published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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No description available.

Vaccinia virus (VV) encodes at least six proteins that are modified by the addition of a 14-carbon saturated fatty acid through an amide linkage and at least eight proteins that are modified post-translationally by the addition of a 16-carbon saturated fatty acid through linkage to cysteine residues. These post-translational modifications are referred to as myristylation and palmitylation, respectively. The purpose of this work was to further characterize the known myristylproteins and to define a consensus motif for the palmitylation of a protein so that we could identify and begin the characterization of new palmitylproteins. Through this work we have identified a loosely conserved consensus motif that directs the palmitylation of a protein. Using the VV palmitylprotein p37, we characterized this motif and then used it in the identification three new VV palmitylproteins. We have also determined the membrane orientation of the VV myristylproteins, L1R, within the intracellular mature virus (IMV) particle. Hydrophobicity plot analysis identified two possible membrane orientations based on two putative transmembrane domains. Through transient expression data, L1R was determined to span the IMV membrane twice, with both the amino and carboxy termini being on the lumen side. Three lac recombinant viruses which are inducible for the A16L, E7R, and G9R open reading frames were created and analyzed using a newly developed vector system that fuses the green fluorescent protein to the neomycin resistance gene. Propagation of these viruses in the absence of the inducer IPTG determined that these genes are essential to VV replication. We have identified and characterized the primary structural determinants specifying the modification of a protein by palmitate, and have identified three new VV palmitylproteins. In addition, the membrane orientation of the VV myristylprotein L1R was deduced, which can enable the construction of better recombinant vaccines through efficient antigen presentation. Lastly, we have developed a technique and vector system to easily create and isolate VV recombinants for characterization. This system enabled us to further characterize the A16L, E7R, and G9R myristylproteins. / Graduation date: 2002

Graduation date: 1994

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 110-120). Also available on the World Wide Web.

Thesis (M. Phil.)--University of Hong Kong, 2006f. / Title proper from title frame. Also available in printed format.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 100-129).

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 93-125). Also available in print.

Influenza has resided with the human race long before we have any written record of it. Its death toll is one of the highest among all other virus. In recent history, pandemic outbreaks of influenza have caused even more deaths. Therefore it is of great importance that we focus our resources on understanding its viral components and functions. In this study, chimeric mutagenesis was used to investigate the antigenic variance of antibodies I50C and I131B on H1N1 and H5N1 NP. It was revealed from previous study that antibodies I50C and I131B can detect H1N1 NP but not H5N1 NP. NP from influenza A strains A/Puerto Rico/8/1934 (PR8), A/Vietnam/3046/2004 (3046) and A/Indonesia/5/2005 (indo) were used to construct the NP chimeric mutants. Nucleotide sequence from the region spanning from bp 484-506 was chosen as template to design the primers for obtaining head and tail fragments which were components of the NP constructs. Results showed that antibodies I50C and I131B can only detect NP constructs with PR8 head fragments regardless of any tail fragments, and cannot detect NP constructs with 3046 or indo head fragments. Therefore the binding epitope on H1N1 NP tested by the antibodies I50C and I131B is deduced to be within bp 1-506. In order to understand the dynamics of host and viral nuclear proteome during the influenza A infection, the pulse SILAC (Stable Isotope Labeling of Amino acids on Cell lines) MS-proteomic approach was adopted. More and more research studies are MS-proteomic based as people recognize that proteins truly define the outcome of a cell, with fewer limitations by solely looking at the genome. The pulse SILAC technique involves incorporating “light” isotope-labeled amino acids such as arginine and lysine into cells’ proteins prior infection experiment. While the cells are under influenza infection, “heavy” isotope-labeled amino acids were used to label the cells 2 hour prior each harvesting time points. Since only proteins synthesized within the 2 hour windows are “heavy” isotope labeled, relative quantification of “heavy” isotope to “light” isotope by mass spectrometry (MS) can be calculated into heavy:light (H/L) ratios. Through this method we can know to what extents are the proteins affected and whether the effect is global or specific. Together with the temporal degree of the data, we can reveal the dynamics of host and viral nuclear proteome during the influenza A infection. MS results of the influenza viral proteins agree with the viral gene expression profile upon infections and corresponded well with time of viral protein expressions during influenza pathogenesis investigated by other research groups. A number of proteins were identified to increase in turnover rate at 8 hpi. This gives a partial view of up-regulated functions inside the nucleus during influenza A infection at that stage. The up-regulated proteins represent cellular functions that are related to: energy homeostasis, microtubule-dependent transport, DNA coiling regulation, transcription regulation, translation regulation and protein folding. The findings of this research present more information to understand influenza virus and provide a stepping stone for fellow influenza researchers. / published_or_final_version / Pathology / Master / Master of Philosophy