Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C1175175 (SARS)
 19,188 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S(e), were not cleaved to any significant degree. They ran at about 180-200kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17-537 and 272-537 but not 17-276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450-454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors.
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PMID:The SARS-CoV S glycoprotein: expression and functional characterization. 1465 94

The aim of the study was to establish the life cycle of severe acute respiratory syndrome-associated coronavirus (SARS CoV) in host cells and determine the pathogenesis of SARS. Vero E6 cells (African green monkey kidney cells) were inoculated with SARS coronavirus for 3, 7, 24, 48, and 72 hr, respectively, and were observed under electron microscope. It was found that the SARS coronavirus entered the cells through membrane fusion instead of endocytosis, and then the nucleocapsids assembled in the RER and matured by budding into the smooth vesicles, which were derived from the Golgi apparatus. The smooth vesicles fused with the cell membrane, and the mature particles were released. A special phenomenon was that some virus-like particles appeared in the nucleus. We propose a scheme of the life cycle of SARS coronavirus and discuss the mechanism of its replication in Vero E6 cells.
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PMID:The life cycle of SARS coronavirus in Vero E6 cells. 1517 Jun 25

The spike (S) protein of severe acute respiratory syndrome-coronavirus (SARS-CoV) is a major virion structural protein. It plays an important role in interaction with receptor and inducing neutralizing antibodies. In the study, six tentative antigenic epitopes (S1 S2 S3 S4 S5 S6) of the spike protein of SARS-CoV were predicted by bio-informatics analysis, and a multi-epitope chimeric gene of S1-S2-S3-S4-S5-S6 was synthesized and fused to downstream GST gene in pGEX-6p-1. The Western blotting demonstrated that SARS patient convalescent serum could recognize the recombinant fusion protein. A number of monoclonal antibodies were developed against the fusion protein. In further, the six predicted epitope genes were individually fused to GST of pGEX-6p-1 and expressed in Escherichia coli BL21, respectively. Among six fusion peptides, S5 reacted with monoclonal antibody D3C5 and S2 reacted with monoclonal antibody D3D1 against spike protein of SARS-CoV. The epitopes recognized by monoclonal antibodies D3C5 and D3D1 are linear, and correspond to 447-458 and 789-799 amino acids of spike protein of SARS-CoV, respectively. Identification of antigenic epitope of spike protein of SARS-CoV could provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic techniques for the control of severe acute respiratory syndrome.
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PMID:Identification of two antigenic epitopes on SARS-CoV spike protein. 1518 71

SARS-CoV (the SARS-Associated Coronavirus) was reported as a novel virus member in the coronavirus family, which was the cause of severe acute respiratory syndrome. Coronavirus replication occurs through a unique mechanism employing Leader sequence in the transcripts when initiating transcription from the genome. Therefore, we cloned the Leader sequence from SARS-CoV(BJ01), which is identical to that identified from SARS-CoV(HKU-39849), and constructed specific siRNA targeting the Leader sequence. Using EGFP and RFP reporter genes fused with the cloned SARS-CoV Leader sequence, we demonstrated that the siRNA targeting the Leader sequence decreased the mRNA abundance and protein expression levels of the reporter genes in 293T cells. By stably expressing the siRNA in Vero E6 cells, we provided data that the siRNA could effectively and specifically decrease the mRNA abundance of SARS-CoV genes as analyzed by RT-PCR and Northern blot. Our data indicated that the siRNA targeting the Leader sequence inhibited the replication of SARS-CoV in Vero E6 cells by silencing gene expression. We further demonstrated, via transient transfection experiments, that the siRNA targeting the Leader sequence had a much stronger inhibitory effect on SARS-CoV replication than the siRNAs targeting the Spike gene or the antisense oligodeoxynucleotides did. This report provides evidence that targeting Leader sequence using siRNA could be a powerful tool in inhibiting SARS-CoV replication.
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PMID:siRNA targeting the leader sequence of SARS-CoV inhibits virus replication. 1577 89

The RNA-dependent RNA polymerase (RdRp) of SARS coronavirus (SARS-CoV) is essential for viral replication and a potential target for anti-SARS drugs. We report here the cloning, expression, and purification of the N-terminal GST-fused SARS-CoV RdRp and its polymerase catalytic domain in Escherichia coli. During purification, the full-length GST-RdRp was found to cleave into three main fragments: an N-terminal p12 fragment, a middle p30 fragment, and a C-terminal p64 fragment comprising the catalytic domain, presumably due to bacterial proteases. Biochemical assays show that the full-length GST-RdRp has RdRp activity and the p64 and p12 fragments form a complex that exhibits comparable RdRp activity, whereas the GST-p64 protein has no activity, suggesting that the p12 domain is required for polymerase activity possibly via involvement in template-primer binding. Nonnucleoside HIV-1 RT inhibitors are shown to have no evident inhibitory effect on SARS-CoV RdRp activity. This work provides a basis for biochemical and structural studies of SARS-CoV RdRp and for development of anti-SARS drugs.
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PMID:Expression, purification, and characterization of SARS coronavirus RNA polymerase. 1584 May 16

The SARS-nsp13 protein was identified as an mRNA cap1 methyltransferase. In this study, the nsp13 gene was cloned from the SARS-CoV PUMC02 strain viral RNA by RT-PCR, and inserted into the expression plasmid pET30a(+). The recombinant plasmid pET30a(+)-nsp13 was confirmed by restriction enzymes and sequencing analysis, and transformed into Escherichia coli BL21(DE3). The His-tag-fused protein was expressed by induction of 0.5mM IPTG and purified by a single Ni(2+) affinity chromatography. The protein was validated by western blot and MS analysis. A large quantity of the nsp13 protein obtained with this method may be useful for further study of its structure and function.
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PMID:Molecular cloning, expression, and purification of SARS-CoV nsp13. 1586 8

The severe acute respiratory syndrome (SARS) is a newly emerging human infectious disease caused by the severe acute respiratory syndrome coronavirus (SARS-CoV). The spike (S) protein of SARS-CoV is a major virion structural protein. It plays an important role in the interaction with receptors and neutralizing antibodies. In this study, the S1 domain of the spike protein and three truncated fragments were expressed by fusion with GST in a pGEX-6p-1 vector. Western blot results demonstrated that the 510-672 fragment of the S1 domain is a linear epitope dominant region. To map the antigenic epitope of this linear epitope dominant region, a set of 16 partially overlapping fragments spanning the fragment were fused with GST and expressed. Four antigenic epitopes S1C3 (539-559), S1C4 (548-567), S1C7/8 (583-606), and S1C10/11 (607-630) were identified. Immunization of mice with each of the four antigenic epitope-fused proteins revealed that all four proteins could elicit spike protein specific antisera. All of them were able to bind to the surface domain of the whole spike protein expressed by recombinant baculovirus in insect cells. Identification of antigenic epitopes of the spike protein of SARS-CoV may provide the basis for the development of immunity-based prophylactic, therapeutic, and diagnostic clinical techniques for the severe acute respiratory syndrome.
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PMID:Identification and antigenic epitope mapping of immunodominant region amino residues 510 to 672 on the spike protein of the severe acute respiratory syndrome coronavirus. 1610 48

The genome of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) contains four structural genes that are homologous to genes found in other coronaviruses, and also contains six subgroup-specific open reading frames (ORFs). Expression of one of these subgroup-specific genes, ORF7a, resulted in apoptosis via a caspase-dependent pathway. Here, we observed that transient expression of ORF7a protein fused with myc or GFP tags at its N or C terminus inhibited cell growth and prevented BrdU incorporation in different cultural cells, suggesting that ORF7a expression may regulate cell cycle progression. Analysis by flow cytometry demonstrated that ORF7a expression was associated with blockage of cell cycle progression at G0/G1 phase in HEK 293 cells after 24 to 60 h post-transfection. Similar results were observed in COS-7 and Vero cells. Mutation analysis of ORF7a revealed that the domain spanning aa 44-82 of 7a protein was essential for its cytoplasmic localization and for induction of the cell cycle arrest. After analyzing the cellular proteins involving in regulation of cell cycle progression, we demonstrated that ORF7a expression was correlated with a significant reduction of cyclin D3 level of mRNA transcription and expression, and phosphorylation of retinoblastoma (Rb) protein at ser795 and ser809/811, not with the expression of cyclin D1, D2, cdk4 and cdk6 in HEK 293 cells. These results suggest that the insufficient expression of cyclin D3 may cause a decreased activity of cyclin D/cdk4/6, resulting in the inhibition of Rb phosphorylation. Accumulation of hypo- or non-phosphorylated pRb thus prevents cell cycle progression at G0/G1 phase.
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PMID:SARS coronavirus 7a protein blocks cell cycle progression at G0/G1 phase via the cyclin D3/pRb pathway. 1630 60

The severe acute respiratory syndrome coronavirus papain-like protease (SARS-CoV PLpro) is involved in the processing of the viral polyprotein and, thereby, contributes to the biogenesis of the virus replication complex. Structural bioinformatics has revealed a relationship for the SARS-CoV PLpro to herpesvirus-associated ubiquitin-specific protease (HAUSP), a ubiquitin-specific protease, indicating potential deubiquitinating activity in addition to its function in polyprotein processing (T. Sulea, H. A. Lindner, E. O. Purisima, and R. Menard, J. Virol. 79:4550-4551, 2005). In order to confirm this prediction, we overexpressed and purified SARS-CoV PLpro (amino acids [aa]1507 to 1858) from Escherichia coli. The purified enzyme hydrolyzed ubiquitin-7-amino-4-methylcoumarin (Ub-AMC), a general deubiquitinating enzyme substrate, with a catalytic efficiency of 13,100 M(-1)s(-1), 220-fold more efficiently than the small synthetic peptide substrate Z-LRGG-AMC, which incorporates the C-terminal four residues of ubiquitin. In addition, SARS-CoV PLpro was inhibited by the specific deubiquitinating enzyme inhibitor ubiquitin aldehyde, with an inhibition constant of 210 nM. The purified SARS-CoV PLpro disassembles branched polyubiquitin chains with lengths of two to seven (Ub2-7) or four (Ub4) units, which involves isopeptide bond cleavage. SARS-CoV PLpro processing activity was also detected against a protein fused to the C terminus of the ubiquitin-like modifier ISG15, both in vitro using the purified enzyme and in HeLa cells by coexpression with SARS-CoV PLpro (aa 1198 to 2009). These results clearly establish that SARS-CoV PLpro is a deubiquitinating enzyme, thereby confirming our earlier prediction. This unexpected activity for a coronavirus papain-like protease suggests a novel viral strategy to modulate the host cell ubiquitination machinery to its advantage.
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PMID:The papain-like protease from the severe acute respiratory syndrome coronavirus is a deubiquitinating enzyme. 1630 91

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a distant member of the Group 2 coronaviruses, has recently been identified as the etiological agent of severe acute respiratory syndrome (SARS). The genome of SARS-CoV contains four structural genes that are homologous to genes found in other coronaviruses, as well as six subgroup-specific open reading frames (ORFs). ORF3 encodes a predicted 154-amino-acid protein that lacks similarity to any known protein, and is designated 3b in this article. We reported previously that SARS-CoV 3b is predominantly localized in the nucleolus, and induces G0/G1 arrest and apoptosis in transfected cells. In this study, we show that SARS-CoV 3b fused with EGFP at its N- or C- terminus co-localized with a mitochondria-specific marker in some transfected cells. Mutation analysis of SARS-CoV 3b revealed that the domain spanning amino acids 80 to 138 was essential for its mitochondria localization. These results provide new directions for studies of the role of SARS-CoV 3b protein in SARS pathogenesis.
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PMID:Mitochondrial location of severe acute respiratory syndrome coronavirus 3b protein. 1668 11


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