published_or_final_version / abstract / Zoology / Master / Master of Philosophy

Abstract not available

CAR (for C&barbelow;oxsackievirus and A&barbelow;denovirus R&barbelow;eceptor) is a novel member of the Ig superfamily, which has recently been identified as a high affinity receptor for both Coxsackievirus and certain adenovirus (AV) serotypes. Virus bound by CAR is believed to be passed to integrins which bind an RGD (Arg-Gly-Asp) sequence in the viral penton base protein and act as secondary receptors responsible for virus internalization. / Recent studies have shown that, in integrin-expressing cells, CAR-mediated AV uptake does not require the cytoplasmic (CP) domain of CAR, presumably because virus bound to the CAR extracellular (EC) domain can be passed to integrins for subsequent internalization. It has however also been reported that CAR can directly mediate AV uptake in the absence of penton base RGD-alphav integrin interactions. I therefore attempted to determine whether the CP domain of CAR is required for CAR-mediated AV uptake in cells which do not express integrins, or in which integrin function has been blocked by RGD-containing peptide. / As CAR is the primary AV receptor and integrins are secondary AV receptors I investigated the possibility that these proteins associate in a functional complex in the cell membrane. (Abstract shortened by UMI.)

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Severe lymphopenia is a clinical feature of Severe Acute Respiratory Syndrome (SARS) patients. However, lymphocytes do not express receptor for SARS-CoV, neither the widely accepted viral receptor angiotensin converting enzyme 2 (ACE2) nor the putative receptors Dendritic Cell- and Liver/lymph-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN and L-SIGN). Our group previously showed in vitro that, SARS-CoV Spike pseudotyped particles (SARSCoVpp) could infect human B cells only when inoculated in presence of anti-SARSCoV Spike immune serum. Such observations raised concerns about the possible occurrence of antibody-dependent enhancement (ADE) of infection, a phenomenon during which a virus bounded by antibodies could gain entry into cells through mechanisms involving complement receptors or Fc receptors. Recently, we have demonstrated the participation of the human Fc gamma receptor II (hFcγRII) molecules in granting SARS-CoV an opportunity to infect human immune cells. The aim of this study was to decipher the molecular mechanism leading to antibodymediated, FcγRII-dependent infection of immune cells by SARS-CoV. By using transduction experiment, I highlighted that different members of the hFcγRII family (namely hFcγRIIA, hFcγRIIB1 and hFcγRIIB2) could confer susceptibility to ADE of SARS-CoVpp infection. I further demonstrated that purified anti-viral immunoglobulin G, but not other soluble factor(s) from heat-inactivated immune serum, was the determinant for occurrence of ADE infection. Additionally, with the development of a cell-cell fusion assay, I illustrated that in contrast to the ACE2- dependent pathway, ADE infection did not occur at the plasma membrane, but rather require internalization of virus/antibodies immune complexes by the target cells. In line with this hypothesis, my results using a panel of FcγRII-expressing mutants demonstrated that binding of immune complexes to cell surface FcγRII was a prerequisite but was not sufficient to trigger ADE infection. In these experiments, only FcγRII signaling-competent constructions conferred susceptibility to ADE of SARS-CoVpp infection. Altogether my results point toward a role of the anti-SARS-CoV Spike IgG in vitro in granting SARS-CoV an opportunity to infect cells bearing signaling-competent FcγRII receptors. If further confirmed, such observations could have implications for understanding SARS-CoV tropism and SARS pathogenesis, as well as warrant for careful design of SARS vaccines and immunotherapy based on anti-viral antibodies. / published_or_final_version / Microbiology / Master / Master of Philosophy

Influenza infections continue to be a global health concern that causing both seasonal epidemics and unpredictable pandemics. Hemagglutinin (HA) and Neuraminidase (NA) are the two major surface glycoproteins of influenza viruses, which are important for their host cell sialic acid (Sia) receptor binding and cleaving activities. Although numerous methods have been developed to study the HA and NA interactions with sialic acid, x-ray crystallography remained the only method to provide detailed information at atomic resolution. The aim of this study is to develop and evaluate a novel strategy for the investigation of influenza virus-receptor interactions, which is able to provide information about an interaction down to atomic resolution. Influenza virus-like particles (VLPs) containing HA and NA separately were developed and it was reported here for the first time that sole expression of NA in mammalian cell led to VLP formation. Characterization of these VLPs demonstrated that they are non-infectious, but morphologically and biochemically mimic the native viruses. Therefore the VLPs can be regarded as an ideal research model to study the HA-Sia interaction without the interference of NA, or vice versa. Saturation transfer difference (STD) NMR spectroscopy is a state-of-the-art technology to determine how a binding-ligand interacts with its target protein. Modification of STD-NMR methodology was performed to adapt the technique to influenza VLP system. HA-Sia interaction was investigated in great detail and group epitope mapping of the interacting ligands was performed by analyzing the STD-NMR spectra. The data obtained are in a good agreement with the well established crystallography technique, reflecting the reliability of the STD-NMR technology. Regarding the NA-Sia interaction, my data demonstrated that substrate-hydrolysis specificity of NA is dependent on the binding of NA to those ligands. In addition, using competition experiments with NA inhibitor, a secondary sialic acid binding site was detected. It is the first direct experimental evidence that confirms avian, seasonal human and human pandemic swine-origin influenza virus N1 neuraminidases exhibit a distinct secondary binding site. In conclusion, here I presented a novel interdisciplinary strategy using VLP and NMR technology to study the interaction of influenza virus with its receptor. This method is unique in its ability to provide detailed information on the HA and NA interactions with sialic acid leading to group epitope mapping of the binding ligands, which will help us not only to understand the virus tropism but also to define new therapeutic targets. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Chuck Chi-pang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 138-160). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Abstract --- p.iii / 摘要 --- p.v / Contents --- p.vii / List of Figures --- p.xi / List of Tables --- p.xiii / Abbreviations --- p.xiv / Acknowledgement --- p.xviii / Introduction / Chapter 1. --- Background / Chapter 1.1 --- SARS / Chapter 1.1.1 --- Outbreak and Influence --- p.1 / Chapter 1.1.2 --- Clinical Features --- p.4 / Chapter 1.2 --- SARS-CoV / Chapter 1.2.1 --- Genomic Organization --- p.5 / Chapter 1.2.2 --- Morphology --- p.7 / Chapter 1.2.3 --- Phylogenetic Analysis --- p.9 / Chapter 1.3 --- S Glycoprotein / Chapter 1.3.1 --- Functional Roles --- p.11 / Chapter 1.3.2 --- Structure and Functional Domains --- p.12 / Chapter 1.3.3 --- Interacting Partners --- p.15 / Chapter 1.3.4 --- Viral Entry Mechanism --- p.17 / Chapter 1.4 --- Aim of Study / Chapter 1.4.1 --- Mismatch of SARS-CoV Tissue Tropism and Tissue Distribution of ACE2 --- p.20 / Chapter 1.4.2 --- Presence of Other Interacting Partner(s) --- p.22 / Chapter 1.4.3 --- Significance of the Study Materials and Methods --- p.22 / Chapter 2. --- Plasmid Construction / Chapter 2.1 --- Fragment Design / Chapter 2.1.1 --- Functional Domain Analysis --- p.23 / Chapter 2.1.2 --- Secondary Structure and Burial Region Predictions --- p.24 / Chapter 2.2 --- Vector Amplification / Chapter 2.2.1 --- E. coli Strain DH5a Competent Cell Preparation --- p.30 / Chapter 2.2.2 --- Transformation of E. coli --- p.30 / Chapter 2.2.3 --- Small-scale Vector Amplification --- p.31 / Chapter 2.3 --- Cloning of DNA Fragments into Various Vectors / Chapter 2.3.1 --- Primer Design --- p.32 / Chapter 2.3.2 --- DNA Amplification --- p.35 / Chapter 2.3.3 --- DNA Purification --- p.35 / Chapter 2.3.4 --- "Restriction Enzyme Digestion, Ligation and Transformation" --- p.36 / Chapter 2.3.5 --- Colony PCR --- p.37 / Chapter 2.4 --- DNA Sequence Analysis / Chapter 2.4.1 --- Primer Design --- p.35 / Chapter 2.4.2 --- DNA Amplification and Purification for DNA Sequence Analysis --- p.39 / Chapter 2.4.3 --- Sequence Detection and Result Analysis --- p.40 / Chapter 3. --- "Protein Expression, Purification and Analysis" / Chapter 3.1 --- Protein Expression in E. coli / Chapter 3.1.1 --- Molecular Weight and pI Predictions --- p.41 / Chapter 3.1.2 --- Glycerol Stock Preparation --- p.41 / Chapter 3.1.3 --- Protein Expression Induction --- p.41 / Chapter 3.1.4 --- Protein Extraction --- p.42 / Chapter 3.1.5 --- Affinity Chromatography --- p.42 / Chapter 3.1.6 --- Removal of GroEL --- p.43 / Chapter 3.1.7 --- Protein Solubilization and Refolding --- p.44 / Chapter 3.2 --- Protein Expression in P. pastoris / Chapter 3.2.1 --- Large-scale Plasmid Amplification --- p.46 / Chapter 3.2.2 --- Restriction Enzyme Digestion and Ethanol Precipitation --- p.47 / Chapter 3.2.3 --- Preparation of KM71H Competent Cells --- p.47 / Chapter 3.2.4 --- Electroporation --- p.48 / Chapter 3.2.5 --- Colony PCR --- p.48 / Chapter 3.2.6 --- Protein Expression Induction and Time Course Study --- p.49 / Chapter 3.2.7 --- Deglycosylation --- p.49 / Chapter 3.3 --- Protein Analysis / Chapter 3.3.1 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.50 / Chapter 3.3.2 --- Western Blotting --- p.50 / Chapter 3.3.3 --- Mass Spectrometry --- p.51 / Chapter 3.3.4 --- N-terminal Sequencing --- p.52 / Chapter 3.3.5 --- Size Exclusion Chromatography --- p.52 / Chapter 4. --- Identification of Interacting Partner(s) / Chapter 4.1 --- VeroE6 Preparation / Chapter 4.1.1 --- Cell Culture --- p.53 / Chapter 4.1.2 --- Protein Extraction and Western Blotting --- p.53 / Chapter 4.2 --- Pull-down Assay --- p.54 / Chapter 4.3 --- Two-dimensional Gel Electrophores --- p.is / Chapter 4.3.1 --- Isoelectric Focusing --- p.56 / Chapter 4.3.2 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.56 / Chapter 4.3.3 --- Silver Staining --- p.57 / Chapter 4.4 --- Mass Spectrometry / Chapter 4.4.1 --- Destaining --- p.58 / Chapter 4.4.2 --- In-gel Digestion --- p.58 / Chapter 4.4.3 --- Desalting by Zip-tip --- p.59 / Chapter 4.4.4 --- Loading Sample --- p.59 / Chapter 4.4.5 --- Peptide Mass Detection and Data Analysis --- p.59 / Results / Chapter 5. --- S Protein Expression / Chapter 5.1 --- Plasmid Construction --- p.61 / Chapter 5.2 --- Molecular Weight and pi Predictions --- p.63 / Chapter 5.3 --- Protein Expression and Optimization in E. coli / Chapter 5.3.1 --- "Comparison of Expression Levels, Solubility and Purities of S Protein Fragments" --- p.64 / Chapter 5.3.2 --- "Alteration of the Solubility in Various Cell Strains, Expression Conditions and Lysis Buffers" --- p.68 / Chapter 5.3.3 --- Identification and Remove of the non-target proteins --- p.72 / Chapter 5.3.4 --- Unfolding and Refolding --- p.79 / Chapter 5.4 --- Protein Expression and Optimization in P. pastoris / Chapter 5.4.1 --- "Expression Levels, Solubility and Purities of Various S Protein Fragments" --- p.85 / Chapter 5.4.2 --- Characterization of De-N-glycosylated Recombinant Proteins --- p.89 / Chapter 6. --- Identification of Interacting partners / Chapter 6.1 --- Practicability of Pull-down Assay / Chapter 6.1.1 --- ACE2 Extraction --- p.95 / Chapter 6.1.2 --- Pull-down of ACE2 by the P. pastoris-expressed recombinant RBD --- p.96 / Chapter 6.2 --- Pull-down Assay and Two-dimensional Gel Electrophoresis --- p.97 / Chapter 6.3 --- Identification of Putative Interacting Partners by MALDI-TOF-TOF --- p.107 / Chapter 7. --- Discussion / Chapter 7.1 --- S Protein Expression in E. coli / Chapter 7.1.1 --- Improving Recombinant Protein Expression Level and Solubility --- p.114 / Chapter 7.1.2 --- S Recombinant Protein Bound by GroEL --- p.117 / Chapter 7.2 --- S Protein Expression in P. pastoris / Chapter 7.2.1 --- Advantages of Using P. pastoris --- p.119 / Chapter 7.2.2 --- Variation of S Fragment Expression Levels --- p.120 / Chapter 7.2.3 --- Sizes of S Protein Fragments --- p.123 / Chapter 7.3 --- Identification of Interacting Partners / Chapter 7.3.1 --- Relationship between S Protein and Putative Interacting Partners --- p.124 / Chapter 7.3.2 --- Failure of Finding ACE2 --- p.125 / Chapter 7.3.2 --- Difficulty in the Identification of Protein Spots --- p.126 / Chapter 7.4 --- Conclusion --- p.131 / Chapter 7.5 --- Future Perspective --- p.132 / Chapter 8. --- Appendix --- p.133 / Chapter 9. --- References --- p.138

Japanese encephalitis virus (JEV) causes viral encephalitis in new born and young adults that is prevalent in different parts of India and other parts of South East Asia with an estimated 6000 deaths per year. JEV is a single stranded RNA virus that belongs to the Flavivirusgenus of the family Flaviviridae. It is a neurotropic virus which infects the central nervous system (CNS). The virus follows a zoonotic life-cycle involving mosquitoes and vertebrates, chiefly pigs and ardeid birds, as amplifying hosts. Humans are dead end hosts. After entry into the host following a mosquito bite, JEV infection leads to acute peripheral leukocytosis in the brain and damage to Blood Brain Barrier (BBB). The exact role of the endothelial cells during CNS infection is still unclear. However, disruption of this endothelial barrier has been shown to be an important step in entry of the virus into the brain. Humoral and cell mediated immune responses during JEV infection have been intensively investigated. Previous studies from our lab have shown the activation of cytotoxic T-cells (CTLs) upon JEV infection. MHC molecules play pivotal role in eliciting both adaptive (T-cells) and innate (NK cells) immune response against viral invasion. Many viruses such as HIV, MCMV, HCMV, AdV and EBV have been found to decrease MHC expression upon infection. On the contrary, flaviviruses like West Nile Virus (WNV) have been found to increase MHC-I and MHC-II expression. More recently, data from our lab has shown that JEV infection can lead to upregulation of mouse non-classical MHC class Ib molecules like Qb1, Qa1 and T-10 along with classical MHC molecules. Non-classical MHC molecules are important components of the innate and adaptive immune systems. Non-classical MHC molecules differ from their classical MHC class I counterparts by their limited polymorphism, restricted tissue distribution and lower levels of cell surface expression. Human classical MHC class I molecules are HLA-A, -B and –C while non-classical MHC Class Ib molecules are HLA-E, -G and –F. HLA-E, the human homologue of the mouse non-classical MHC molecule, Qa-1b has been shown to be the ligand for the inhibitory NK, NKG2A/CD94 and may bridge innate and adaptive immune responses. In this thesis, we have studied the expression of human classical class I molecules HLA-A, -B, -C and the non-classical HLA molecule, HLA-E in immortalized human brain microvascular endothelial cells (HBMEC), human endothelial like cell line ECV304 (ECV), human glioblastoma cell line U87MG and human foreskin fibroblast cells (HFF). We observed an upregulation of classical HLA molecules and HLA-E mRNA in endothelial and fibroblast cells upon JEV infection. This mRNA increase also resulted in upregulation of cell surface classical HLA molecules and HLA-E in HFF cells but not in both the human endothelial cell lines, ECV and HBMECs. Release of soluble classical HLA molecules upon cytokine treatment has been a long known phenomenon. Recently HLA-E has also been shown to be released as a 37 kDa protein from endothelial cells upon cytokine treatments. Our study suggests that JEV mediated upregulation of classical HLA and HLA-E upregulation leads to release of both Classical HLA molecules and HLA-E as soluble forms in the human endothelial cell lines, ECV and HBMEC. This shedding of sHLA-E from human endothelial cells was found to be mediated by matrix metalloproteinase (MMP) proteolytic activity. MMP-9, a protease implicated in release of sHLA molecules was also found to be upregulated upon JEV infection only in endothelial cell lines but not in HFF cells. Our study provides evidence that the JEV mediated solubilisation of HLA-E could be mediated by MMP-9. Further, we have tried to understand the role of the MAPK pathway and NF-κB pathway in the process of HLA-E solubilisation by using specific inhibitors of these pathways during JEV infection of ECV cells. Our data suggests that release of sHLA-E is dependent on p38 and JNK pathways while ERK 1/2 and NF-κB pathway only had a minor role to play in this process. Treatment of endothelial cells with TNF-α, IL-1β and IFN-γ is known to result in release of sHLA-E. In addition to TNF-α and IFNtreatment, we observed that activating agents like poly (I:C), LPS and PMA also resulted in the shedding of sHLA-E from ECV as well as U87MG but not from HFF cells. Treatment of endothelial cells with IFN-β, a type-I interferon also led to release of sHLA-E. IFN-γ, a type II interferon and TNF-α are known to show additive increase in solubilisation of HLA-E. We studied the interaction between type I interferon, IFN-β and TNF-α with regard to shedding of sHLA- E. Both IFNand TNF, when present together caused an additive increase in the shedding of sHLA-E. These two cytokines were also found to potentiate the HLA-E and MMP-9 mRNA expression. Hence, our data suggest that these two cytokines could be working conjunctly to release HLA-E, when these two cytokines are present together as in the case of virus infection of endothelial cells. HLA-E is known to be a ligand for NKG2A/CD94 inhibitory receptors present on NK and a subset of T cells. Previous reports have suggested that NKG2A/CD94 mediated signaling events could inhibit ERK 1/2 phosphorylation leading to inhibition of NK cell activation. IL-2 mediated ERK 1/2 phosphorylation is known to play a very important role in maintenance and activation of NK cells. We studied the effects of sHLA-E that was released, either by JEV infection or IFN-γ treatment on IL-2 mediated ERK 1/2 phosphorylation in two NK cell lines, Nishi and NKL. The soluble HLA-E that was released upon JEV infection was functionally active since it inhibited IL-2 and PMA induced phosphorylation of ERK 1/2 in NKL and Nishi cells. Virus infected or IFN-γ treated ECV cell culture supernatants containing sHLA-E was also found to partially inhibit IL-2 mediated induction of CD25 molecules on NKL cells. CD25 is a component of the high affinity IL-2 receptor and hence could play an important role in proliferation and activation of NK cells. sHLA-E was also found to inhibit IL-2 induced [3H]-thymidine incorporation suggesting that, similar to cell surface expressed HLA-E, sHLA-E could also inhibit the proliferation and activation of NK cells. In summary, we found that establishment of JEV infection and production of cytokines like IFN-β, TNF-α, IL-6 along with MMP-9 in human endothelial cells. These cytokines may also indirectly lead to the reported damage and leukocyte infiltration across infected and uninfected vicinal endothelial cells. The increased surface expression of HLA-E in fibroblast and release of sHLA and sHLA-E molecules from endothelial cells may have an important immunoregulatory role. HLA-E is an inhibitory ligand for NKG2A/CD94 positive CD8+ T and NK cells. Hence our finding that sHLA-E can inhibit NK cell proliferation suggests an immune evasive strategy by JEV.