Recombinant murine coronaviruses, differing only in the spike gene and containing the strain A59 (moderately hepatotropic) and JHM (neurotropic) spike genes in the background of the JHM genome, were compared for the ability to replicate in the liver and induce hepatitis in weanling C57BL/6 mice. Interestingly, expression of the A59 spike glycoprotein within the background of the neurotropic JHM strain does not reproduce the A59 hepatotropic phenotype. Thus, the JHM genetic background plays a dominant role over the spike in the determination of hepatotropism. (+info)
Characterization of a novel coronavirus associated with severe acute respiratory syndrome.
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In March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The genome of SARS-CoV is 29,727 nucleotides in length and has 11 open reading frames, and its genome organization is similar to that of other coronaviruses. Phylogenetic analyses and sequence comparisons showed that SARS-CoV is not closely related to any of the previously characterized coronaviruses. (+info)
The Genome sequence of the SARS-associated coronavirus.
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We sequenced the 29,751-base genome of the severe acute respiratory syndrome (SARS)-associated coronavirus known as the Tor2 isolate. The genome sequence reveals that this coronavirus is only moderately related to other known coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E. Phylogenetic analysis of the predicted viral proteins indicates that the virus does not closely resemble any of the three previously known groups of coronaviruses. The genome sequence will aid in the diagnosis of SARS virus infection in humans and potential animal hosts (using polymerase chain reaction and immunological tests), in the development of antivirals (including neutralizing antibodies), and in the identification of putative epitopes for vaccine development. (+info)
Mass spectrometric characterization of proteins from the SARS virus: a preliminary report.
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A new coronavirus has been implicated as the causative agent of severe acute respiratory syndrome (SARS). We have used convalescent sera from several SARS patients to detect proteins in the culture supernatants from cells exposed to lavage another SARS patient. The most prominent protein in the supernatant was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a approximately 46-kDa species. This was found to be a novel nucleocapsid protein that matched almost exactly one predicted by an open reading frame in the recently published nucleotide sequence of the same virus isolate (>96% coverage). A second viral protein corresponding to the predicted approximately 139-kDa spike glycoprotein has also been examined by MALDI-TOF MS (42% coverage). After peptide N-glycosidase F digestion, 12 glycosylation sites in this protein were confirmed. The sugars attached to four of the sites were also identified. These results suggest that the nucleocapsid protein is a major immunogen that may be useful for early diagnostics, and that the spike glycoprotein may present a particularly attractive target for prophylactic intervention in combating SARS. (+info)
Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease.
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An investigation into the causes of canine infectious respiratory disease was carried out in a large rehoming kennel. Tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using RT-PCR with conserved primers for the polymerase gene. Sequence analysis of four positive samples showed the presence of a coronavirus with high similarity to both bovine and human coronavirus (strain OC43) in their polymerase and spike genes, whereas there was a low similarity to comparable genes in the enteric canine coronavirus. This canine respiratory coronavirus (CRCV) was detected by RT-PCR in 32/119 tracheal and 20/119 lung samples, with the highest prevalence being detected in dogs with mild clinical symptoms. Serological analysis showed that the presence of antibodies against CRCV on the day of entry into the kennel decreased the risk of developing respiratory disease. (+info)
Putative hAPN receptor binding sites in SARS_CoV spike protein.
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AIM: To obtain the information of ligand-receptor binding between the S protein of SARS-CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites in the S protein of SARS-CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS-CoV in validating the bioinformatics predictions. RESULTS: Possible binding sites in the SARS-CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS-CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION: CD13 may be a possible receptor of the SARS-CoV S protein, which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome. (+info)
Emergence of a coronavirus infectious bronchitis virus mutant with a truncated 3b gene: functional characterization of the 3b protein in pathogenesis and replication.
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The subgenomic RNA 3 of IBV has been shown to be a tricistronic mRNA, encoding three products in IBV-infected cells. To explore if the least expressed ORF, ORF 3b, which encodes a nonstructural protein, is evolutionarily conserved and functionally indispensable for viral propagation in cultured cells, the Beaudette strain of IBV was propagated in chicken embryonated eggs for three passages and then adapted to a monkey kidney cell line, Vero. The 3b gene of passage 3 in embryonated eggs and passages 7, 15, 20, 25, 30, 35 50, and 65 in Vero cells were amplified by reverse transcription-polymerase chain reaction and sequenced. The results showed that viral RNA extracted from passages 35, 50, and 65 contained a single A insertion in a 6A stretch of the 3b gene between nucleotides 24075 and 24080, whereas the early passages carried the normal 3b gene. This insertion resulted in a frameshift event and therefore, if expressed, a C-terminally truncated protein. We showed that the frameshifting product, cloned in a plasmid, was expressed in vitro and in cells transfected with the mutant construct. The normal product of the 3b gene is 64 amino acids long, whereas the frameshifting product is 34 amino acids long with only 17 homogeneous amino acid residues at the N-terminal half. Immunofluorescent studies revealed that the normal 3b protein was localized to the nucleus and the truncated product showed a "free" distribution pattern, indicating that the C-terminal portion of 3b was responsible for its nuclear localization. Comparison of the complete genome sequences (27.6 kb) of isolates p20c22 and p36c12 (from passages 20 and 36, respectively) revealed that p36c12 contains three amino acid substitutions, two in the 195-kDa protein (encoded by gene 1) and one in the S protein, in addition to the frameshifting 3b product. Further characterization of the two isolates demonstrated that p36c12 showed growth advantage over p20c22 in both Vero cells and chicken embryos and was more virulent in chicken embryos than p20c22. These results suggest that the 3b gene product is not essential for the replication of IBV. (+info)
The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex.
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Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein. (+info)