, although coexpression of El seemed to increase the rate of transport. Conversely, El was retained in a Golgi-like region and was not found on the plasma membrane in the absence of E2. Oligonucleotide-directed mutagenesis of El and E2 cDNAs showed that El andE2 both contain three N-linked glycans respectively. Lack of glycosylation did not appear to affect the intracellular localization of the RV glycoproteins in COS cells. A number of significant differences between RV and SFV/SV structural protein expression strategies were discovered, and their possible relationship to RV virion assembly are discussed. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Picornaviruses replicate in association with cytoplasmic membranes of infected cells. Poliovirus 2C and 2BC play an important role in the formation of membranous vesicles, and induce dramatic changes in membrane trafficking. Theiler’s murine encephalomyelitis virus protein 2C was localized in infected cells using an anti-TMEV-2C antibody. Early upon infection, TMEV 2C was localized in the cytoplasm in an ER-like pattern. At later stages, 2C redistributed to a juxtanuclear site, which represents the viral replication site. Co-localization with the Golgi complex could not be observed. TMEV 2C seems to interact in vitro with reticulon 3, a highly conserved ER-associated protein. It was not possible to confirm a previously identified interaction with AKAP10, a protein kinase anchoring protein, presumably reflecting conformational constraints of the interaction. Two mutations in the AKAP10 binding site of TMEV 2C were identified, which inhibit the completion of the infectious cycle of TMEV. The intracellular changes that occur during TMEV infection were observed. Both actin filaments and microtubules may be used at early stages of infection; however both cytoskeleton components accumulate at the periphery of the cell during late stages of infection. A computer- based analysis has demonstrated that TMEV 2C is highly similar to katanin, a microtubule- severing protein, and may play a similar role in the reorganization of microtubules during infection. The Golgi complex turns from a solid, crescent-shaped organelle, into a series of punctuate fluorescent points forming an expanding balloon-like structure surrounding the concomitantly expanding site of virus replication. The remnants of the Golgi complex are finally dispersed throughout the cytoplasm. Live imaging confirmed these findings. It was observed that PKA also undergoes displacement to the cell periphery during infection. However, BIG1 seems to locate to the viral replication site during infection, suggesting it may play a role during viral replication. The localization of PKA and BIG1 in the infected cell may in part explain the observed dispersion of the Golgi complex.

Bunyamwera virus (BUNV) is the prototype of the family Bunyaviridae. It has a tripartite genome consisting of negative sense RNA segments called large (L), medium (M) and small (S). The S segment encodes the nucleocapsid protein (N) of 233 amino acids. The N protein encapsidates all three segments to form transcriptionally active ribonucleoproteins (RNPs). The aim of this project was to determine the domain map of BUNV N protein. To investigate residues in BUNV N crucial for its functionality, random and site- specific mutagenesis were performed on a cDNA clone encoding the BUNV N protein. In total, 102 single amino acid substitutions were generated in the BUNV N protein sequence. All mutant N proteins were used in a BUNV minigenome system to compare their activity to wt BUNV N. The mutant proteins displayed a wide-range of activity, from parental-like to essentially inactive. The most disruptive mutations were R94A, I118N, W134A, Y141C, L177A, K179I and W193A. Sixty-four clones carrying single substitutions in the BUNV N protein were used in the BUNV rescue system in an attempt to recover viable mutant viruses. Fifty recombinant mutant viruses were rescued and 14 N genes were nonrescuable. The 50 mutant viruses were characterized by: titration, protein labelling, western blotting, temperature sensitivity and host-restriction. Mutant viruses displayed a wide range of titers between 10³ -10⁸ pfu/ml, and three different plaque sizes large, medium and small. Protein labelling and western blotting showed that mutations in the N gene did not affect expression of the other viral genes as much as affecting N protein expression. It was demonstrated that single amino acid substitutions could alter N protein electrophoretic mobility in SDS- PAGE (e.g. P19Q and L53F). Temperature sensitivity tests showed that recombinant viruses N74S, S96S, K228T and G230R were ts, growing at 33˚C but not at 37˚C or 38˚C, while the parental virus grew at all temperatures. Using the northern blotting technique, mutant viruses N74S and S96G were shown to have a ts defect in genome-synthesis (late replication step), while mutant viruses K228T and G230R had a ts defect in antigenome- synthesis (early replication step). Host-restriction experiments were performed using 5 different cell lines (Vero-E6, BHK-21, 2FTGH-V, A549-V and 293-V). Overall, the parental virus grew similarly in all cell lines. Likewise, the majority of mutant viruses follow this pattern except mutant virus Y23A. It showed a 100-fold reduction in titer in 2FTGH-V cells. Comparing the ratios of intracellular and extracellular particles revealed that only 15% of the total virus particles of mutant Y23A was released as extracellular particles compared to 30% of the parental virus. Fourteen N genes were nonrescuable. They were characterized by (i) their activity in the BUNV minigenome system, (ii) their activity in BUNV packaging assay, (iii) their ability to form multimers, (iv) their ability to interact with L protein, and (v) their impact on RNA synthesis. In summary, BUNV N protein was shown to be multi-functional and involved in the regulation of virus transcription and replication, RNA synthesis and assembly, via interactions with the viral L polymerase, RNA backbone, itself or the viral glycoproteins.

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Master / Master of Philosophy