Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage

Snijder, Eric J.; Bredenbeek, Peter J.; Dobbe, Jessika C.; Thiel, Volker; Ziebuhr, John; Poon, Leo L. M.; Guan, Yi; Rozanov, Mikhail; Spaan, Willy J. M.; Gorbalenya, Alexander E.

doi:10.1016/s0022-2836(03)00865-9

Cited by 1,137 publications

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“…aries of the gene were 3241Lys-Gln3240 [19]. The gene was ligated into pET11d plasmid and the resultant plasmid electroporated into TOP10 cells for propagation.…”

Section: Synthesis and Purification Of The Fluorogenic Substratementioning

confidence: 99%

Enzymatic activity of the SARS coronavirus main proteinase dimer

et al. 2006

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The enzymatic activity of the SARS coronavirus main proteinase dimer was characterized by a sensitive, quantitative assay. The new, fluorogenic substrate, (Ala-Arg-Leu-Gln-NH) 2 -Rhodamine, contained a severe acute respiratory syndrome coronavirus (SARS CoV) main proteinase consensus cleavage sequence and Rhodamine 110, one of the most detectable compounds known, as the reporter group. The gene for the enzyme was cloned in the absence of purification tags, expressed in Escherichia coli and the enzyme purified. Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K m , and is unusually sensitive to ionic strength and reducing agents.

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“…aries of the gene were 3241Lys-Gln3240 [19]. The gene was ligated into pET11d plasmid and the resultant plasmid electroporated into TOP10 cells for propagation.…”

Section: Synthesis and Purification Of The Fluorogenic Substratementioning

confidence: 99%

Enzymatic activity of the SARS coronavirus main proteinase dimer

et al. 2006

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“…proteins, S, E, M and N, and a set of accessory proteins whose number and sequence vary among different coronavirus species . The extraordinary size of the coronavirus replicase (poly)proteins, their generally large phylogenetic distance from those of other RNA viruses, and the presence of several predicted RNA processing activities which are not found in other positive-strand RNA viruses (Gorbalenya, 2001;Snijder et al, 2003), indicate that coronavirus replicases are of an unparalleled complexity. The underlying biological mechanisms and functional constraints that determine the evolution and conservation of these unique activities remain to be elucidated.…”

Section: Introductionmentioning

confidence: 99%

“…The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al, 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et al, 2000;Gorbalenya, 2001), SARS-CoV is predicted to encode only two proteinases (Rota et al, 2003;Snijder et al, 2003). The replicase complex mediates both genome replication and transcription of a 'nested' set of subgenomic mRNAs.…”

Section: Introductionmentioning

confidence: 99%

Mechanisms and enzymes involved in SARS coronavirus genome expression

Thiel

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et al. 2003

Journal of General Virology

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A novel coronavirus is the causative agent of the current epidemic of severe acute respiratory syndrome (SARS). Coronaviruses are exceptionally large RNA viruses and employ complex regulatory mechanisms to express their genomes. Here, we determined the sequence of SARS coronavirus (SARS-CoV), isolate Frankfurt 1, and characterized key RNA elements and protein functions involved in viral genome expression. Important regulatory mechanisms, such as the (discontinuous) synthesis of eight subgenomic mRNAs, ribosomal frameshifting and posttranslational proteolytic processing, were addressed. Activities of three SARS coronavirus enzymes, the helicase and two cysteine proteinases, which are known to be critically involved in replication, transcription and/or post-translational polyprotein processing, were characterized. The availability of recombinant forms of key replicative enzymes of SARS coronavirus should pave the way for high-throughput screening approaches to identify candidate inhibitors in compound libraries. INTRODUCTIONSevere acute respiratory syndrome (SARS) is a lifethreatening form of pneumonia (Peiris et al., 2003a). In the course of a few months in 2003, an epidemic emerged that has spread from its likely origin in Guangdong Province, China, to 32 countries. By 11 June 2003 more than 8400 cases and 789 deaths had been recorded by the World Health Organization. The rapid transmission by aerosols (and probably also the faecal-oral route) and the high mortality rate make SARS a global threat for which no efficacious therapy is available. There is now clear evidence that SARS is caused by a previously unknown coronavirus, provisionally termed SARS coronavirus (SARS-CoV) (Peiris et al., 2003b;Drosten et al., 2003;Ksiazek et al., 2003;Fouchier et al., 2003). Genome sequences of SARS-CoV isolates obtained from a number of index patients have been published recently and provide important information on the organization, phylogeny and variability of the 29?7 kb positive-strand RNA genome of SARS-CoV (Rota et al., 2003;Marra et al., 2003;Ruan et al., 2003). By analogy with other coronaviruses (Lai & Holmes, 2001;Gorbalenya, 2001), SARS-CoV gene expression is expected to involve complex transcriptional, translational and post-translational regulatory mechanisms, whose molecular details remain to be determined. SARS-CoV genome expression starts with the translation of two large replicative polyproteins, pp1a (486 kDa) and pp1ab (790 kDa), which are encoded by the viral replicase gene (21 221 nt) that comprises ORFs 1a and 1b (Fig. 1). Expression of the ORF1b-encoded region of pp1ab is predicted to involve ribosomal frameshifting into the 21 frame just upstream of the ORF1a translation termination codon (Brierley et al., 1989). The pp1a and pp1ab polyproteins are processed by viral proteinases to yield the functional components of the membrane-bound replicase complex (Ziebuhr et al., 2000). In contrast to most other coronaviruses, which use three proteinase activities for replicase polyprotein processing (Ziebuhr et ...

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“…[32][33][34]. Phylogenetic data suggest that the SARS-CoV is a distinct group 2 coronavirus [35,36]. Previous studies have shown that IFNs-α/β provide protection from MHV-A59 and MHV-2 infections in vivo [37,38].…”

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confidence: 99%

Characterization of the antiviral effects of interferon-α against a SARS-like coronoavirus infection in vitro

et al. 2006

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Cell Research | www.cell-research.com Antiviral effects of IFN-α against MHV-1 220 npg ORIGINAL ARTICLE IntroductionIn March 2003, a number of laboratories independently reported the isolation and characterization of a novel coronavirus (CoV) from specimens of patients with severe acute respiratory syndrome (SARS) [1,2]. Although an effective therapeutic strategy against SARS has yet to be developed, interferons (IFN) are recognized to play critical roles in host resistance to viral infection [3][4][5][6], thereby implicating them as potential candidates for SARS CoV treatment. IFNs inhibit viral infection by directly interfering with viral replication and by inducing both an innate and adaptive immune response to infection. IFN-αs are effective in the treatment of hepatitis B and C [7,8] and respiratory coronavirus infections unrelated to the Urbani strain of SARS-CoV [9][10][11][12]. , and combinations of 18] are effective in inhibiting SARS-CoV replication in vitro. Moreover, it was reported that macacque monkeys were protected from infection with SARS CoV by treatment with .IFN alfacon-1 (Infergen, Intermune Corp, Brisbane, California), a synthetic IFN-α designed to represent a consensus , has been shown in both cell culture systems [21] and comparative clinical trials [22] to inhibit viral replication more potently than other IFN-αs. Based on these findings, a pilot study to evaluate the potential clinical benefit and safety of IFN alfacon-1 in SARS treatment was conducted by our laboratory. Interferon (IFN)-αs bind to and activate their cognate cell surface receptor to invoke an antiviral response in target cells. Well-described receptor-mediated signaling events result in transcriptional regulation of IFN sensitive genes, effectors of this antiviral response. Results from a pilot study to evaluate the clinical efficacy of IFN-α treatment of SARS patients provided evidence for IFN-inducible resolution of disease. In this report we examined the contribution of IFN-inducible phosphorylation-activation of specific signaling effectors to protection from infection by a SARS-related murine coronavirus, MHV-1. As anticipated, the earliest receptor-activation event, Jak1 phosphorylation, is critical for IFN-inducible protection from MHV-1 infection. Additionally, we provide evidence for the contribution of two kinases, the MAP kinase p38MAPK, and protein kinase C (PKC) d to antiviral protection from MHV-1 infection. Notably, our data suggest that MHV-1 infection, as for the Urbani SARS coronoavirus, inhibits an IFN response, inferred from the lack of activation of pkr and 2'5'-oas, genes associated with mediating the antiviral activities of IFN-αs. To identify potential target genes that are activated downstream of the IFN-inducible signaling effectors we identified, and that mediate protection from coronavirus infection, we examined the gene expression profiles in the peripheral blood mononuclear cells of SARS patients who received IFN treatment. A subset of differentially regulated genes were distinguished with...

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Unique and Conserved Features of Genome and Proteome of SARS-coronavirus, an Early Split-off From the Coronavirus Group 2 Lineage

Cited by 1,137 publications

References 70 publications

Enzymatic activity of the SARS coronavirus main proteinase dimer

Enzymatic activity of the SARS coronavirus main proteinase dimer

Mechanisms and enzymes involved in SARS coronavirus genome expression

Characterization of the antiviral effects of interferon-α against a SARS-like coronoavirus infection in vitro

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