Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity

Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase poly-proteins 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 identif...

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Main Authors: Harcourt B.H., Jukneliene D., Kanjanahaluethai A., Bechill J., Severson K.M., Smith C.M., Rota P.A., Baker S.C.
Format: Article
Language:English
Published: 2014
Online Access:http://www.scopus.com/inward/record.url?eid=2-s2.0-10044268025&partnerID=40&md5=7c5b29b2581178033f18b4b4f2886707
http://cmuir.cmu.ac.th/handle/6653943832/3744
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spelling th-cmuir.6653943832-37442014-08-30T02:35:16Z Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity Harcourt B.H. Jukneliene D. Kanjanahaluethai A. Bechill J. Severson K.M. Smith C.M. Rota P.A. Baker S.C. Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase poly-proteins 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 aminoterminal replicase products nsp1, 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 nsp1/2 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. 2014-08-30T02:35:16Z 2014-08-30T02:35:16Z 2004 Article 0022538X 10.1128/JVI.78.24.13600-13612.2004 15564471 JOVIA http://www.scopus.com/inward/record.url?eid=2-s2.0-10044268025&partnerID=40&md5=7c5b29b2581178033f18b4b4f2886707 http://cmuir.cmu.ac.th/handle/6653943832/3744 English
institution Chiang Mai University
building Chiang Mai University Library
country Thailand
collection CMU Intellectual Repository
language English
description Gene 1 of the coronavirus associated with severe acute respiratory syndrome (SARS) encodes replicase poly-proteins 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 aminoterminal replicase products nsp1, 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 nsp1/2 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.
format Article
author Harcourt B.H.
Jukneliene D.
Kanjanahaluethai A.
Bechill J.
Severson K.M.
Smith C.M.
Rota P.A.
Baker S.C.
spellingShingle Harcourt B.H.
Jukneliene D.
Kanjanahaluethai A.
Bechill J.
Severson K.M.
Smith C.M.
Rota P.A.
Baker S.C.
Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
author_facet Harcourt B.H.
Jukneliene D.
Kanjanahaluethai A.
Bechill J.
Severson K.M.
Smith C.M.
Rota P.A.
Baker S.C.
author_sort Harcourt B.H.
title Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
title_short Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
title_full Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
title_fullStr Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
title_full_unstemmed Identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
title_sort identification of severe acute respiratory syndrome coronavirus replicase products and characterization of papain-like protease activity
publishDate 2014
url http://www.scopus.com/inward/record.url?eid=2-s2.0-10044268025&partnerID=40&md5=7c5b29b2581178033f18b4b4f2886707
http://cmuir.cmu.ac.th/handle/6653943832/3744
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