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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
, 2003
"... *Corresponding authors The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be ex ..."
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Cited by 146 (25 self)
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*Corresponding authors The genome organization and expression strategy of the newly identified severe acute respiratory syndrome coronavirus (SARS-CoV) were predicted using recently published genome sequences. Fourteen putative open reading frames were identified, 12 of which were predicted to be expressed from a nested set of eight subgenomic mRNAs. The synthesis of these mRNAs in SARS-CoV-infected cells was confirmed experimentally. The 4382- and 7073 amino acid residue SARS-CoV replicase polyproteins are predicted to be cleaved into 16 subunits by two viral proteinases (bringing the total number of SARS-CoV proteins to 28). A phylogenetic analysis of the replicase gene, using a distantly related torovirus as an outgroup, demonstrated that, despite a number of unique features, SARS-CoV is most closely related to group 2 coronaviruses. Distant homologs of cellular RNA processing enzymes were identified in group 2 coronaviruses, with four of them being conserved in SARS-CoV. These newly recognized viral enzymes place the mechanism of coronavirus RNA synthesis in a completely new perspective. Furthermore, together with previously described viral enzymes, they will be important targets for the design of antiviral strategies aimed at controlling the further spread of SARS-CoV.
Virus-encoded proteinases and proteolytic processing in the Nidovirales
- J. Gen. Virol
, 2000
"... On the basis of similarities in their genome organization and replication strategy, RNA viruses can now be classified into ‘supergroups ’ that often include both animal and plant ..."
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Cited by 105 (29 self)
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On the basis of similarities in their genome organization and replication strategy, RNA viruses can now be classified into ‘supergroups ’ that often include both animal and plant
The genome organization of the Nidovirales: similarities and differences between arteri-, toro-, and coronaviruses
- Semin Virol
, 1997
"... Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonseg-mented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organizatio ..."
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Cited by 49 (11 self)
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Viruses in the families Arteriviridae and Coronaviridae have enveloped virions which contain nonseg-mented, positive-stranded RNA, but the constituent genera differ markedly in genetic complexity and virion structure. Nevertheless, there are striking resemblances among the viruses in the organization and expression of their genomes, and sequence conservation among the polymerase polyproteins strongly suggests that they have a common ancestry. On this basis, the International Committee on Taxonomy of Viruses recently established a new order, Nidovirales, to contain the two families. Here, the common traits and distinguishing features of the Nidovirales are reviewed. r 1997 Academic Press KEY WORDS: arterivirus; coronavirus; torovirus; polyprotein processing; RNA recombination.
Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59
- J
, 1995
"... Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predic ..."
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Cited by 33 (7 self)
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Gene 1 of the murine coronavirus, MHV-A59, encodes approximately 800 kDa of protein products within two overlapping open reading frames (ORFs 1a and 1b). The gene is expressed as a polyprotein that is processed into individual proteins, presumably by virus-encoded proteinases. ORF 1a has been predicted to encode proteins with similarity to viral and cellular proteinases, such as papain, and to the 3C proteinases of the
Characterization of a human coronavirus (strain 229E) 3C-like proteinase activity
- J
, 1995
"... Characterization of a human coronavirus ..."
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Functional and genetic analysis of coronavirus replicase-transcriptase proteins. PLoS Pathog 1:e39. ACCEPTED Downloaded from jvi.asm.org at Penn State Univ on February 12
, 2005
"... The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis viru ..."
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Cited by 22 (4 self)
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The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (nonstructural proteins nsp1–nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12–nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II–VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicasetranscriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the nonpermissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive- and negative-strand RNA. On the basis of these results, we
Characterization of the Rubella Virus Nonstructural Protease Domain and Its Cleavage Site
, 1995
"... Characterization of the rubella virus nonstructural protease domain and its site. ..."
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Cited by 17 (5 self)
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Characterization of the rubella virus nonstructural protease domain and its site.
Localization of mouse hepatitis virus open reading frame 1A derived proteins
- Journal of Neurovirology
, 1998
"... We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5 ' portion of mouse hepatitis virus (MHV) gene 1. Immuno¯uorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewha ..."
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Cited by 10 (0 self)
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We have investigated the intracellular localization of proteolytic cleavage products encoded in the 5 ' portion of mouse hepatitis virus (MHV) gene 1. Immuno¯uorescent labeling of cells with an antiserum which recognizes p28, the ORF1a N-terminal cleavage product, resulted in widespread somewhat granular cytoplasmic staining, indicating that this protein is widely distributed in the cytoplasm of MHV-infected, but not control uninfected cells. Immuno-¯uorescent staining of infected cells with antisera which recognize the downstream polypeptides, p65, p240 and p290 labeled discrete vesicular perinuclear structures. Double immuno¯uorescent labeling of BHK cells expressing the MHV receptor (BHKMHVR1) and infected with MHV-A59 with a Golgi-speci®c anti-mannosidase II monoclonal antibody and with antiserum recognizing each of these anti-MHV ORF1a polypeptides, showed that the p240 and p290 polypeptides were localized in discrete vesicular structures that overlapped the Golgi complex. Labeling with antibodies speci®c for p65 colocalized with the Golgi region, and showed staining of the perinuclear
In vitro reconstitution of SARS-coronavirus mRNA cap methylation
- PLoS Pathog
, 2010
"... SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 59 end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase com ..."
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Cited by 7 (2 self)
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SARS-coronavirus (SARS-CoV) genome expression depends on the synthesis of a set of mRNAs, which presumably are capped at their 59 end and direct the synthesis of all viral proteins in the infected cell. Sixteen viral non-structural proteins (nsp1 to nsp16) constitute an unusually large replicase complex, which includes two methyltransferases putatively involved in viral mRNA cap formation. The S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) activity was recently attributed to nsp14, whereas nsp16 has been predicted to be the AdoMet-dependent (nucleoside-29O)-methyltransferase. Here, we have reconstituted complete SARS-CoV mRNA cap methylation in vitro. We show that mRNA cap methylation requires a third viral protein, nsp10, which acts as an essential trigger to complete RNA cap-1 formation. The obligate sequence of methylation events is initiated by nsp14, which first methylates capped RNA transcripts to generate cap-0 7Me GpppA-RNAs. The latter are then selectively 29O-methylated by the 29O-MTase nsp16 in complex with its activator nsp10 to give rise to cap-1 7Me GpppA29OMe-RNAs. Furthermore, sensitive in vitro inhibition assays of both activities show that aurintricarboxylic acid, active in SARS-CoV infected cells, targets both MTases with IC50 values in the micromolar range, providing a validated basis for anti-coronavirus drug design.
Papain-Like Protease 1 from Transmissible Gastroenteritis Virus: Crystal Structure and Enzymatic Activity toward Viral
, 2010
"... Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to faci ..."
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Coronaviruses encode two classes of cysteine proteases, which have narrow substrate specificities and either a chymotrypsin- or papain-like fold. These enzymes mediate the processing of the two precursor polyproteins of the viral replicase and are also thought to modulate host cell functions to facilitate infection. The papain-like protease 1 (PL1pro) domain is present in nonstructural protein 3 (nsp3) of alphacoronaviruses and subgroup 2a betacoronaviruses. It participates in the proteolytic processing of the N-terminal region of the replicase polyproteins in a manner that varies among different coronaviruses and remains poorly understood. Here we report the first structural and biochemical characterization of a purified coronavirus PL1pro domain, that of transmissible gastroenteritis virus (TGEV). Its tertiary structure is compared with that of severe acute respiratory syndrome (SARS) coronavirus PL2pro, a downstream paralog that is conserved in the nsp3’s of all coronaviruses. We identify both conserved and unique structural features likely controlling the interaction of PL1pro with cofactors and substrates, including the tentative mapping of substrate pocket residues. The purified recombinant TGEV PL1pro was shown to cleave a peptide mimicking the cognate nsp2nsp3 cleavage site. Like its PL2pro paralogs from several coronaviruses, TGEV PL1pro was also found to have deubiquiti-nating activity in an in vitro cleavage assay, implicating it in counteracting ubiquitin-regulated host cell pathways, likely including innate immune responses. In combination with the prior characterization of PL2pro
