Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
Identifieur interne : 000756 ( PascalFrancis/Corpus ); précédent : 000755; suivant : 000757Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
Auteurs : Brian H. Harcourt ; Dalia Jukneliene ; Amornrat Kanjanahaluethai ; John Bechill ; Kari M. Severson ; Catherine M. Smith ; Paul A. Rota ; Susan C. BakerSource :
- Journal of virology [ 0022-538X ] ; 2004.
Descripteurs français
- Pascal (Inist)
English descriptors
- KwdEn :
Abstract
Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nspl, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nspl (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nspl/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.
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Format Inist (serveur)
NO : | PASCAL 05-0016080 INIST |
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ET : | Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity |
AU : | HARCOURT (Brian H.); JUKNELIENE (Dalia); KANJANAHALUETHAI (Amornrat); BECHILL (John); SEVERSON (Kari M.); SMITH (Catherine M.); ROTA (Paul A.); BAKER (Susan C.) |
AF : | Centers for Disease Control and Prevention/Atlanta, Georgia/Etats-Unis (1 aut., 7 aut.); Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago/Maywood, Illinois/Etats-Unis (2 aut., 3 aut., 4 aut., 5 aut., 6 aut., 8 aut.); Department of Microbiology, Faculty of Medicine, Chiang Mai University/Chiang Mai/Thaïlande (3 aut.) |
DT : | Publication en série; Niveau analytique |
SO : | Journal of virology; ISSN 0022-538X; Etats-Unis; Da. 2004; Vol. 78; No. 24; Pp. 13600-13612; Bibl. 35 ref. |
LA : | Anglais |
EA : | Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nspl, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nspl (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nspl/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site. |
CC : | 002A05C10 |
FD : | Coronavirus; Identification; Grave; Malade état grave; Aigu; Voie respiratoire; RNA-directed RNA polymerase; Caractérisation; Papain; Syndrome respiratoire aigu sévère; Microbiologie; Virologie; Forme grave |
FG : | Coronaviridae; Nidovirales; Virus; Nucleotidyltransferases; Transferases; Enzyme; Cysteine endopeptidases; Peptidases; Hydrolases; Virose; Infection; Appareil respiratoire pathologie; Poumon pathologie; Appareil respiratoire |
ED : | Coronavirus; Identification; Severe; Critically ill; Acute; Respiratory tract; RNA-directed RNA polymerase; Characterization; Papain; Severe acute respiratory syndrome; Microbiology; Virology |
EG : | Coronaviridae; Nidovirales; Virus; Nucleotidyltransferases; Transferases; Enzyme; Cysteine endopeptidases; Peptidases; Hydrolases; Viral disease; Infection; Respiratory disease; Lung disease; Respiratory system |
SD : | Coronavirus; Identificación; Grave; Enfermo estado grave; Agudo; Vía respiratoria; RNA-directed RNA polymerase; Caracterización; Papain; Síndrome respiratorio agudo severo; Microbiología; Virología |
LO : | INIST-13592.354000122800130220 |
ID : | 05-0016080 |
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Pascal:05-0016080Le document en format XML
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<front><div type="abstract" xml:lang="en">Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nspl, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nspl (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nspl/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.</div>
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<s5>01</s5>
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<fC03 i1="01" i2="X" l="SPA"><s0>Coronavirus</s0>
<s2>NW</s2>
<s5>01</s5>
</fC03>
<fC03 i1="02" i2="X" l="FRE"><s0>Identification</s0>
<s5>05</s5>
</fC03>
<fC03 i1="02" i2="X" l="ENG"><s0>Identification</s0>
<s5>05</s5>
</fC03>
<fC03 i1="02" i2="X" l="SPA"><s0>Identificación</s0>
<s5>05</s5>
</fC03>
<fC03 i1="03" i2="X" l="FRE"><s0>Grave</s0>
<s5>06</s5>
</fC03>
<fC03 i1="03" i2="X" l="ENG"><s0>Severe</s0>
<s5>06</s5>
</fC03>
<fC03 i1="03" i2="X" l="SPA"><s0>Grave</s0>
<s5>06</s5>
</fC03>
<fC03 i1="04" i2="X" l="FRE"><s0>Malade état grave</s0>
<s5>07</s5>
</fC03>
<fC03 i1="04" i2="X" l="ENG"><s0>Critically ill</s0>
<s5>07</s5>
</fC03>
<fC03 i1="04" i2="X" l="SPA"><s0>Enfermo estado grave</s0>
<s5>07</s5>
</fC03>
<fC03 i1="05" i2="X" l="FRE"><s0>Aigu</s0>
<s5>08</s5>
</fC03>
<fC03 i1="05" i2="X" l="ENG"><s0>Acute</s0>
<s5>08</s5>
</fC03>
<fC03 i1="05" i2="X" l="SPA"><s0>Agudo</s0>
<s5>08</s5>
</fC03>
<fC03 i1="06" i2="X" l="FRE"><s0>Voie respiratoire</s0>
<s5>09</s5>
</fC03>
<fC03 i1="06" i2="X" l="ENG"><s0>Respiratory tract</s0>
<s5>09</s5>
</fC03>
<fC03 i1="06" i2="X" l="SPA"><s0>Vía respiratoria</s0>
<s5>09</s5>
</fC03>
<fC03 i1="07" i2="X" l="FRE"><s0>RNA-directed RNA polymerase</s0>
<s2>FE</s2>
<s5>10</s5>
</fC03>
<fC03 i1="07" i2="X" l="ENG"><s0>RNA-directed RNA polymerase</s0>
<s2>FE</s2>
<s5>10</s5>
</fC03>
<fC03 i1="07" i2="X" l="SPA"><s0>RNA-directed RNA polymerase</s0>
<s2>FE</s2>
<s5>10</s5>
</fC03>
<fC03 i1="08" i2="X" l="FRE"><s0>Caractérisation</s0>
<s5>11</s5>
</fC03>
<fC03 i1="08" i2="X" l="ENG"><s0>Characterization</s0>
<s5>11</s5>
</fC03>
<fC03 i1="08" i2="X" l="SPA"><s0>Caracterización</s0>
<s5>11</s5>
</fC03>
<fC03 i1="09" i2="X" l="FRE"><s0>Papain</s0>
<s2>FE</s2>
<s5>12</s5>
</fC03>
<fC03 i1="09" i2="X" l="ENG"><s0>Papain</s0>
<s2>FE</s2>
<s5>12</s5>
</fC03>
<fC03 i1="09" i2="X" l="SPA"><s0>Papain</s0>
<s2>FE</s2>
<s5>12</s5>
</fC03>
<fC03 i1="10" i2="X" l="FRE"><s0>Syndrome respiratoire aigu sévère</s0>
<s2>NM</s2>
<s5>14</s5>
</fC03>
<fC03 i1="10" i2="X" l="ENG"><s0>Severe acute respiratory syndrome</s0>
<s2>NM</s2>
<s5>14</s5>
</fC03>
<fC03 i1="10" i2="X" l="SPA"><s0>Síndrome respiratorio agudo severo</s0>
<s2>NM</s2>
<s5>14</s5>
</fC03>
<fC03 i1="11" i2="X" l="FRE"><s0>Microbiologie</s0>
<s5>68</s5>
</fC03>
<fC03 i1="11" i2="X" l="ENG"><s0>Microbiology</s0>
<s5>68</s5>
</fC03>
<fC03 i1="11" i2="X" l="SPA"><s0>Microbiología</s0>
<s5>68</s5>
</fC03>
<fC03 i1="12" i2="X" l="FRE"><s0>Virologie</s0>
<s5>69</s5>
</fC03>
<fC03 i1="12" i2="X" l="ENG"><s0>Virology</s0>
<s5>69</s5>
</fC03>
<fC03 i1="12" i2="X" l="SPA"><s0>Virología</s0>
<s5>69</s5>
</fC03>
<fC03 i1="13" i2="X" l="FRE"><s0>Forme grave</s0>
<s4>INC</s4>
<s5>79</s5>
</fC03>
<fC07 i1="01" i2="X" l="FRE"><s0>Coronaviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="01" i2="X" l="ENG"><s0>Coronaviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="01" i2="X" l="SPA"><s0>Coronaviridae</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="FRE"><s0>Nidovirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="ENG"><s0>Nidovirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="02" i2="X" l="SPA"><s0>Nidovirales</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="FRE"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="ENG"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="03" i2="X" l="SPA"><s0>Virus</s0>
<s2>NW</s2>
</fC07>
<fC07 i1="04" i2="X" l="FRE"><s0>Nucleotidyltransferases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="04" i2="X" l="ENG"><s0>Nucleotidyltransferases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="04" i2="X" l="SPA"><s0>Nucleotidyltransferases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="05" i2="X" l="FRE"><s0>Transferases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="05" i2="X" l="ENG"><s0>Transferases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="05" i2="X" l="SPA"><s0>Transferases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="06" i2="X" l="FRE"><s0>Enzyme</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="06" i2="X" l="ENG"><s0>Enzyme</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="06" i2="X" l="SPA"><s0>Enzima</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="07" i2="X" l="FRE"><s0>Cysteine endopeptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="07" i2="X" l="ENG"><s0>Cysteine endopeptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="07" i2="X" l="SPA"><s0>Cysteine endopeptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="08" i2="X" l="FRE"><s0>Peptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="08" i2="X" l="ENG"><s0>Peptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="08" i2="X" l="SPA"><s0>Peptidases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="09" i2="X" l="FRE"><s0>Hydrolases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="09" i2="X" l="ENG"><s0>Hydrolases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="09" i2="X" l="SPA"><s0>Hydrolases</s0>
<s2>FE</s2>
</fC07>
<fC07 i1="10" i2="X" l="FRE"><s0>Virose</s0>
<s2>NM</s2>
</fC07>
<fC07 i1="10" i2="X" l="ENG"><s0>Viral disease</s0>
<s2>NM</s2>
</fC07>
<fC07 i1="10" i2="X" l="SPA"><s0>Virosis</s0>
<s2>NM</s2>
</fC07>
<fC07 i1="11" i2="X" l="FRE"><s0>Infection</s0>
<s2>NM</s2>
</fC07>
<fC07 i1="11" i2="X" l="ENG"><s0>Infection</s0>
<s2>NM</s2>
</fC07>
<fC07 i1="11" i2="X" l="SPA"><s0>Infección</s0>
<s2>NM</s2>
</fC07>
<fC07 i1="12" i2="X" l="FRE"><s0>Appareil respiratoire pathologie</s0>
<s5>19</s5>
</fC07>
<fC07 i1="12" i2="X" l="ENG"><s0>Respiratory disease</s0>
<s5>19</s5>
</fC07>
<fC07 i1="12" i2="X" l="SPA"><s0>Aparato respiratorio patología</s0>
<s5>19</s5>
</fC07>
<fC07 i1="13" i2="X" l="FRE"><s0>Poumon pathologie</s0>
<s5>20</s5>
</fC07>
<fC07 i1="13" i2="X" l="ENG"><s0>Lung disease</s0>
<s5>20</s5>
</fC07>
<fC07 i1="13" i2="X" l="SPA"><s0>Pulmón patología</s0>
<s5>20</s5>
</fC07>
<fC07 i1="14" i2="X" l="FRE"><s0>Appareil respiratoire</s0>
<s5>22</s5>
</fC07>
<fC07 i1="14" i2="X" l="ENG"><s0>Respiratory system</s0>
<s5>22</s5>
</fC07>
<fC07 i1="14" i2="X" l="SPA"><s0>Aparato respiratorio</s0>
<s5>22</s5>
</fC07>
<fN21><s1>004</s1>
</fN21>
<fN44 i1="01"><s1>OTO</s1>
</fN44>
<fN82><s1>OTO</s1>
</fN82>
</pA>
</standard>
<server><NO>PASCAL 05-0016080 INIST</NO>
<ET>Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity</ET>
<AU>HARCOURT (Brian H.); JUKNELIENE (Dalia); KANJANAHALUETHAI (Amornrat); BECHILL (John); SEVERSON (Kari M.); SMITH (Catherine M.); ROTA (Paul A.); BAKER (Susan C.)</AU>
<AF>Centers for Disease Control and Prevention/Atlanta, Georgia/Etats-Unis (1 aut., 7 aut.); Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago/Maywood, Illinois/Etats-Unis (2 aut., 3 aut., 4 aut., 5 aut., 6 aut., 8 aut.); Department of Microbiology, Faculty of Medicine, Chiang Mai University/Chiang Mai/Thaïlande (3 aut.)</AF>
<DT>Publication en série; Niveau analytique</DT>
<SO>Journal of virology; ISSN 0022-538X; Etats-Unis; Da. 2004; Vol. 78; No. 24; Pp. 13600-13612; Bibl. 35 ref.</SO>
<LA>Anglais</LA>
<EA>Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase polyproteins that are predicted to be processed into 16 nonstructural proteins (nsps 1 to 16) by two viral proteases, a papain-like protease (PLpro) and a 3C-like protease (3CLpro). Here, we identify SARS coronavirus amino-terminal replicase products nspl, nsp2, and nsp3 and describe trans-cleavage assays that characterize the protease activity required to generate these products. We generated polyclonal antisera to glutathione S-transferase-replicase fusion proteins and used the antisera to detect replicase intermediates and products in pulse-chase experiments. We found that nspl (p20) is rapidly processed from the replicase polyprotein. In contrast, processing at the nsp2/3 site is less efficient, since a 300-kDa intermediate (NSP2-3) is detected, but ultimately nsp2 (p71) and nsp3 (p213) are generated. We found that SARS coronavirus replicase products can be detected by 4 h postinfection in the cytoplasm of infected cells and that nsps 1 to 3 colocalize with newly synthesized viral RNA in punctate, perinuclear sites consistent with their predicted role in viral RNA synthesis. To determine if PLpro is responsible for processing these products, we cloned and expressed the PLpro domain and the predicted substrates and established PLpro trans-cleavage assays. We found that the PLpro domain is sufficient for processing the predicted nspl/2 and nsp2/3 sites. Interestingly, expression of an extended region of PLpro that includes the downstream hydrophobic domain was required for processing at the predicted nsp3/4 site. We found that the hydrophobic domain is inserted into membranes and that the lumenal domain is glycosylated at asparagine residues 2249 and 2252. Thus, the hydrophobic domain may anchor the replication complex to intracellular membranes. These studies revealed that PLpro can cleave in trans at the three predicted cleavage sites and that it requires membrane association to process the nsp3/4 cleavage site.</EA>
<CC>002A05C10</CC>
<FD>Coronavirus; Identification; Grave; Malade état grave; Aigu; Voie respiratoire; RNA-directed RNA polymerase; Caractérisation; Papain; Syndrome respiratoire aigu sévère; Microbiologie; Virologie; Forme grave</FD>
<FG>Coronaviridae; Nidovirales; Virus; Nucleotidyltransferases; Transferases; Enzyme; Cysteine endopeptidases; Peptidases; Hydrolases; Virose; Infection; Appareil respiratoire pathologie; Poumon pathologie; Appareil respiratoire</FG>
<ED>Coronavirus; Identification; Severe; Critically ill; Acute; Respiratory tract; RNA-directed RNA polymerase; Characterization; Papain; Severe acute respiratory syndrome; Microbiology; Virology</ED>
<EG>Coronaviridae; Nidovirales; Virus; Nucleotidyltransferases; Transferases; Enzyme; Cysteine endopeptidases; Peptidases; Hydrolases; Viral disease; Infection; Respiratory disease; Lung disease; Respiratory system</EG>
<SD>Coronavirus; Identificación; Grave; Enfermo estado grave; Agudo; Vía respiratoria; RNA-directed RNA polymerase; Caracterización; Papain; Síndrome respiratorio agudo severo; Microbiología; Virología</SD>
<LO>INIST-13592.354000122800130220</LO>
<ID>05-0016080</ID>
</server>
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