RESUMO
Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.
Assuntos
Coronaviridae , Domínios Proteicos , RNA Polimerase Dependente de RNA , Humanos , Nucleotídeos/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , SARS-CoV-2/enzimologia , Proteínas não Estruturais Virais/metabolismo , Coronaviridae/enzimologia , Coronaviridae/genética , Domínios Proteicos/fisiologia , Proteínas Virais/metabolismo , Sequência Conservada , Estrutura Secundária de Proteína/genética , Células VeroRESUMO
Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.
Assuntos
Coronaviridae/enzimologia , Infecções por Coronavirus/imunologia , Endonucleases/imunologia , Evasão da Resposta Imune/fisiologia , Proteínas Virais/imunologia , Animais , Coronaviridae/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo RealRESUMO
White bream virus (WBV), a poorly characterized plus-strand RNA virus infecting freshwater fish of the Cyprinidae family, is the prototype species of the genus Bafinivirus in the subfamily Torovirinae (family Coronaviridae, order Nidovirales). In common with other nidoviruses featuring >20 kilobase genomes, bafiniviruses have been predicted to encode an exoribonuclease (ExoN) in their replicase gene. Here, we used information on the substrate specificity of bafinivirus 3C-like proteases to express WBV ExoN in an active form in Escherichia coli. The 374-residue protein displayed robust 3'-to-5' exoribonuclease activity in the presence of Mg2+ ions and, unlike its coronavirus homologues, did not require a protein cofactor for activity. Characterization of mutant forms of ExoN provided support for predictions on putative active-site and conserved zinc-binding residues. WBV ExoN was revealed to be most active on double-stranded RNA substrates containing one or two non-paired 3'-terminal nucleotides, supporting its presumed role in increasing the fidelity of the bafinivirus RNA-dependent RNA polymerase.
Assuntos
Coronaviridae/enzimologia , Exorribonucleases/metabolismo , Proteínas Virais/metabolismo , Coronaviridae/metabolismo , Exorribonucleases/genética , Regulação Enzimológica da Expressão Gênica , Regulação Viral da Expressão Gênica , RNA de Cadeia Dupla , RNA Viral , Especificidade por Substrato , Replicação ViralRESUMO
The emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe pulmonary disease in humans and represents the second example of a highly pathogenic coronavirus (CoV) following severe acute respiratory syndrome coronavirus (SARS-CoV). Genomic studies revealed that two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), process the polyproteins encoded by the MERS-CoV genomic RNA. We previously reported that SARS-CoV PLpro acts as both deubiquitinase (DUB) and IFN antagonist, but the function of the MERS-CoV PLpro was poorly understood. In this study, we characterized MERS-CoV PLpro, which is a protease and can recognize and process the cleavage sites (CS) of nsp1-2, nsp2-3 and nsp3-4. The LXGG consensus cleavage sites in the N terminus of pp1a/1ab, which is generally essential for CoV PLpro-mediated processing, were also characterized in MERS-CoV. MERS-CoV PLpro, like human SARS-CoV PLpro and NL63-CoV PLP2, is a viral deubiquitinating enzyme. It acts on both K48- and K63-linked ubiquitination and ISG15-linked ISGylation. We confirmed that MERS-CoV PLpro acts as an IFN antagonist through blocking the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3). These findings indicate that MERS-CoV PLpro acts as a viral DUB and suppresses production of IFN-ß by an interfering IRF3-mediated signalling pathway, in addition to recognizing and processing the CS at the N terminus of replicase polyprotein to release the non-structural proteins. The characterization of proteolytic processing, DUB and IFN antagonist activities of MERS-CoV PLpro would reveal the interactions between MERS-CoV and its host, and be applicable to develop strategies targeting PLpro for the effective control of MERS-CoV infection.
Assuntos
Infecções por Coronaviridae/metabolismo , Coronaviridae/enzimologia , Interferon beta/antagonistas & inibidores , Papaína/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Coronaviridae/química , Coronaviridae/genética , Infecções por Coronaviridae/virologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/metabolismo , Dados de Sequência Molecular , Papaína/química , Papaína/genética , Fosforilação , Poliproteínas/genética , Poliproteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Alinhamento de Sequência , Ubiquitina , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The production of functional nidovirus replication-transcription complexes involves extensive proteolytic processing by virus-encoded proteases. In this study, we characterized the viral main protease (M(pro)) of the type species, White bream virus (WBV), of the newly established genus Bafinivirus (order Nidovirales, family Coronaviridae, subfamily Torovirinae). Comparative sequence analysis and mutagenesis data confirmed that the WBV M(pro) is a picornavirus 3C-like serine protease that uses a Ser-His-Asp catalytic triad embedded in a predicted two-ß-barrel fold, which is extended by a third domain at its C terminus. Bacterially expressed WBV M(pro) autocatalytically released itself from flanking sequences and was able to mediate proteolytic processing in trans. Using N-terminal sequencing of autoproteolytic processing products we tentatively identified Gln↓(Ala, Thr) as a substrate consensus sequence. Mutagenesis data provided evidence to suggest that two conserved His and Thr residues are part of the S1 subsite of the enzyme's substrate-binding pocket. Interestingly, we observed two N-proximal and two C-proximal autoprocessing sites in the bacterial expression system. The detection of two major forms of M(pro), resulting from processing at two different N-proximal and one C-proximal site, in WBV-infected epithelioma papulosum cyprini cells confirmed the biological relevance of the biochemical data obtained in heterologous expression systems. To our knowledge, the use of alternative M(pro) autoprocessing sites has not been described previously for other nidovirus M(pro) domains. The data presented in this study lend further support to our previous conclusion that bafiniviruses represent a distinct group of viruses that significantly diverged from other phylogenetic clusters of the order Nidovirales.
Assuntos
Coronaviridae/enzimologia , Serina Proteases/metabolismo , Proteínas Virais/metabolismo , Sítios de Ligação , Domínio Catalítico , Análise Mutacional de DNA , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina Proteases/genética , Proteínas Virais/genéticaRESUMO
Inherited genetic factors can influence the severity of COVID-19, but the molecular explanation underpinning a genetic association is often unclear. Intracellular antiviral defenses can inhibit the replication of viruses and reduce disease severity. To better understand the antiviral defenses relevant to COVID-19, we used interferon-stimulated gene (ISG) expression screening to reveal that 2'-5'-oligoadenylate synthetase 1 (OAS1), through ribonuclease L, potently inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that a common splice-acceptor single-nucleotide polymorphism (Rs10774671) governs whether patients express prenylated OAS1 isoforms that are membrane-associated and sense-specific regions of SARS-CoV-2 RNAs or if they only express cytosolic, nonprenylated OAS1 that does not efficiently detect SARS-CoV-2. In hospitalized patients, expression of prenylated OAS1 was associated with protection from severe COVID-19, suggesting that this antiviral defense is a major component of a protective antiviral response.
Assuntos
2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , COVID-19/genética , COVID-19/fisiopatologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/metabolismo , SARS-CoV-2/fisiologia , Regiões 5' não Traduzidas , Células A549 , Animais , COVID-19/enzimologia , COVID-19/imunologia , Quirópteros/genética , Quirópteros/virologia , Coronaviridae/enzimologia , Coronaviridae/genética , Coronaviridae/fisiologia , Endorribonucleases/metabolismo , Humanos , Interferons/imunologia , Isoenzimas/genética , Isoenzimas/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Polimorfismo de Nucleotídeo Único , Prenilação de Proteína , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/genética , RNA Viral/química , RNA Viral/genética , Retroelementos , SARS-CoV-2/genética , Índice de Gravidade de Doença , Replicação ViralRESUMO
Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-ß), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-ß production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses.
Assuntos
Infecções por Coronaviridae/veterinária , Coronaviridae/enzimologia , Cisteína Endopeptidases/metabolismo , Quinase I-kappa B/metabolismo , Interferon beta/metabolismo , Doenças dos Suínos/enzimologia , Proteínas não Estruturais Virais/metabolismo , Animais , Coronaviridae/genética , Infecções por Coronaviridae/enzimologia , Infecções por Coronaviridae/metabolismo , Infecções por Coronaviridae/virologia , Proteases 3C de Coronavírus , Cisteína Endopeptidases/genética , Interações Hospedeiro-Patógeno , Quinase I-kappa B/genética , Interferon beta/genética , Processamento de Proteína Pós-Traducional , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/metabolismo , Doenças dos Suínos/virologia , Proteínas não Estruturais Virais/genéticaRESUMO
A computer-assisted comparative analysis of the amino acid sequences of (putative) thiol proteases encoded by the genomes of several diverse groups of positive-stranded RNA viruses and distantly related to the family of cellular papain-like proteases is presented. A high level of similarity was detected between the leader protease of foot-and-mouth-disease virus and the protease of murine hepatitis coronavirus which cleaves the N-terminal p28 protein from the polyprotein. Statistically significant alignment of a portion of the rubella virus polyprotein with cellular papain-like proteases was obtained, leading to tentative identification of the papain-like protease as the enzyme mediating processing of the non-structural proteins of this virus. Specific grouping between the sequences of the proteases of alpha-viruses, and poty- and bymoviruses was revealed. It was noted that papain-like proteases of positive-stranded RNA viruses are much more variable both in their sequences and in genomic locations than chymotrypsin-related proteases found in the same virus class. A novel conserved domain of unknown function has also been identified which flanks the papain-like proteases of alpha-, rubi- and coronaviruses.
Assuntos
Cisteína Endopeptidases/química , Papaína/química , Vírus de RNA/enzimologia , Alphavirus/enzimologia , Sequência de Aminoácidos , Aphthovirus/enzimologia , Coronaviridae/enzimologia , Dados de Sequência Molecular , Vírus de Plantas/enzimologia , Sinais Direcionadores de Proteínas/química , Vírus da Rubéola/enzimologia , Alinhamento de SequênciaRESUMO
In this paper we demonstrate that the organization of the polymerase gene of toroviruses and coronaviruses is similar. The polymerase gene of both virus families consists of at least two large ORFs (1a and 1b). Four domains of conserved amino acid sequences have been identified in nearly identical positions in the 3' ORF of the pol gene of toroviruses and coronaviruses. The most 3' conserved domain which is still unique for these viruses encodes a 33-kDA protein in MHV-A59, which is cleaved from a precursor protein. Expression of ORF1b of the pol gene of both virus families occurs by ribosomal frameshifting. A predicted stem-loop structure and pseudoknot are conserved in the ORF1a/ORF1b overlap of toro- and coronaviruses. On the basis of these results we postulate that toro- and coronaviruses are ancestrally more related to each other than to other families of positive stranded RNA viruses.
Assuntos
Evolução Biológica , Coronaviridae/genética , RNA Polimerases Dirigidas por DNA/genética , Vírus da Hepatite Murina/genética , Vírus de RNA/genética , RNA Viral/genética , Sequência de Aminoácidos , Animais , Composição de Bases , Sequência de Bases , Calorimetria , Coronaviridae/enzimologia , Dados de Sequência Molecular , Vírus da Hepatite Murina/enzimologia , Conformação de Ácido Nucleico , Fases de Leitura Aberta , Biossíntese de Proteínas , Vírus de RNA/enzimologia , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
Coronaviruses encode papain-like proteases (PLpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (DUB)/deISGylating activity, which is hypothesized to modify the innate immune response to infection. Here, we investigate the predicted DUB activity of the PLpro domain of the recently described Middle East Respiratory Syndrome Coronavirus (MERS-CoV). We found that expression of MERS-CoV PLpro reduces the levels of ubiquitinated and ISGylated host cell proteins; consistent with multifunctional PLpro activity. Further, we compared the ability of MERS-CoV PLpro and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) PLpro to block innate immune signaling of proinflammatory cytokines. We show that expression of SARS-CoV and MERS-CoV PLpros blocks upregulation of cytokines CCL5, IFN-ß and CXCL10 in stimulated cells. Overall these results indicate that the PLpro domains of MERS-CoV and SARS-CoV have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis.
Assuntos
Infecções por Coronaviridae/virologia , Coronaviridae/enzimologia , Papaína/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Coronaviridae/química , Coronaviridae/genética , Infecções por Coronaviridae/genética , Infecções por Coronaviridae/metabolismo , Citocinas/genética , Citocinas/metabolismo , Glicosilação , Humanos , Dados de Sequência Molecular , Papaína/química , Papaína/genética , Estrutura Terciária de Proteína , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/enzimologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Ubiquitinação , Ubiquitinas/genética , Ubiquitinas/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaAssuntos
Acetilesterase/isolamento & purificação , Coronaviridae/enzimologia , Acetilesterase/metabolismo , Animais , Linhagem Celular , Galinhas , Coronaviridae/imunologia , Coronaviridae/isolamento & purificação , Eritrócitos/microbiologia , Hemaglutinação , Cinética , Peso Molecular , Receptores Virais/fisiologia , SuínosAssuntos
Infecções por Coronaviridae/veterinária , Coronaviridae , Hemaglutinação por Vírus , Doenças dos Roedores/microbiologia , Animais , Coronaviridae/enzimologia , Coronaviridae/isolamento & purificação , Infecções por Coronaviridae/microbiologia , Diarreia/microbiologia , Diarreia/veterinária , Camundongos , RatosRESUMO
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 (non-structural 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 replicase-transcriptase 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 non-permissive 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 propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved.
Assuntos
Coronaviridae/enzimologia , Coronaviridae/genética , RNA Polimerases Dirigidas por DNA/genética , Mutação , DNA Polimerase Dirigida por RNA/genética , Proteínas Virais/genéticaRESUMO
Many viruses achieve reversible attachment to sialic acid (Sia) by encoding envelope glycoproteins with receptor-binding and receptor-destroying activities. Toroviruses and group 2 coronaviruses bind to O-acetylated Sias, presumably via their spike proteins (S), whereas other glycoproteins, the hemagglutinin-esterases (HE), destroy Sia receptors by de-O-acetylation. Here, we present a comprehensive study of these enzymes. Sialate-9-O-acetylesterases specific for 5-N-acetyl-9-O-acetylneuraminic acid, described for bovine and human coronaviruses, also occur in equine coronaviruses and in porcine toroviruses. Bovine toroviruses, however, express novel sialate-9-O-acetylesterases, which prefer the di-O-acetylated substrate 5-N-acetyl-7(8),9-di-O-acetylneuraminic acid. Whereas most rodent coronaviruses express sialate-4-O-acetylesterases, the HE of murine coronavirus DVIM cleaves 9-O-acetylated Sias. Under the premise that HE specificity reflects receptor usage, we propose that two types of Sias serve as initial attachment factors for coronaviruses in mice. There are striking parallels between orthomyxo- and nidovirus biology. Reminiscent of antigenic shifts in orthomyxoviruses, rodent coronaviruses exchanged S and HE sequences through recombination to extents not appreciated before. As for orthomyxovirus reassortants, the fitness of nidovirus recombinant offspring probably depends both on antigenic properties and on compatibility of receptor-binding and receptor-destroying activities.
Assuntos
Acetiltransferases/fisiologia , Evolução Molecular , Nidovirales/enzimologia , Animais , Sequência de Bases , Coronaviridae/enzimologia , Coronaviridae/genética , Coronaviridae/patogenicidade , Hemaglutininas Virais/fisiologia , Humanos , Dados de Sequência Molecular , Nidovirales/genética , Nidovirales/patogenicidade , Receptores Virais/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Torovirus/enzimologia , Torovirus/genética , Torovirus/patogenicidade , Proteínas Virais de Fusão/fisiologiaRESUMO
Human coronavirus RNA, prepared by extraction of purified virions with phenol-chloroform, consists of a major 15 to 55S class and a minor 4S class of RNA fragments. Polyadenylic acid [poly (A)] sequences are present in 15 to 55S but not in 4S RNA, suggesting different functions for each class. A stretch of poly (A) of approximately 19 adenosine monophosphate residues was obtained in sizing experiments after digesting OC-43 RNA with pancreatic and T1 ribonucleases. An OC-43 virion RNA transcriptase could not be detected with systems optimal for detecting the transcriptases of influenza and Newcastle disease virus.
Assuntos
Coronaviridae/análise , RNA Polimerases Dirigidas por DNA/metabolismo , Poli A/análise , RNA Viral/análise , Sequência de Bases , Coronaviridae/enzimologiaRESUMO
The murine coronavirus polymerase gene is 22 kb in length with the potential to encode a polyprotein of approximately 750 kDa. The polyprotein has been proposed to encode three proteinase domains which are responsible for the processing of the polyprotein into mature proteins. The proteolytic activity of the first proteinase domain has been characterized and resembles the papain family of cysteine proteinases. This proteinase domain acts autoproteolytically to cleave the amino terminal portion of the polymerase polyprotein, releasing a 28-kDa protein designated p28. To identify the cleavage site of this papain-like cysteine proteinase, we isolated the peptide adjacent to p28 and determined the amino terminus sequence by Edman degradation reaction. We report that proteolysis occurs between the Gly-247 and Val-248 dipeptide bond. To determine the role of the amino acid residues surrounding the cleavage site, we introduced a total of 42 site-specific mutations at the residues spanning the P5 to P3' positions and assessed the effects of the mutations on the processing of p28 in an in vitro transcription and translation system. The substitutions of Gly-247 at the P1 position or Arg-246 at the P2 position resulted in a dramatic decrease of proteolytic activity, and the mutations of Arg-243 at P5 position also led to considerable reduction in p28 cleavage. In contrast, the substitutions of amino acids Gly-244 (P4), Tyr-245 (P3), Val-248 (P1'), Lys-249 (P2'), and Pro-250 (P3') had little or no effect on the amount of p28 that was released. This work had identified Gly-247-Val-248 as the cleavage site for the release of p28, the amino-terminal protein of the murine coronavirus polymerase polyprotein. Additionally, we conclude that the Gly-247 and Arg-246 are the major determinants for the cleavage site recognition by the first papain-like cysteine proteinase of murine coronavirus.
Assuntos
Coronaviridae/enzimologia , RNA Polimerases Dirigidas por DNA/metabolismo , Papaína/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
The nucleotide sequence of the human coronavirus 229E (HCV 229E) RNA polymerase gene and the 5' region of the genome has been determined. The polymerase gene is comprised of two large open reading frames, ORF1a and ORF1b, that contain 4086 and 2687 codons, respectively. ORF1b overlaps ORF1a by 43 bases in the (-1) reading frame. The in vitro translation of SP6 transcripts which include HCV 229E sequences encompassing the ORF1a/ORF1b junction show that expression of ORF1b can be mediated by ribosomal frame-shifting. The predicted translation products of ORF1a (454,200 molecular weight) and ORF1a/1b (754,200 molecular weight) have been compared to the predicted RNA polymerase gene products of infectious bronchitis virus (IBV) and murine hepatitis virus (MHV) and conserved structural features and putative functional domains have been identified. This analysis completes the nucleotide sequence of the HCV 229E genome.
Assuntos
Coronaviridae/genética , RNA Polimerases Dirigidas por DNA/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Coronaviridae/enzimologia , DNA Viral , RNA Polimerases Dirigidas por DNA/metabolismo , Genoma Viral , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , RNA ViralRESUMO
The polymerase-encoding region of the genomic RNA of the coronavirus infectious bronchitis virus (IBV) contains two very large, briefly overlapping open reading frames (ORF), F1 and F2, and it has been suggested on the basis of sequence analysis that expression of the downstream ORF, F2, might be mediated through ribosomal frame-shifting. To examine this possibility a cDNA fragment containing the F1/F2 overlap region was cloned within a marker gene and placed under the control of the bacteriophage SP6 promoter in a recombinant plasmid. Messenger RNA transcribed from this plasmid, when translated in cell-free systems, specified the synthesis of polypeptides whose size was entirely consistent with the products predicted by an efficient ribosomal frame-shifting event within the overlap region. The nature of the products was confirmed by their reactivity with antisera raised against defined portions of the flanking marker gene. This is the first non-retroviral example of ribosomal frame-shifting in higher eukaryotes.
Assuntos
Coronaviridae/genética , RNA Polimerases Dirigidas por DNA/genética , Genes Virais , Genes , RNA Viral/genética , Ribossomos/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Coronaviridae/enzimologia , Vetores Genéticos , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Biossíntese de Proteínas , RNA Mensageiro/genética , Transcrição GênicaRESUMO
An enzymatic activity which incorporates [3H]UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with the mitochondrial fraction of infected cells, suggesting that the enzyme is membrane bound. The activity required the presence of all three ribonucleoside triphosphates in addition to [3H]UTP, and it was not inhibited by actinomycin D. The heated product was digested by RNase but not by DNase. Mg2+ was required for enzymatic activity, and its optimal concentration was approximately 5 mM. The size of the in vitro products was compared by electrophoresis with that of in vivo-synthesized virus-specified RNA to confirm the viral specificity of the polymerase activity. Virus-specified RNA from infected cells consisted of 10 species of single-stranded, polyadenylated RNA with molecular weights of 6.8 X 10(6), 6.2 X 10(6), 3.15 X 10(6), 1.40 X 10(6), 1.05 X 10(6), 0.94 X 10(6), 0.66 X 10(6), 0.39 X 10(6), 0.34 X 10(6), and 0.24 X 10(6). In vitro synthesized RNA consisted of a high-molecular-weight species, of apparently higher molecular weight than genomic RNA, and two single-stranded species that electrophoretically comigrated with the species of 1.40 X 10(6) and 0.66 X 10(6) molecular weight made in vivo.
Assuntos
Coronaviridae/enzimologia , RNA Nucleotidiltransferases/análise , RNA Polimerase Dependente de RNA/análise , Vírus da Gastroenterite Transmissível/enzimologia , Replicação Viral , Animais , Fracionamento Celular , Linhagem Celular , Humanos , Masculino , Poli A/análise , RNA Viral/biossíntese , Suínos , Testículo , Vírion/análiseRESUMO
Coronavirus JHM contains six major proteins, one of which, the 60 000 mol. wt. nucleocapsid protein pp60, is phosphorylated. In JHM-infected cells ip 60K, the intracellular precursor to pp60 is also phosphorylated. Associated with purified JHM virions is a protein kinase which will phosphorylate pp60 and a variety of exogenous substances in vitro. The enzyme has the characteristics of a cyclic nucleotide-independent protein kinase. Both the in vivo reaction and the enzyme activity in vitro transferred the gamma-phosphate of ATP to serine residues on the nucleocapsid protein.