Browsing by Author "Chou, Chih-Fong"

Now showing 1 - 8 of 8
  • Thumbnail Image
    Item
    Amino acids 1055 to 1192 in the S2 Region of severe acute respiratory syndrome Coronavirus S Protein induce neutralizing antibodies: Implications for the development of vaccines and antiviral agents
    (American Society for Microbiology, 2005) Keng, Choong-Tat; Zhang, Aihua; Shen, Shuo; Lip, Kuo-Ming; Fielding, Burtram C.; Tan, Timothy H.P.; Chou, Chih-Fong; Loh, Chay Boon; Wang, Sifang; Fu, Jianlin; Yang, Xiaoming; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
    The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-S 10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
  • Thumbnail Image
    Item
    Genetic lesions within the 3a gene of SARS-CoV
    (BioMed Central, 2005) Tan, Timothy H.P.; Barkham, Timothy; Fielding, Burtram C.; Chou, Chih-Fong; Shen, Shuo; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
    A series of frameshift mutations within the 3a gene has been observed in culture-derived severe acute respiratory syndrome coronavirus (SARS-CoV). We report here that viral RNA from clinical samples obtained from SARS-CoV infected patients also contains a heterogeneous population of wild-type and mutant 3a transcripts.
  • Thumbnail Image
    Item
    Monoclonal Antibodies targeting the HR2 Domain and the region immediately upstream of the HR2 of the S Protein neutralize in Vitro Infection of Severe Acute Respiratory Syndrome Coronavirus
    (American Society for Microbiology, 2006) Lip, Kuo-Ming; Shen, Shuo; Yang, Xiaoming; Keng, Choong-Tat; Zhang, Aihua; Oh, Hsueh-Ling Janice; Li, Zhi-Hong; Hwang, Le-Ann; Chou, Chih-Fong; Fielding, Burtram C.; Tan, Timothy H.P.; Mayrhofer, Josef; Falkner, Falko G.; Fu, Jianlin; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
    We have previously shown that an Escherichia coli-expressed, denatured spike (S) protein fragment of the severe acute respiratory coronavirus, containing residues 1029 to 1192 which include the heptad repeat 2 (HR2) domain, was able to induce neutralizing polyclonal antibodies (C. T. Keng, A. Zhang, S. Shen, K. M. Lip, B. C. Fielding, T. H. Tan, C. F. Chou, C. B. Loh, S. Wang, J. Fu, X. Yang, S. G. Lim, W. Hong, and Y. J. Tan, J. Virol. 79:3289–3296, 2005). In this study, monoclonal antibodies (MAbs) were raised against this fragment to identify the linear neutralizing epitopes in the functional domain and to investigate the mechanisms involved in neutralization. Eighteen hybridomas secreting the S protein-specific MAbs were obtained. Binding sites of these MAbs were mapped to four linear epitopes. Two of them were located within the HR2 region and two immediately upstream of the HR2 domain. MAbs targeting these epitopes showed in vitro neutralizing activities and were able to inhibit cell-cell membrane fusion. These results provide evidence of novel neutralizing epitopes that are located in the HR2 domain and the spacer region immediately upstream of the HR2 of the S protein.
  • No Thumbnail Available
    Item
    A novel cell-based binding assay system reconstituting interaction between SARS-CoV S protein and its cellular receptor
    (Elsevier, 2005) Chou, Chih-Fong; Shen, Shuo; Tan, Yee-Joo; Fielding, Burtram C.; Tan, Timothy H.P.; Fu, Jianlin; Xu, Qiurong; Lim, Seng Gee; Hong, Wanjin
    Severe acute respiratory syndrome (SARS), a life-threatening disease, is caused by the newly identified virus SARS coronavirus (SARSCoV). In order to study the spike (S) protein of this highly contagious virus, we established a clonal cell-line, CHO-SG, from the Chinese hamster ovary cells that stably expresses C-terminally EGFP-tagged SARS-CoV S protein (S-EGFP). The ectodomain of the S glycoprotein is localized on the surface of CHO-SG cells with N-acetyl-glucosamine-terminated carbohydrate structure. CHO-SG cells associated tightly with Vero E6 cells, a SARS-CoV receptor (ACE2) expressing cell-line, and the interaction remained stable under highly stringent condition (1MNaCl). This interaction could be blocked by either the serum from a SARS convalescent patient or a goat anti-ACE2 antibody, indicating that the interaction is specific. A binding epitope with lesser degree of glycosylation and native conformation was localized by using rabbit anti-sera raised against five denatured recombinant S protein fragments expressed in Escherichia coli. One of the sera obtained from the fragment encompassing amino acids 48-358 significantly blocked the interaction between CHO-SG and Vero E6 cells. The region is useful for studying neutralizing antibodies in future vaccine development. This paper describes an easy and safe cell-based assay suitable for studying the binding between SARS-CoV S protein and its receptor.
  • Thumbnail Image
    Item
    Over-expression of severe acute respiratory syndrome coronavirus 3b protein induces both apoptosis and necrosis in Vero E6 cells
    (Elsevier, 2006) Khan, Sehaam; Fielding, Burtram C.; Tan, Timothy H.P.; Chou, Chih-Fong; Shou, Shen; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
    The genome of the severe acute respiratory syndrome coronavirus encodes for eight accessory viral proteins with no known homologues in other coronaviruses. One of these is the 3b protein, which is encoded by the second open reading frame in subgenomic RNA 3 and contains 154 amino acids. Here, a detailed time-course study was performed to compare the apoptosis and necrosis profiles induced by full-length 3b, a 3b mutant that was deleted by 30 amino acids from the C terminus (3b 124-154) and the classical apoptosis inducer, Bax. Our results showed that Vero E6 cells transfected with a construct for expressing 3b underwent necrosis as early as 6 h after transfection and underwent simultaneous necrosis and apoptosis at later time-points. At all the time-points analysed, the apoptosis induced by the expression of 3b was less than the level induced by Bax but the level of necrosis was comparable. The 3b 124-154 mutant behaves in a similar manner indicating that the localization of the 3b protein does not seems to be important for the cell-death pathways since full-length 3b is localized predominantly to the nucleolus, while the mutant is found to be concentrated in the peri-nuclear regions. To our knowledge, this is the first report of the induction of necrosis by a SARS-CoV protein.
  • Thumbnail Image
    Item
    Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers
    (American Society for Microbiology, 2004) Tan, Yee-Joo; Goh, Phuay-Yee; Fielding, Burtram C.; Shen, Shuo; Chou, Chih-Fong; Fu, Jianlin; Leong, Hoe Nam; Leo, Yee Sin; Ooi, Eng-Eong; Ling, Ai Ee; Lim, Seng Gee; Hong, Wanjin
  • Thumbnail Image
    Item
    The Severe Acute Respiratory Syndrome Coronavirus 3a Protein Up-Regulates Expression of Fibrinogen in Lung Epithelial Cells
    (American Society for Microbiology, 2005) Tan, Yee-Joo; Tham, Puay-Yoke; Chan, Daphne Z. L.; Chou, Chih-Fong; Shen, Shuo; Fielding, Burtram C.; Tan, Timothy H.P.; Lim, Seng Gee; Hong, Wanjin
    Here we analyzed the gene expression profile of cells that stably express the severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein to determine its effects on host functions. A lung epithelial cell-line, A549, was chosen for this study because the lung is the primary organ infected by SARS-CoV and fatalities resulted mainly from pulmonary complications. Our results showed that the expression of 3a up-regulates the mRNA levels of all three subunits, A , B , and , of fibrinogen. Consequently, the intracellular levels as well as the secretion of fibrinogen were increased. We also observed increased fibrinogen levels in SARS-CoV-infected Vero E6 cells.
  • Thumbnail Image
    Item
    Severe acute respiratory syndrome coronavirus protein 7a interacts with hSGT
    (Elsevier, 2006) Fielding, Burtram C.; Gunalan, Vithiagaran; Tan, Timothy H.P.; Chou, Chih-Fong; Shen, Shuo; Khan, Sehaam; Lim, Seng Gee; Hong, Wanjin; Tan, Yee-Joo
    Severe acute respiratory syndrome coronavirus (SARS-CoV) 7a is an accessory protein with no known homologues. In this study, we report the interaction of a SARS-CoV 7a and small glutamine-rich tetratricopeptide repeat-containing protein (SGT). SARS-CoV 7a and human SGT interaction was identified using a two-hybrid system screen and confirmed with interaction screens in cell culture and cellular co-localization studies. The SGT domain of interaction was mapped by deletion mutant analysis and results indicated that tetratricopeptide repeat 2 (aa 125-158) was essential for interaction. We also showed that 7a interacted with SARS-CoV structural proteins M (membrane) and E (envelope), which have been shown to be essential for virus-like particle formation. Taken together, our results coupled with data from studies of the interaction between SGT and HIV-1 vpu indicated that SGT could be involved in the life-cycle, possibly assembly of SARS-CoV.