Browsing by Author "Lim, Seng Gee"

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    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.
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    Cellular Characterization of SARS Coronavirus Nucleocapsid
    (Leibniz Research Centre for Working Environment and Human Factors, 2004) Goh, Phuay-Yee; Choi, Yook-Wah; Shen, Shuo; Tan, Yee-Joo; Fielding, Burtram C.; Tan, Timothy H.P.; O, Eng-Eong; Lim, Seng Gee; Hong, Wanjin
    The Severe and Acute Respiratory Syndrome coronavirus (SARS CoV) is a newly-emerged virus that caused an outbreak of atypical pneumonia in the winter of 2002-2003. Polyclonal antibodies raised against the nucleocapsid (N) of the SARS CoV showed the localization of N to the cytoplasm and the nucleolus in virus-infected and N-expressing Vero E6 cells. Like other coronavirus N proteins, the SARS N is probably a phosphoprotein. N protein expressed in mammalian cells is apparently able to “spread” to neighboring cells. For N to spread to neighboring cells, it must be exported out of the expressing cells. This is shown by the immunoprecipitation of N from the culture medium of a stable cell line expressing myc-N. Deletion studies showed that the 27 kD C-terminal domain of N (C1/2) is the minimal region of N that can spread to other cells. The nucleolar localization and spreading of N are artefacts of fixation, reminiscent of other protein-transduction domain (PTD)-containing proteins
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    Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus
    (American Society for Microbiology, 2004) Fielding, Burtram C.; Tan, Yee-Joo; Shen, Shuo; Tan, Timothy H.P.; Ooi, Eng-Eong; Lim, Seng Gee; Hong, Wanjin; Goh, Phuay-Yee
    A novel coronavirus (CoV) has been identified as the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV genome encodes the characteristic essential CoV replication and structural proteins. Additionally, the genome contains six group-specific open reading frames (ORFs) larger than 50 amino acids, with no known homologues. As with the group-specific genes of the other CoVs, little is known about the SARS-CoV group-specific genes. SARS-CoV ORF7a encodes a putative unique 122-amino-acid protein, designated U122 in this study. The deduced sequence contains a probable cleaved signal sequence and a C-terminal transmembrane helix, indicating that U122 is likely to be a type I membrane protein. The C-terminal tail also contains a typical endoplasmic reticulum (ER) retrieval motif, KRKTE. U122 was expressed in SARS-CoVinfected Vero E6 cells, as it could be detected by Western blot and immunofluorescence analyses. U122 is localized to the perinuclear region of both SARS-CoV-infected and transfected cells and colocalized with ER and intermediate compartment markers. Mutational analyses showed that both the signal peptide sequence and ER retrieval motif were functional.
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    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.
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    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.
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    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.
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    A Novel Severe Acute Respiratory Syndrome Coronavirus Protein, U274, is transported to the Cell Surface and undergoes Endocytosis
    (American Society for Microbiology, 2004) Tan, Yee-Joo; Teng, Eileen; Shen, Shuo; Tan, Timothy H.P.; Goh, Phuay-Yee; Fielding, Burtram C.; Ooi, Eng-Eong; Tan, Hwee-Cheng; Lim, Seng Gee; Hong, Wanjin
    The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxx and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.
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    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.
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    Overexpression of 7a, a Protein Specifically Encoded by the Severe Acute Respiratory Syndrome Coronavirus, Induces Apoptosis via a Caspase-Dependent Pathway
    (American Society for Microbiology, 2004) Tan, Yee-Joo; Fielding, Burtram C.; Goh, Phuay-Yee; Shen, Shuo; Tan, Timothy H.P.; Lim, Seng Gee; Hong, Wanjin
    Besides genes that are homologous to proteins found in other coronaviruses, the severe acute respiratory syndrome coronavirus genome also contains nine other potential open reading frames. Previously, we have characterized the expression and cellular localization of two of these “accessory” viral proteins, 3a (previously termed U274) and 7a (previously termed U122). In this study, we further examined whether they can induce apoptosis, which has been observed clinically. We showed that the overexpression of 7a, but not of 3a or the viral structural proteins, nucleocapsid, membrane, and envelope, induces apoptosis. 7a induces apoptosis via a caspase-dependent pathway and in cell lines derived from different organs, including lung, kidney, and liver.
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    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
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    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.
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    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.