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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Severe Acute Respiratory Syndrome Coronavirus nsp1 Suppresses Host Gene Expression, Including That of Type I Interferon, in Infected Cells<xref ref-type="fn" rid="fn2">▿</xref>
</title>
<author><name sortKey="Narayanan, Krishna" sort="Narayanan, Krishna" uniqKey="Narayanan K" first="Krishna" last="Narayanan">Krishna Narayanan</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Huang, Cheng" sort="Huang, Cheng" uniqKey="Huang C" first="Cheng" last="Huang">Cheng Huang</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lokugamage, Kumari" sort="Lokugamage, Kumari" uniqKey="Lokugamage K" first="Kumari" last="Lokugamage">Kumari Lokugamage</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kamitani, Wataru" sort="Kamitani, Wataru" uniqKey="Kamitani W" first="Wataru" last="Kamitani">Wataru Kamitani</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ikegami, Tetsuro" sort="Ikegami, Tetsuro" uniqKey="Ikegami T" first="Tetsuro" last="Ikegami">Tetsuro Ikegami</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tseng, Chien Te K" sort="Tseng, Chien Te K" uniqKey="Tseng C" first="Chien-Te K." last="Tseng">Chien-Te K. Tseng</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Makino, Shinji" sort="Makino, Shinji" uniqKey="Makino S" first="Shinji" last="Makino">Shinji Makino</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">18305050</idno>
<idno type="pmc">2293030</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2293030</idno>
<idno type="RBID">PMC:2293030</idno>
<idno type="doi">10.1128/JVI.02472-07</idno>
<date when="2008">2008</date>
<idno type="wicri:Area/Pmc/Corpus">000663</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000663</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Severe Acute Respiratory Syndrome Coronavirus nsp1 Suppresses Host Gene Expression, Including That of Type I Interferon, in Infected Cells<xref ref-type="fn" rid="fn2">▿</xref>
</title>
<author><name sortKey="Narayanan, Krishna" sort="Narayanan, Krishna" uniqKey="Narayanan K" first="Krishna" last="Narayanan">Krishna Narayanan</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Huang, Cheng" sort="Huang, Cheng" uniqKey="Huang C" first="Cheng" last="Huang">Cheng Huang</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Lokugamage, Kumari" sort="Lokugamage, Kumari" uniqKey="Lokugamage K" first="Kumari" last="Lokugamage">Kumari Lokugamage</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Kamitani, Wataru" sort="Kamitani, Wataru" uniqKey="Kamitani W" first="Wataru" last="Kamitani">Wataru Kamitani</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ikegami, Tetsuro" sort="Ikegami, Tetsuro" uniqKey="Ikegami T" first="Tetsuro" last="Ikegami">Tetsuro Ikegami</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Tseng, Chien Te K" sort="Tseng, Chien Te K" uniqKey="Tseng C" first="Chien-Te K." last="Tseng">Chien-Te K. Tseng</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Makino, Shinji" sort="Makino, Shinji" uniqKey="Makino S" first="Shinji" last="Makino">Shinji Makino</name>
<affiliation><nlm:aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">Journal of Virology</title>
<idno type="ISSN">0022-538X</idno>
<idno type="eISSN">1098-5514</idno>
<imprint><date when="2008">2008</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p>The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.</p>
</div>
</front>
</TEI>
<pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Virol</journal-id>
<journal-id journal-id-type="publisher-id">jvi</journal-id>
<journal-title>Journal of Virology</journal-title>
<issn pub-type="ppub">0022-538X</issn>
<issn pub-type="epub">1098-5514</issn>
<publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">18305050</article-id>
<article-id pub-id-type="pmc">2293030</article-id>
<article-id pub-id-type="publisher-id">2472-07</article-id>
<article-id pub-id-type="doi">10.1128/JVI.02472-07</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Virus-Cell Interactions</subject>
</subj-group>
</article-categories>
<title-group><article-title>Severe Acute Respiratory Syndrome Coronavirus nsp1 Suppresses Host Gene Expression, Including That of Type I Interferon, in Infected Cells<xref ref-type="fn" rid="fn2">▿</xref>
</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Narayanan</surname>
<given-names>Krishna</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
<xref ref-type="fn" rid="fn1">†</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Huang</surname>
<given-names>Cheng</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
<xref ref-type="fn" rid="fn1">†</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Lokugamage</surname>
<given-names>Kumari</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
<xref ref-type="fn" rid="fn1">†</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Kamitani</surname>
<given-names>Wataru</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ikegami</surname>
<given-names>Tetsuro</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Tseng</surname>
<given-names>Chien-Te K.</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
</contrib>
<contrib contrib-type="author"><name><surname>Makino</surname>
<given-names>Shinji</given-names>
</name>
<xref ref-type="aff" rid="aff0"></xref>
<xref ref-type="corresp" rid="cor1">*</xref>
</contrib>
</contrib-group>
<aff id="aff0">Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, Texas 77555-1019</aff>
<author-notes><fn id="cor1"><label>*</label>
<p>Corresponding author. Mailing address: Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-1019. Phone: (409) 772-2323. Fax: (409) 772-5065. E-mail: <email>shmakino@utmb.edu</email>
</p>
</fn>
<fn id="fn1"><label>†</label>
<p>K.N., C.H., and K.L. contributed equally to this study.</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><month>5</month>
<year>2008</year>
</pub-date>
<pub-date pub-type="epub"><day>27</day>
<month>2</month>
<year>2008</year>
</pub-date>
<volume>82</volume>
<issue>9</issue>
<fpage>4471</fpage>
<lpage>4479</lpage>
<history><date date-type="received"><day>16</day>
<month>11</month>
<year>2007</year>
</date>
<date date-type="accepted"><day>15</day>
<month>2</month>
<year>2008</year>
</date>
</history>
<permissions><copyright-statement>Copyright © 2008, American Society for Microbiology</copyright-statement>
</permissions>
<self-uri xlink:title="pdf" xlink:href="zjv00908004471.pdf"></self-uri>
<abstract><p>The severe acute respiratory syndrome coronavirus (SARS-CoV) nsp1 protein has unique biological functions that have not been described in the viral proteins of any RNA viruses; expressed SARS-CoV nsp1 protein has been found to suppress host gene expression by promoting host mRNA degradation and inhibiting translation. We generated an nsp1 mutant (nsp1-mt) that neither promoted host mRNA degradation nor suppressed host protein synthesis in expressing cells. Both a SARS-CoV mutant virus, encoding the nsp1-mt protein (SARS-CoV-mt), and a wild-type virus (SARS-CoV-WT) replicated efficiently and exhibited similar one-step growth kinetics in susceptible cells. Both viruses accumulated similar amounts of virus-specific mRNAs and nsp1 protein in infected cells, whereas the amounts of endogenous host mRNAs were clearly higher in SARS-CoV-mt-infected cells than in SARS-CoV-WT-infected cells, in both the presence and absence of actinomycin D. Further, SARS-CoV-WT replication strongly inhibited host protein synthesis, whereas host protein synthesis inhibition in SARS-CoV-mt-infected cells was not as efficient as in SARS-CoV-WT-infected cells. These data revealed that nsp1 indeed promoted host mRNA degradation and contributed to host protein translation inhibition in infected cells. Notably, SARS-CoV-mt infection, but not SARS-CoV-WT infection, induced high levels of beta interferon (IFN) mRNA accumulation and high titers of type I IFN production. These data demonstrated that SARS-CoV nsp1 suppressed host innate immune functions, including type I IFN expression, in infected cells and suggested that SARS-CoV nsp1 most probably plays a critical role in SARS-CoV virulence.</p>
</abstract>
</article-meta>
</front>
<floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label>
<caption><p>The Nsp1 C-terminal region is important for inhibition of reporter gene activity. (A) 293 cells were independently cotransfected with pRL-SV40 and pCAGGS (EV), pRL-SV40 and pCAGGS-Nsp1-WT (Nsp1 WT), or pRL-SV40 and pCAGGS-Nsp1Δ160-173 (Nsp1Δ160-173). (A) Total intracellular proteins were extracted at 30 h posttransfection, and Western blot analysis was performed using anti-myc antibody. (B) At 30 h after transfection, cells were lysed in <italic>Renilla</italic>
luciferase lysis buffer, and aliquots of lysates were used to measure rLuc activities.</p>
</caption>
<graphic xlink:href="zjv0090805340001"></graphic>
</fig>
<fig position="float" id="f2"><label>FIG. 2.</label>
<caption><p>Mutations in the C-terminal region of Nsp1 abrogate its ability to inhibit reporter gene expression. (A) 293 cells were transfected with pCAGGS (EV), pCAGGS-Nsp1-WT (WT), or pCAGGS-Nsp1-mt (mt). At 30 h posttransfection, total intracellular proteins were extracted and Western blot analysis was performed using anti-myc antibody. (B) 293 cells were cotransfected with pIFNβ-luc and pCAGGS (EV), pIFNβ-luc and pCAGGS-Nsp1-WT (WT), or pIFNβ-luc and pCAGGS-Nsp1-mt (mt). At 24 h posttransfection, cells were infected with SeV (+) or mock infected (-). At 16 h p.i., firefly luciferase (Luc) activities were measured. (C and D) 293 cells were independently cotransfected with pRL-SV40 and pCAGGS (EV), pRL-SV40 and pCAGGS-Nsp1-WT (WT), or pRL-SV40 and pCAGGS-Nsp1-mt (mt). At 30 h posttransfection, cell extracts were prepared and used to measure rLuc activities (C), or total RNAs were extracted and Northern blot analysis was performed using a riboprobe specific for rLuc (D).</p>
</caption>
<graphic xlink:href="zjv0090805340002"></graphic>
</fig>
<fig position="float" id="f3"><label>FIG. 3.</label>
<caption><p>Nsp1 mutant protein expression does not promote host endogenous mRNA degradation and host protein synthesis inhibition. 293 cells were transfected independently with in vitro-synthesized CAT RNA transcripts (CAT), Nsp1-WT RNA transcripts (WT), or Nsp1-mt RNA transcripts (mt). One hour after RNA transfection, cells were incubated in the absence of ActD (ActD-) or presence of ActD (ActD+). (A) Total proteins were extracted at 8 h after ActD addition, and Western blot analysis was performed using anti-myc antibody to demonstrate the expression of CAT, Nsp1 WT, and Nsp1 mt proteins. (B) Total RNAs were also extracted at 0 or 8 h after ActD addition. The abundance of endogenous GAPDH mRNA was examined by Northern blot analysis using a riboprobe specific for GAPDH. (C) Cells were radiolabeled with 20 μCi of Tran<sup>35</sup>
S-label from 8.5 to 9.5 h after ActD addition. Equivalent amounts of intracellular proteins were analyzed by 12% SDS-PAGE and visualized by autoradiography. The molecular masses of marker proteins (in kilodaltons) are shown to the left of the gel.</p>
</caption>
<graphic xlink:href="zjv0090805340003"></graphic>
</fig>
<fig position="float" id="f4"><label>FIG. 4.</label>
<caption><p>Characterization of the SARS-CoV Nsp1 mutant. 293/ACE2 (A to D) and Vero E6 (A to C) cells were infected with SARS-CoV-WT (WT) or SARS-CoV-mt (mt) at an MOI of 1 (A), 0.01 (B), or 3 (C and D). (A and B) Culture supernatants were collected at the indicated times, and virus titers were determined by TCID<sub>50</sub>
analysis in Vero E6 cells. The results represent the averages of three independent experiments. (C) At 16 h p.i., total RNAs were extracted. The viral mRNAs were detected by Northern blot analysis using a probe that binds to the 3′-end of the SARS-CoV genome. (D) At 16 h p.i., total proteins were extracted, and Western blot analysis was performed to detect the Nsp1 protein by using an anti-nsp1 peptide antibody.</p>
</caption>
<graphic xlink:href="zjv0090805340004"></graphic>
</fig>
<fig position="float" id="f5"><label>FIG. 5.</label>
<caption><p>Effect of SARS-CoV-mt replication on abundance of host endogenous mRNAs. 293/ACE2 cells (A and B) and Vero E6 cells (A and C) were either mock infected or infected with SARS-CoV-WT (WT) or SARS-CoV-mt (mt) at an MOI of 3. (A) Intracellular RNAs were extracted at 1 and 16 h p.i. The amount of endogenous GAPDH mRNA was determined by Northern blot analysis. (B and C) At 1 h p.i., intracellular RNAs were extracted (1 h) or ActD was added to the culture medium. Intracellular RNAs were extracted at 15 h after ActD addition (16 h). The abundance of GAPDH (B, upper panel) and β-actin (B, lower panel, and C) mRNAs was determined using Northern blot analysis.</p>
</caption>
<graphic xlink:href="zjv0090805340005"></graphic>
</fig>
<fig position="float" id="f6"><label>FIG. 6.</label>
<caption><p>Effect of SARS-CoV-mt replication on host protein synthesis. 293/ACE2 and Vero E6 cells were either mock infected (M) or infected with SARS-CoV-WT (WT) or SARS-CoV-mt (mt) at an MOI of 3. (A) Cells were radiolabeled for 1 h with 100 μCi of Tran<sup>35</sup>
S-label, and extracts were prepared at the indicated times postinfection. Equivalent amounts of the intracellular proteins were analyzed on a 12% SDS-PAGE gel and visualized by autoradiography (top panel, 293/ACE2 cells; bottom panel, Vero E6 cells). Phosphorimager analysis was used to determine the level of host protein synthesis, and the numbers below the lanes represent the percent radioactivity relative to mock-infected cells. The boxes represent the region of the gel used for phosphorimager analysis. Representative data from two independent experiments are shown. (B) Western blot analysis was performed with extracts prepared at the indicated times postinfection to detect Nsp1 protein accumulation using anti-nsp1 peptide antibody.</p>
</caption>
<graphic xlink:href="zjv0090805340006"></graphic>
</fig>
<fig position="float" id="f7"><label>FIG. 7.</label>
<caption><p>Effect of SARS-CoV-mt replication on type I IFN and ISG induction. 293/ACE2 cells were either mock infected (M) or infected with SARS-CoV-WT (WT) or SARS-CoV-mt (mt) at an MOI of 3. SeV infection (100 HA units) was used as a positive control (A and B). (A) Total intracellular RNAs were extracted at the indicated times postinfection, and the amounts of endogenous IFN-β (top panel), ISG15 (middle panel), and ISG56 (bottom panel) mRNAs were determined by Northern blot analysis using riboprobes specific for the IFN-β, ISG15, and ISG56 genes, respectively. The identity of the band indicated by an asterisk in the bottom panel is unknown. (B) Culture supernatants were collected from mock-infected cells (M) and SARS-CoV-infected cells at 24 and 48 h p.i. and from SeV-infected cells at 24 h p.i. A type I IFN bioassay was performed to measure the concentration of type I IFN protein (in IU/ml) produced by infected cells. The data represent the averages of two independent experiments. (C) At the indicated times postinfection, total proteins were extracted and Western blot analysis was performed to detect ISG15 protein using anti-ISG15 antibody.</p>
</caption>
<graphic xlink:href="zjv0090805340007"></graphic>
</fig>
</floats-wrap>
</pmc>
</record>
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