published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Vaccinia virus encodes at least eight proteins that are modified post-translationally by the addition of a 16-carbon saturated fatty acid through thioester linkage to cysteine residues. This is referred to as palmitylation of proteins. The purpose of this work was to gain an understanding of palmitylation, focusing on what defined the substrate for the modification, and the biological relevance of protein palmitylation in the vaccinia virus life cycle. A systematic approach was taken to identify the genes in vaccinia virus that encode these proteins. We found that vaccinia virus palmitylproteins are of the "late" temporal class, associate with intracellular membranes, and are specific for a particular form of the infectious virion. These criteria were used to narrow the number of genes expressed by vaccinia virus that potentially encode palmitylproteins. The "candidate" palmitylprotein genes were cloned and transiently expressed in mammalian tissue culture cells and analyzed for incorporation of palmitic acid. In addition to three previously identified vaccinia virus palmitylproteins, three new palmitylproteins were identified. The six known palmitylprotein genes were mutated to determine the site of modification, leading to the identification of the modification site for four of the six proteins. One of the proteins, p37, was analyzed further for biological significance of the palmitate modification. A recombinant vaccinia virus was constructed that did not express the wild-type palmitylated form of p37, but expressed a nonpalmitylated form of the protein instead. This virus was severely inhibited from proceeding past a particular morphogenetic stage, leading to an attenuated phenotype in tissue culture systems. Although the expression of the nonpalmitylated protein appeared normal compared to the wild-type protein, the lack of the palmityl moiety resulted in the loss of a targeting signal that directed the protein to its normal intracellular location. By this work, significant contributions have been made toward understanding the process of protein palmitylation. We have identified, at least for vaccinia virus, primary structural determinants specifying the modification, leading to the identification of a palmitylation motif. Considering the attenuated phenotype of the mutant virus, our conclusion is that palmitylation is necessary for biological function, at least for p37. / Graduation date: 1999

The attachment of myristic acid to the N-terminal glycine residue of many eukaryotic and viral proteins is often essential for the acquisition of the protein's biological activity. Vaccinia virus (VV), the prototype member of the Poxviridae, expresses several myristylated proteins during the course of infection. Only one of these proteins, L1R, has been identified and characterized. Experiments were performed to identify and analyze four additional VV myristylproteins. These proteins were identified as the A-type inclusion protein (92 kDa), G9R (39 kDa), A16L (36 kDa), and E7R (17 kDa). The latter three proteins were shown to be myristylated on an N-terminal glycine residue. Additional studies demonstrated that both A16L and E7R are soluble proteins, unlike L1R, which is a constituent of the viral envelope. Furthermore, A16L could not be detected in either purified extracellular enveloped virus (EEV) or in intracellular mature virus (IMV). These are the two predominant forms of infectious virions produced during a VV infection. E7R was detected in EEV and, to a lesser extent, in IMV. Unlike the other proteins, the amino terminal sequence of the A-type inclusion protein did not fit the consensus sequence for N-myristylation (M-G-X-X-X-S/T/A/C/N), suggesting that it was internally myristylated. A combination of studies revealed that the protein is both myristylated and palmitylated. Addition of each acyl group could be separated temporally: myristylation occured co-translationally, while palmitylation occurred post-translationally. Genetic analyses of lysine doublets and arginine/lysine doublets within the A-type inclusion protein indicated that these sites are not utilized for myristylation. This is in contrast to the precursors of TNFoc and Ilia which are internally-myristylated on a lysine doublet. It is not clear why this protein would be both myristylated and palmitylated. Only class four palmitylproteins, such as the Src family of proteins, have been shown to be both myristylated and palmitylated. The A-type inclusion protein expressed by cowpox virus forms a large symmetrical matix in the cytoplasm of infected cells and generally contains mature virions. It is possible, therefore, that the function of acylation may be to stabilize the protein matrix or to assist in occlusion of enveloped virus particles. / Graduation date: 1998

Acquired Immunodeficiency Syndrome (AIDS) and tuberculosis (TB) have posed diagnostic and therapeutic challenges globally. Nowadays, it is estimated that 34 millions people are living with Human Immunodeficiency Virus (HIV). About 2 millions of people die from AIDS-related causes currently in each year. Tuberculosis is the most common presenting illness and leading cause of death among AIDS patients. Emerging studies suggest that HIV and Mycobacterium tuberculosis (Mtb), the causative pathogen of TB, act synergistically to accelerate decline of immune functions and cause the death. Mtb infection usually remains latent. Only small portion of infected individuals develops active TB. However HIV infection boosts the risk of reactivation of TB and susceptibility to new Mtb infection. In contrast, Mtb infection dysregulates cytokines production and induces HIV viral replication. Although it is well-known that HIV and Mtb potentiate each other in disease development, mechanisms of interaction of the two pathogens remain not well-elucidated. The aim of this study is to investigate the interaction of HIV viral protein Tat with mycobacteria infection, which may provide insights in the interplay between HIV and Mtb infections. HIV viral transactivator protein, Tat, plays a critical role in HIV replication; and its induction of apoptosis in CD4+ T cells contributes to immune defects. In this study, Tat was demonstrated to dysregulate immune responses against mycobacteria such as autophagy, a tightly regulated bacterial clearance mechanism. With pretreatment of the primary human blood monocyte-derived macrophages with Tat, the interferon-γ (IFN-γ)-induced Signal Transducer and Activator of Transcription-1 (STAT-1) phosphorylation was suppressed. Inhibition of STAT-1 phosphorylation ultimately led to downregulation of autophagy-associated gene, microtubule-associated protein light chain 3 (LC3) expressions. Of note, Tat was demonstrated to inhibit the colocalization of Bacillus Calmette Guerin (BCG) and IFN-γ-induced autophagosomes under fluorescent microscopy examination. In addition to the inhibition of bactericidal autophagy, Tat was found to manipulate cytokines production. Tat was demonstrated to enhance mycobacteria-induced tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production. TNF-α and IL-1β have been well-demonstrated in literatures that can limit bacterial growth. They, however, have been also shown as important contributors to the increase of HIV viral replication in HIV and mycobacteria coinfection. Mtb-induced TNF-α production can induce transcriptional activation of the HIV long terminal repeat (LTR) promoter while blocking of IL-1β production decreases HIV replication. Tat enhancement of these cytokines production may therefore contribute to the knowledge of the increased viral replication in HIV and mycobacteria coinfection. Furthermore, new microRNAs, up-and-coming fine-tuners of innate immunity, were discovered. MicroRNAs, a family of non-coding RNAs, can regulate gene expressions post-transcriptionally and control various developmental and cellular processes. They can target mRNAs of cellular signaling molecules, transcription factors or cytokines as to regulate the immunity. Herein, microRNA-1303, originally with unknown function, was shown to regulate mycobacteria-induced TNF-α production and affect the Tat enhancement of TNF-α production. Taken together, the results of this study demonstrated that HIV viral protein, Tat could dysregulate immune responses to mycobacteria. The study of the dysregulation may further elucidate the interplay between HIV and mycobacteria infections. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xv, 155 p.; also includes graphics (some col.) Includes bibliographical references (p. 147-155). Available online via OhioLINK's ETD Center

<p>The long-range aim of this thesis was to further the understanding of the mechanisms involved in virus interference. To this end, biochemical and immunological procedures were used to characterize and define the virus-specific proteins of VSV. Biological studies of homotypic and heterotypic ·interference were also carried out. </p> / Thesis / Doctor of Philosophy (PhD)

3C-like protease (3CLpro) of severe acute respiratory syndrome-coronavirus (SARS-CoV) is required for autoprocessing of the polyproteins 1a and 1ab, and is a potential target for treating coronaviral infection. To obtain a thorough understanding of its substrate preference, we created a substrate library of 19 x 8 variants by performing saturation mutagenesis on the autocleavage sequence at P5 to P3' positions. The substrate sequences were inserted between cyan and yellow fluorescent proteins so that the cleavage rates were monitored by in vitro fluorescence resonance energy transfer (FRET). The relative cleavage rate for different substrate sequences was correlated with various structural properties. P5 and P3 positions prefer residues with high beta-sheet propensity P4 prefers small hydrophobic residues: P2 prefers hydrophobic residues without beta-branch. Gln is the best residue at P1 position, but observable cleavage can be detected with His and Met substitutions. P1' position prefers small residues, while P2' and P3' positions have no strong preference on residue substitutions. Noteworthy, solvent exposed sites such as P5, P3 and P3' positions favour positively charged residues over negatively charged one, suggesting that electrostatic interactions may play a role in catalysis. A super-active substrate, which combined the preferred residues at P5 to P1 positions, was found to have 2.8 fold higher activity than the wild-type sequence. / Inhibition of SARS-CoV 3CLpro proteolytic activity suppresses virion replication and virus-induced cytopathic effects. Peptidomimetic inhibitors with nitrile warheads, which inhibit Cys protease activity, have been applied for clinical therapy. To investigate whether the nitrile group can target 3CLpro, a series of nitrile-based peptidomimetic inhibitors with various protective groups, peptide length and peptide sequences were synthesized. Inhibitor potency in terms of IC50 and Ki values was determined by FRET assay. Most of these nitrile-based inhibitors in micromolar range can significantly reduce 3CLpro activity. The most potent inhibitor is the tetrapeptidomimetie inhibitor linked with carbobenzyloxy (cbz) group 'cbz-AVLQ-CN' with IC50 and Ki values of 5.9 +/- 0.6 muM and 0.62 +/- 0.11 muM respectively. Crystal structures of 3CLpro-inhibitor complexes demonstrated that nitrite warhead covalently bonded to Cys145, while P1 -- P4 residues interacted with 3CLpro as substrate bound. The cbz group in 'cbz-AVLQ-CN' flipped into a cavity of Gu166 -- Pro168, providing an extra binding force to enhance inhibitor potency. In conclusion, the nitrile-based peptidomimetic inhibitor with cbz group is a convincing model for drug development. / Substrate specificities of various 3CLpro were further investigated by using the substrate library of SARS-CoV 3CLpro. Among various viral strains, the proteases of HCoV-NL63, HCoV-OC43 and infectious bronchitis virus (IBV) were selected from group I, IIa and III respectively for specificity profiling. Their proteolytic rates against 19 x 8 variants were obtained by FRET assay, and correlated with structural properties of substituting residues. Like SARS-CoV 3CLpro in group IIb, these 3CLpro consistently prefer small hydrophobic P4 residues, positively charged P3 residues, hydrophobic P2 residues without beta-branch, P1-Gln and small P1' residues. These proteases also tend to accommodate P5 and P3' residues with positive charge, and P2' residues with small size. In contrast, their preferences on secondary structure are diverse. Correlation was found between IBV 3Clpro activity and beta-sheet propensity at P5 position, while no strong correlation with secondary structure propensities was observed in HCoV-NL63 and HCoV-0C43. Collectively, all 3CLpro share universal preferences on charge, side chain volume and hydrophobicity, but not secondary structure. Their relative activities against universal and specific super-active substrates were elevated to 1.4 -- 4.3, showing synergetic effects by combining preferred residues. These substrates were examined by group I HCoV-229E and group IIa HCoV-HKU1 in parallel. Their activities were highly comparable to those of other group members. / Chuck, Chi Pang. / Adviser: Chi-Cheong Wan. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves [179]-187). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.