RESUMO
We report a complete genomic sequence of rare isolates (minor genotype) of the SARS-CoV from SARS patients in Guangdong, China, where the first few cases emerged. The most striking discovery from the isolate is an extra 29-nucleotide sequence located at the nucleotide positions between 27,863 and 27,864 (referred to the complete sequence of BJ01) within an overlapped region composed of BGI-PUP5 (BGI-postulated uncharacterized protein 5) and BGI-PUP6 upstream of the N (nucleocapsid) protein. The discovery of this minor genotype, GD-Ins29, suggests a significant genetic event and differentiates it from the previously reported genotype, the dominant form among all sequenced SARS-CoV isolates. A 17-nt segment of this extra sequence is identical to a segment of the same size in two human mRNA sequences that may interfere with viral replication and transcription in the cytosol of the infected cells. It provides a new avenue for the exploration of the virus-host interaction in viral evolution, host pathogenesis, and vaccine development.
Assuntos
Sequência de Bases , China , Análise por Conglomerados , Componentes do Gene , Variação Genética , Genoma Viral , Genótipo , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Genética , Análise de Sequência de DNA , Síndrome Respiratória Aguda Grave , GenéticaRESUMO
We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To investigate its immunogenicity, we synthesized eight oligopeptides covering 69.2% of the entire ORF and screened them by using ELISA (enzyme-linked immunosorbent assay) with sera from SARS patients. The results confirmed our predictions on antigenic sites.
Assuntos
Sequência de Aminoácidos , Sequência de Bases , Análise por Conglomerados , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Dados de Sequência Molecular , Mutação , Genética , Oligopeptídeos , Filogenia , Estrutura Terciária de Proteína , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Genética , Alinhamento de Sequência , Análise de Sequência de DNA , Proteínas da Matriz Viral , Química , Genética , Alergia e ImunologiaRESUMO
The Coronaviridae family is characterized by a nucleocapsid that is composed of the genome RNA molecule in combination with the nucleoprotein (N protein) within a virion. The most striking physiochemical feature of the N protein of SARS-CoV is that it is a typical basic protein with a high predicted pI and high hydrophilicity, which is consistent with its function of binding to the ribophosphate backbone of the RNA molecule. The predicted high extent of phosphorylation of the N protein on multiple candidate phosphorylation sites demonstrates that it would be related to important functions, such as RNA-binding and localization to the nucleolus of host cells. Subsequent study shows that there is an SR-rich region in the N protein and this region might be involved in the protein-protein interaction. The abundant antigenic sites predicted in the N protein, as well as experimental evidence with synthesized polypeptides, indicate that the N protein is one of the major antigens of the SARS-CoV. Compared with other viral structural proteins, the low variation rate of the N protein with regards to its size suggests its importance to the survival of the virus.
Assuntos
Motivos de Aminoácidos , Genética , Sequência de Aminoácidos , Antígenos Virais , Alergia e Imunologia , Composição de Bases , Sequência de Bases , Análise por Conglomerados , Biologia Computacional , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Variação Genética , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Genética , Alergia e Imunologia , Metabolismo , Fosforilação , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Genética , Análise de Sequência de DNARESUMO
The R (replicase) protein is the uniquely defined non-structural protein (NSP) responsible for RNA replication, mutation rate or fidelity, regulation of transcription in coronaviruses and many other ssRNA viruses. Based on our complete genome sequences of four isolates (BJ01-BJ04) of SARS-CoV from Beijing, China, we analyzed the structure and predicted functions of the R protein in comparison with 13 other isolates of SARS-CoV and 6 other coronaviruses. The entire ORF (open-reading frame) encodes for two major enzyme activities, RNA-dependent RNA polymerase (RdRp) and proteinase activities. The R polyprotein undergoes a complex proteolytic process to produce 15 function-related peptides. A hydrophobic domain (HOD) and a hydrophilic domain (HID) are newly identified within NSP1. The substitution rate of the R protein is close to the average of the SARS-CoV genome. The functional domains in all NSPs of the R protein give different phylogenetic results that suggest their different mutation rate under selective pressure. Eleven highly conserved regions in RdRp and twelve cleavage sites by 3CLP (chymotrypsin-like protein) have been identified as potential drug targets. Findings suggest that it is possible to obtain information about the phylogeny of SARS-CoV, as well as potential tools for drug design, genotyping and diagnostics of SARS.
Assuntos
Sequência de Aminoácidos , Composição de Bases , Sequência de Bases , Análise por Conglomerados , Biologia Computacional , Sequência Conservada , Genética , Evolução Molecular , Componentes do Gene , Genoma Viral , Dados de Sequência Molecular , Mutação , Genética , Filogenia , Estrutura Terciária de Proteína , RNA Polimerase Dependente de RNA , Genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Genética , Análise de Sequência de DNARESUMO
Beijing has been one of the epicenters attacked most severely by the SARS-CoV (severe acute respiratory syndrome-associated coronavirus) since the first patient was diagnosed in one of the city's hospitals. We now report complete genome sequences of the BJ Group, including four isolates (Isolates BJ01, BJ02, BJ03, and BJ04) of the SARS-CoV. It is remarkable that all members of the BJ Group share a common haplotype, consisting of seven loci that differentiate the group from other isolates published to date. Among 42 substitutions uniquely identified from the BJ group, 32 are non-synonymous changes at the amino acid level. Rooted phylogenetic trees, proposed on the basis of haplotypes and other sequence variations of SARS-CoV isolates from Canada, USA, Singapore, and China, gave rise to different paradigms but positioned the BJ Group, together with the newly discovered GD01 (GD-Ins29) in the same clade, followed by the H-U Group (from Hong Kong to USA) and the H-T Group (from Hong Kong to Toronto), leaving the SP Group (Singapore) more distant. This result appears to suggest a possible transmission path from Guangdong to Beijing/Hong Kong, then to other countries and regions.