The Experts below are selected from a list of 5178 Experts worldwide ranked by ideXlab platform
Stuart G. Siddell - One of the best experts on this subject based on the ideXlab platform.
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The ADP-ribose-1 -monophosphatase domains of severe acute respiratory syndrome Coronavirus and Human Coronavirus 229E mediate resistance to antiviral interferon responses
Journal of General Virology, 2011Co-Authors: Thomas Kuri, Andrew D. Davidson, Ákos Putics, Klara K Eriksson, Roland Züst, John Ziebuhr, Volker Thiel, Stuart G. Siddell, Friedemann WeberAbstract:Several plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1″-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The relevance of these activities in the viral life cycle has not yet been resolved. Here, we report that genetically engineered mutants of severe acute respiratory syndrome Coronavirus (SARS-CoV) and Human Coronavirus 229E (HCoV-229E) expressing ADRP-deficient macrodomains displayed an increased sensitivity to the antiviral effect of alpha interferon compared with their wild-type counterparts. The data suggest that macrodomain-associated ADRP activities may have a role in viral escape from the innate immune responses of the host.
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selective replication of Coronavirus genomes that express nucleocapsid protein
Journal of Virology, 2005Co-Authors: Barbara Schelle, Nadja Karl, Stuart G. Siddell, Burkhard Ludewig, Volker ThielAbstract:The Coronavirus nucleocapsid (N) protein is a structural protein that forms a ribonucleoprotein complex with genomic RNA. In addition to its structural role, it has been described as an RNA-binding protein that might be involved in Coronavirus RNA synthesis. Here, we report a reverse genetic approach to elucidate the role of N in Coronavirus replication and transcription. We found that Human Coronavirus 229E (HCoV-229E) vector RNAs that lack the N gene were greatly impaired in their ability to replicate, whereas the transcription of subgenomic mRNA from these vectors was easily detectable. In contrast, vector RNAs encoding a functional N protein were able to carry out both replication and transcription. Furthermore, modification of the transcription signal required for the synthesis of N protein mRNAs in the HCoV-229E genome resulted in the selective replication of genomes that are able to express the N protein. This genetic evidence leads us to conclude that at least one Coronavirus structural protein, the N protein, is involved in Coronavirus replication.
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Multigene RNA Vector Based on Coronavirus Transcription
Journal of Virology, 2003Co-Authors: Volker Thiel, Barbara Schelle, Petra Disterer, Ingo M. Klagge, Nadja Karl, Stuart G. SiddellAbstract:Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on Human Coronavirus 229E. We have constructed a prototype RNA vector containing the 5′ and 3′ ends of the Human Coronavirus genome, the entire Human Coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a Human Coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and Human Coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of Coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that Human Coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature Human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the Human Coronavirus 229E and have the ability to transduce Human dendritic cells.
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viral replicase gene products suffice for Coronavirus discontinuous transcription
Journal of Virology, 2001Co-Authors: Volker Thiel, Barbara Schelle, Jens Herold, Stuart G. SiddellAbstract:We have used vaccinia virus as a vector to clone a 22.5-kbp cDNA that represents the 5′ and 3′ ends of the Human Coronavirus 229E (HCoV 229E) genome, the HCoV 229E replicase gene, and a single reporter gene (coding for green fluorescent protein [GFP]) located downstream of a regulatory element for Coronavirus mRNA transcription. When RNA transcribed from this cDNA was transfected into BHK-21 cells, a small percentage of cells displayed strong fluorescence. A region of the mRNA encoding GFP was amplified by PCR and shown to have the unique mRNA leader-body junction indicative of Coronavirus-mediated transcription. These data show that the Coronavirus replicase gene products suffice for discontinuous subgenomic mRNA transcription.
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infectious rna transcribed in vitro from a cdna copy of the Human Coronavirus genome cloned in vaccinia virus
Journal of General Virology, 2001Co-Authors: Volker Thiel, Barbara Schelle, Jens Herold, Stuart G. SiddellAbstract:The Coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant Coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the Human Coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of Coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.
John Ziebuhr - One of the best experts on this subject based on the ideXlab platform.
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identification and characterization of a Human Coronavirus 229E nonstructural protein 8 associated rna 3 terminal adenylyltransferase activity
Journal of Virology, 2019Co-Authors: Jana Tvarogova, Nadja Karl, Ramakanth Madhugiri, Ganesh Bylapudi, Lyndsey J Ferguson, John ZiebuhrAbstract:: Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the Human Coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5' oligo(U) sequence, while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5' overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other Coronavirus nsp8 proteins, suggesting that the activity is conserved among Coronaviruses. Replacement of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a nonviable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5' end of Coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3' polyadenylation of viral plus-strand RNAs.IMPORTANCE Previously, Coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies have suggested an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits Coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three Coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3' polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce cRNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in Coronavirus replication, the study amends the list of activities mediated by Coronavirus nsp8 proteins in the absence of other proteins.
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The ADP-ribose-1 -monophosphatase domains of severe acute respiratory syndrome Coronavirus and Human Coronavirus 229E mediate resistance to antiviral interferon responses
Journal of General Virology, 2011Co-Authors: Thomas Kuri, Andrew D. Davidson, Ákos Putics, Klara K Eriksson, Roland Züst, John Ziebuhr, Volker Thiel, Stuart G. Siddell, Friedemann WeberAbstract:Several plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1″-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The relevance of these activities in the viral life cycle has not yet been resolved. Here, we report that genetically engineered mutants of severe acute respiratory syndrome Coronavirus (SARS-CoV) and Human Coronavirus 229E (HCoV-229E) expressing ADRP-deficient macrodomains displayed an increased sensitivity to the antiviral effect of alpha interferon compared with their wild-type counterparts. The data suggest that macrodomain-associated ADRP activities may have a role in viral escape from the innate immune responses of the host.
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major genetic marker of nidoviruses encodes a replicative endoribonuclease
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Konstantin A Ivanov, Alexander E Gorbalenya, Volker Thiel, Tobias Hertzig, Mikhail Rozanov, Sonja Bayer, John ZiebuhrAbstract:Coronaviruses are important pathogens that cause acute respiratory diseases in Humans. Replication of the ≈30-kb positive-strand RNA genome of Coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The Coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome Coronavirus and Human Coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn2+-dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that double-stranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2′-3′ cyclic phosphate ends. 2′-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2′-O-ribose methyltransferase located adjacent to NendoU in the Coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length Human Coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription.
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Human Coronavirus 229E nonstructural protein 13 characterization of duplex unwinding nucleoside triphosphatase and rna 5 triphosphatase activities
Journal of Virology, 2004Co-Authors: Konstantin A Ivanov, John ZiebuhrAbstract:The Human Coronavirus 229E (HCoV-229E) replicase gene-encoded nonstructural protein 13 (nsp13) contains an N-terminal zinc-binding domain and a C-terminal superfamily 1 helicase domain. A histidine-tagged form of nsp13, which was expressed in insect cells and purified, is reported to unwind efficiently both partial-duplex RNA and DNA of up to several hundred base pairs. Characterization of the nsp13-associated nucleoside triphosphatase (NTPase) activities revealed that all natural ribonucleotides and nucleotides are substrates of nsp13, with ATP, dATP, and GTP being hydrolyzed most efficiently. Using the NTPase active site, HCoV-229E nsp13 also mediates RNA 5′-triphosphatase activity, which may be involved in the capping of viral RNAs.
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the autocatalytic release of a putative rna virus transcription factor from its polyprotein precursor involves two paralogous papain like proteases that cleave the same peptide bond
Journal of Biological Chemistry, 2001Co-Authors: John Ziebuhr, Volker Thiel, Alexander E GorbalenyaAbstract:The largest replicative protein of Coronaviruses is known as p195 in the avian infectious bronchitis virus (IBV) and p210 (p240) in the mouse hepatitis virus. It is autocatalytically released from the precursors pp1a and pp1ab by one zinc finger-containing papain-like protease (PLpro) in IBV and by two paralogous PLpros, PL1pro and PL2pro, in mouse hepatitis virus. The PLpro-containing proteins have been recently implicated in the control of Coronavirus subgenomic mRNA synthesis (transcription). By using comparative sequence analysis, we now show that the respective proteins of all sequenced Coronaviruses are flanked by two conserved PLpro cleavage sites and share a complex (multi)domain organization with PL1pro being inactivated in IBV. Based upon these predictions, the processing of the Human Coronavirus 229E p195/p210 N terminus was studied in detail. First, an 87-kDa protein (p87), which is derived from a pp1a/pp1ab region immediately upstream of p195/p210, was identified in Human Coronavirus 229E-infected cells. Second, in vitro synthesized proteins representing different parts of pp1a were autocatalytically processed at the predicted site. Surprisingly, both PL1pro and PL2pro cleaved between p87 and p195/p210. The PL1pro-mediated cleavage was slow and significantly suppressed by a non-proteolytic activity of PL2pro. In contrast, PL2pro, whose proteolytic activity and specificity were established in this study, cleaved the same site efficiently in the presence of the upstream domains. Third, a correlation was observed between the overlapping substrate specificities and the parallel evolution of PL1pro and PL2pro. Collectively, our results imply that the p195/p210 autoprocessing mechanisms may be conserved among Coronaviruses to an extent not appreciated previously, with PL2pro playing a major role. A large subset of Coronaviruses may employ two proteases to cleave the same site(s) and thus regulate the expression of the viral genome in a unique way.
Volker Thiel - One of the best experts on this subject based on the ideXlab platform.
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cyclosporin a inhibits the replication of diverse Coronaviruses
Journal of General Virology, 2011Co-Authors: Adriaan H De Wilde, Volker Thiel, Jessika C Zevenhovendobbe, Yvonne Van Der Meer, Krishna Narayanan, Shinji Makino, Eric J Snijder, Martijn J Van HemertAbstract:Low micromolar, non-cytotoxic concentrations of cyclosporin A (CsA) strongly affected the replication of severe acute respiratory syndrome Coronavirus (SARS-CoV), Human Coronavirus 229E and mouse hepatitis virus in cell culture, as was evident from the strong inhibition of GFP reporter gene expression and a reduction of up to 4 logs in progeny titres. Upon high-multiplicity infection, CsA treatment rendered SARS-CoV RNA and protein synthesis almost undetectable, suggesting an early block in replication. siRNA-mediated knockdown of the expression of the prominent CsA targets cyclophilin A and B did not affect SARS-CoV replication, suggesting either that these specific cyclophilin family members are dispensable or that the reduced expression levels suffice to support replication.
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The ADP-ribose-1 -monophosphatase domains of severe acute respiratory syndrome Coronavirus and Human Coronavirus 229E mediate resistance to antiviral interferon responses
Journal of General Virology, 2011Co-Authors: Thomas Kuri, Andrew D. Davidson, Ákos Putics, Klara K Eriksson, Roland Züst, John Ziebuhr, Volker Thiel, Stuart G. Siddell, Friedemann WeberAbstract:Several plus-strand RNA viruses encode proteins containing macrodomains. These domains possess ADP-ribose-1″-phosphatase (ADRP) activity and/or bind poly(ADP-ribose), poly(A) or poly(G). The relevance of these activities in the viral life cycle has not yet been resolved. Here, we report that genetically engineered mutants of severe acute respiratory syndrome Coronavirus (SARS-CoV) and Human Coronavirus 229E (HCoV-229E) expressing ADRP-deficient macrodomains displayed an increased sensitivity to the antiviral effect of alpha interferon compared with their wild-type counterparts. The data suggest that macrodomain-associated ADRP activities may have a role in viral escape from the innate immune responses of the host.
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selective replication of Coronavirus genomes that express nucleocapsid protein
Journal of Virology, 2005Co-Authors: Barbara Schelle, Nadja Karl, Stuart G. Siddell, Burkhard Ludewig, Volker ThielAbstract:The Coronavirus nucleocapsid (N) protein is a structural protein that forms a ribonucleoprotein complex with genomic RNA. In addition to its structural role, it has been described as an RNA-binding protein that might be involved in Coronavirus RNA synthesis. Here, we report a reverse genetic approach to elucidate the role of N in Coronavirus replication and transcription. We found that Human Coronavirus 229E (HCoV-229E) vector RNAs that lack the N gene were greatly impaired in their ability to replicate, whereas the transcription of subgenomic mRNA from these vectors was easily detectable. In contrast, vector RNAs encoding a functional N protein were able to carry out both replication and transcription. Furthermore, modification of the transcription signal required for the synthesis of N protein mRNAs in the HCoV-229E genome resulted in the selective replication of genomes that are able to express the N protein. This genetic evidence leads us to conclude that at least one Coronavirus structural protein, the N protein, is involved in Coronavirus replication.
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major genetic marker of nidoviruses encodes a replicative endoribonuclease
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Konstantin A Ivanov, Alexander E Gorbalenya, Volker Thiel, Tobias Hertzig, Mikhail Rozanov, Sonja Bayer, John ZiebuhrAbstract:Coronaviruses are important pathogens that cause acute respiratory diseases in Humans. Replication of the ≈30-kb positive-strand RNA genome of Coronaviruses and discontinuous synthesis of an extensive set of subgenome-length RNAs (transcription) are mediated by the replicase-transcriptase, a barely characterized protein complex that comprises several cellular proteins and up to 16 viral subunits. The Coronavirus replicase-transcriptase was recently predicted to contain RNA-processing enzymes that are extremely rare or absent in other RNA viruses. Here, we established and characterized the activity of one of these enzymes, replicative nidoviral uridylate-specific endoribonuclease (NendoU). It is considered a major genetic marker that discriminates nidoviruses (Coronaviridae, Arteriviridae, and Roniviridae) from all other RNA virus families. Bacterially expressed forms of NendoU of severe acute respiratory syndrome Coronavirus and Human Coronavirus 229E were revealed to cleave single-stranded and double-stranded RNA in a Mn2+-dependent manner. Single-stranded RNA was cleaved less specifically and effectively, suggesting that double-stranded RNA is the biologically relevant NendoU substrate. Double-stranded RNA substrates were cleaved upstream and downstream of uridylates at GUU or GU sequences to produce molecules with 2′-3′ cyclic phosphate ends. 2′-O-ribose-methylated RNA substrates proved to be resistant to cleavage by NendoU, indicating a functional link with the 2′-O-ribose methyltransferase located adjacent to NendoU in the Coronavirus replicative polyprotein. A mutagenesis study verified potential active-site residues and allowed us to inactivate NendoU in the full-length Human Coronavirus 229E clone. Substitution of D6408 by Ala was shown to abolish viral RNA synthesis, demonstrating that NendoU has critical functions in viral replication and transcription.
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Multigene RNA Vector Based on Coronavirus Transcription
Journal of Virology, 2003Co-Authors: Volker Thiel, Barbara Schelle, Petra Disterer, Ingo M. Klagge, Nadja Karl, Stuart G. SiddellAbstract:Coronavirus genomes are the largest known autonomously replicating RNAs with a size of ca. 30 kb. They are of positive polarity and are translated to produce the viral proteins needed for the assembly of an active replicase-transcriptase complex. In addition to replicating the genomic RNA, a key feature of this complex is a unique transcription process that results in the synthesis of a nested set of six to eight subgenomic mRNAs. These subgenomic mRNAs are produced in constant but nonequimolar amounts and, in general, each is translated to produce a single protein. To take advantage of these features, we have developed a multigene expression vector based on Human Coronavirus 229E. We have constructed a prototype RNA vector containing the 5′ and 3′ ends of the Human Coronavirus genome, the entire Human Coronavirus replicase gene, and three reporter genes (i.e., the chloramphenicol acetyltransferase [CAT] gene, the firefly luciferase [LUC] gene, and the green fluorescent protein [GFP] gene). Each reporter gene is located downstream of a Human Coronavirus transcription-associated sequence, which is required for the synthesis of individual subgenomic mRNAs. The transfection of vector RNA and Human Coronavirus nucleocapsid protein mRNA into BHK-21 cells resulted in the expression of the CAT, LUC, and GFP reporter proteins. Sequence analysis confirmed the synthesis of Coronavirus-specific mRNAs encoding CAT, LUC, and GFP. In addition, we have shown that Human Coronavirus-based vector RNA can be packaged into virus-like particles that, in turn, can be used to transduce immature and mature Human dendritic cells. In summary, we describe a new class of eukaryotic, multigene expression vectors that are based on the Human Coronavirus 229E and have the ability to transduce Human dendritic cells.
Shutoku Matsuyama - One of the best experts on this subject based on the ideXlab platform.
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the inhaled steroid ciclesonide blocks sars cov 2 rna replication by targeting the viral replication transcription complex in cultured cells
Journal of Virology, 2020Co-Authors: Shutoku Matsuyama, Miyuki Kawase, Naganori Nao, Kazuya Shirato, Makoto Ujike, Wataru Kamitani, Masayuki Shimojima, Shuetsu FukushiAbstract:Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome Coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other Coronaviruses, including severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2), the cause of Coronavirus disease 2019 (COVID-19), in cultured cells. The 90% effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated Human bronchial tracheal epithelial cells was 0.55 µM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double-stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to Coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients.IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses the replication of Coronaviruses, including betaCoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphaCoronavirus (Human Coronavirus 229E [HCoV-229E]), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the Coronavirus family. It could be prescribed for the treatment of MERS and COVID-19.
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clinical isolates of Human Coronavirus 229E bypass the endosome for cell entry
Journal of Virology, 2017Co-Authors: Kazuya Shirato, Miyuki Kawase, Kazuhiko Kanou, Shutoku MatsuyamaAbstract:Human Coronavirus 229E (HCoV-229E), a causative agent of the common cold, enters host cells via two distinct pathways: one is mediated by cell surface proteases, particularly transmembrane protease serine 2 (TMPRSS2), and the other by endosomal cathepsin L. Thus, specific inhibitors of these proteases block virus infection. However, it is unclear which of these pathways is actually utilized by HCoV-229E in the Human respiratory tract. Here, we examined the mechanism of cell entry used by a pseudotyped virus bearing the HCoV-229E spike (S) protein in the presence or absence of protease inhibitors. We found that, compared with a laboratory strain isolated in 1966 and passaged for a half century, clinical isolates of HCoV-229E were less likely to utilize cathepsin L; rather, they showed a preference for TMPRSS2. Two amino acid substitutions (R642M and N714K) in the S protein of HCoV-229E clinical isolates altered their sensitivity to a cathepsin L inhibitor, suggesting that these amino acids were responsible for cathepsin L use. After 20 passages in HeLa cells, the ability of the isolate to use cathepsin increased so that it was equal to that of the laboratory strain; this increase was caused by an amino acid substitution (I577S) in the S protein. The passaged virus showed a reduced ability to replicate in differentiated airway epithelial cells cultured at an air-liquid interface. These results suggest that the endosomal pathway is disadvantageous for HCoV-229E infection of Human airway epithelial cells; therefore, clinical isolates are less able to use cathepsin. IMPORTANCE Many enveloped viruses enter cells through endocytosis. Viral spike proteins drive the fusion of viral and endosomal membranes to facilitate insertion of the viral genome into the cytoplasm. Human Coronavirus 229E (HCoV-229E) utilizes endosomal cathepsin L to activate the spike protein after receptor binding. Here, we found that clinical isolates of HCoV-229E preferentially utilize the cell surface protease TMPRSS2 rather than endosomal cathepsin L. The endosome is a main site of Toll-like receptor recognition, which then triggers an innate immune response; therefore, HCoV-229E presumably evolved to bypass the endosome by entering the cell via TMPRSS2. Thus, the virus uses a simple mechanism to evade the host innate immune system. Therefore, therapeutic agents for Coronavirus-mediated diseases, such as severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), should target cell surface TMPRSS2 rather than endosomal cathepsin.
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possible involvement of infection with Human Coronavirus 229E but not nl63 in kawasaki disease
Journal of Medical Virology, 2014Co-Authors: Kazuya Shirato, Shutoku Matsuyama, Miyuki Kawase, Yoshio Imada, Keiko Nakagaki, Fumihiro TaguchiAbstract:Although Human Coronavirus (HCoV)-NL63 was once considered a possible causative agent of Kawasaki disease based on RT-PCR analyses, subsequent studies could not confirm the result. In this study, this possibility was explored using serological tests. To evaluate the role of HCoV infection in patients with Kawasaki disease, immunofluorescence assays and virus neutralizing tests were performed. Paired serum samples were obtained from patients with Kawasaki disease who had not been treated with γ-globulin. HCoV-NL63 and two antigenically different isolates of HCoV-229E (ATCC-VR740 and a new isolate, Sendai-H) were examined as controls. Immunofluorescence assays detected no difference in HCoV-NL63 antibody positivity between the patients with Kawasaki disease and controls, whereas the rate of HCoV-229E antibody positivity was higher in the patients with Kawasaki disease than that in controls. The neutralizing tests revealed no difference in seropositivity between the acute and recovery phases of patients with Kawasaki disease for the two HCoV-229Es. However, the Kawasaki disease specimens obtained from patients in recovery phase displayed significantly higher positivity for Sendai-H, but not for ATCC-VR740, as compared to the controls. The serological test supported no involvement of HCoV-NL63 but suggested the possible involvement of HCoV-229E in the development of Kawasaki disease.
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protease mediated entry via the endosome of Human Coronavirus 229E
Journal of Virology, 2009Co-Authors: Miyuki Kawase, Shutoku Matsuyama, Kazuya Shirato, Fumihiro TaguchiAbstract:Human Coronavirus 229E, classified as a group I Coronavirus, utilizes Human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome Coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine Coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.
Fumihiro Taguchi - One of the best experts on this subject based on the ideXlab platform.
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possible involvement of infection with Human Coronavirus 229E but not nl63 in kawasaki disease
Journal of Medical Virology, 2014Co-Authors: Kazuya Shirato, Shutoku Matsuyama, Miyuki Kawase, Yoshio Imada, Keiko Nakagaki, Fumihiro TaguchiAbstract:Although Human Coronavirus (HCoV)-NL63 was once considered a possible causative agent of Kawasaki disease based on RT-PCR analyses, subsequent studies could not confirm the result. In this study, this possibility was explored using serological tests. To evaluate the role of HCoV infection in patients with Kawasaki disease, immunofluorescence assays and virus neutralizing tests were performed. Paired serum samples were obtained from patients with Kawasaki disease who had not been treated with γ-globulin. HCoV-NL63 and two antigenically different isolates of HCoV-229E (ATCC-VR740 and a new isolate, Sendai-H) were examined as controls. Immunofluorescence assays detected no difference in HCoV-NL63 antibody positivity between the patients with Kawasaki disease and controls, whereas the rate of HCoV-229E antibody positivity was higher in the patients with Kawasaki disease than that in controls. The neutralizing tests revealed no difference in seropositivity between the acute and recovery phases of patients with Kawasaki disease for the two HCoV-229Es. However, the Kawasaki disease specimens obtained from patients in recovery phase displayed significantly higher positivity for Sendai-H, but not for ATCC-VR740, as compared to the controls. The serological test supported no involvement of HCoV-NL63 but suggested the possible involvement of HCoV-229E in the development of Kawasaki disease.
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protease mediated entry via the endosome of Human Coronavirus 229E
Journal of Virology, 2009Co-Authors: Miyuki Kawase, Shutoku Matsuyama, Kazuya Shirato, Fumihiro TaguchiAbstract:Human Coronavirus 229E, classified as a group I Coronavirus, utilizes Human aminopeptidase N (APN) as a receptor; however, its entry mechanism has not yet been fully elucidated. We found that HeLa cells infected with 229E via APN formed syncytia when treated with trypsin or other proteases but not in a low-pH environment, a finding consistent with syncytium formation by severe acute respiratory syndrome Coronavirus (SARS-CoV). In addition, trypsin induced cleavage of the 229E S protein. By using infectious viruses and pseudotyped viruses bearing the 229E S protein, we found that its infection was profoundly blocked by lysosomotropic agents as well as by protease inhibitors that also prevented infection with SARS-CoV but not that caused by murine Coronavirus mouse hepatitis virus strain JHMV, which enters cells directly from the cell surface. We found that cathepsin L (CPL) inhibitors blocked 229E infection the most remarkably among a variety of protease inhibitors tested. Furthermore, 229E infection was inhibited in CPL knockdown cells by small interfering RNA, compared with what was seen for a normal counterpart producing CPL. However, its inhibition was not so remarkable as that found with SARS-CoV infection, which seems to indicate that while CPL is involved in the fusogenic activation of 229E S protein in endosomal infection, not-yet-identified proteases could also play a part in that activity. We also found 229E virion S protein to be cleaved by CPL. Furthermore, as with SARS-CoV, 229E entered cells directly from the cell surface when cell-attached viruses were treated with trypsin. These findings suggest that 229E takes an endosomal pathway for cell entry and that proteases like CPL are involved in this mode of entry.