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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">A conserved region of nonstructural protein 1 from alphacoronaviruses
inhibits host gene expression and is critical for viral
virulence</title>
<author><name sortKey="Shen, Zhou" sort="Shen, Zhou" uniqKey="Shen Z" first="Zhou" last="Shen">Zhou Shen</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Gang" sort="Wang, Gang" uniqKey="Wang G" first="Gang" last="Wang">Gang Wang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yang, Yiling" sort="Yang, Yiling" uniqKey="Yang Y" first="Yiling" last="Yang">Yiling Yang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Shi, Jiale" sort="Shi, Jiale" uniqKey="Shi J" first="Jiale" last="Shi">Jiale Shi</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Fang, Liurong" sort="Fang, Liurong" uniqKey="Fang L" first="Liurong" last="Fang">Liurong Fang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name>
<affiliation><nlm:aff id="aff3">Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota 55108</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Xiao, Shaobo" sort="Xiao, Shaobo" uniqKey="Xiao S" first="Shaobo" last="Xiao">Shaobo Xiao</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Fu, Zhen F" sort="Fu, Zhen F" uniqKey="Fu Z" first="Zhen F." last="Fu">Zhen F. Fu</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4">Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Peng, Guiqing" sort="Peng, Guiqing" uniqKey="Peng G" first="Guiqing" last="Peng">Guiqing Peng</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt><idno type="wicri:source">PMC</idno>
<idno type="pmid">31350335</idno>
<idno type="pmc">6746460</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746460</idno>
<idno type="RBID">PMC:6746460</idno>
<idno type="doi">10.1074/jbc.RA119.009713</idno>
<date when="2019">2019</date>
<idno type="wicri:Area/Pmc/Corpus">000D83</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000D83</idno>
</publicationStmt>
<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">A conserved region of nonstructural protein 1 from alphacoronaviruses
inhibits host gene expression and is critical for viral
virulence</title>
<author><name sortKey="Shen, Zhou" sort="Shen, Zhou" uniqKey="Shen Z" first="Zhou" last="Shen">Zhou Shen</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Gang" sort="Wang, Gang" uniqKey="Wang G" first="Gang" last="Wang">Gang Wang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Yang, Yiling" sort="Yang, Yiling" uniqKey="Yang Y" first="Yiling" last="Yang">Yiling Yang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Shi, Jiale" sort="Shi, Jiale" uniqKey="Shi J" first="Jiale" last="Shi">Jiale Shi</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Fang, Liurong" sort="Fang, Liurong" uniqKey="Fang L" first="Liurong" last="Fang">Liurong Fang</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Li, Fang" sort="Li, Fang" uniqKey="Li F" first="Fang" last="Li">Fang Li</name>
<affiliation><nlm:aff id="aff3">Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota 55108</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Xiao, Shaobo" sort="Xiao, Shaobo" uniqKey="Xiao S" first="Shaobo" last="Xiao">Shaobo Xiao</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Fu, Zhen F" sort="Fu, Zhen F" uniqKey="Fu Z" first="Zhen F." last="Fu">Zhen F. Fu</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff4">Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602</nlm:aff>
</affiliation>
</author>
<author><name sortKey="Peng, Guiqing" sort="Peng, Guiqing" uniqKey="Peng G" first="Guiqing" last="Peng">Guiqing Peng</name>
<affiliation><nlm:aff id="aff1">State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff2">Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff5">College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China</nlm:aff>
</affiliation>
</author>
</analytic>
<series><title level="j">The Journal of Biological Chemistry</title>
<idno type="ISSN">0021-9258</idno>
<idno type="eISSN">1083-351X</idno>
<imprint><date when="2019">2019</date>
</imprint>
</series>
</biblStruct>
</sourceDesc>
</fileDesc>
<profileDesc><textClass></textClass>
</profileDesc>
</teiHeader>
<front><div type="abstract" xml:lang="en"><p>Coronaviruses are enveloped, single-stranded RNA viruses that are distributed
worldwide. They include transmissible gastroenteritis virus (TGEV), porcine
epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute
respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome
coronavirus (MERS-CoV), many of which seriously endanger human health and
well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural
protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host
responses. The role of the C terminus of betacoronavirus nsp1 in virulence has
been characterized, but the location of the alphacoronavirus nsp1 region that is
important for virulence remains unclear. Here, using TGEV nsp1 as a model to
explore the function of this protein in alphacoronaviruses, we demonstrate that
alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal
structure of full-length TGEV at 1.85-Å resolution and conducting several
biochemical analyses, we observed that a specific motif (amino acids
91–95) of alphacoronavirus nsp1 is a conserved region that inhibits host
protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9
technology to construct a recombinant TGEV in which this specific nsp1 motif was
altered, we found that this mutation does not affect virus replication in cell
culture but significantly reduces TGEV pathogenicity in pigs. Taken together,
our findings suggest that alphacoronavirus nsp1 is an essential virulence
determinant, providing a potential paradigm for the development of a new
attenuated vaccine based on modified nsp1.</p>
</div>
</front>
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<pmc article-type="research-article"><pmc-dir>properties open_access</pmc-dir>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Biol Chem</journal-id>
<journal-id journal-id-type="iso-abbrev">J. Biol. Chem</journal-id>
<journal-id journal-id-type="hwp">jbc</journal-id>
<journal-id journal-id-type="pmc">jbc</journal-id>
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<journal-title-group><journal-title>The Journal of Biological Chemistry</journal-title>
</journal-title-group>
<issn pub-type="ppub">0021-9258</issn>
<issn pub-type="epub">1083-351X</issn>
<publisher><publisher-name>American Society for Biochemistry and Molecular
Biology</publisher-name>
<publisher-loc>11200 Rockville Pike, Suite 302, Rockville, MD 20852-3110,
U.S.A.</publisher-loc>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">31350335</article-id>
<article-id pub-id-type="pmc">6746460</article-id>
<article-id pub-id-type="publisher-id">RA119.009713</article-id>
<article-id pub-id-type="doi">10.1074/jbc.RA119.009713</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Microbiology</subject>
</subj-group>
</article-categories>
<title-group><article-title>A conserved region of nonstructural protein 1 from alphacoronaviruses
inhibits host gene expression and is critical for viral
virulence</article-title>
<alt-title alt-title-type="short">A conserved virulence region within
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<xref ref-type="aff" rid="aff3"><sup>¶</sup>
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<xref ref-type="aff" rid="aff1"><sup>‡</sup>
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</xref>
<xref ref-type="aff" rid="aff4"><sup>‖</sup>
</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Peng</surname>
<given-names>Guiqing</given-names>
</name>
<xref ref-type="aff" rid="aff1"><sup>‡</sup>
</xref>
<xref ref-type="aff" rid="aff2"><sup>§</sup>
</xref>
<xref ref-type="aff" rid="aff5">**</xref>
<xref ref-type="corresp" rid="cor1"><sup>1</sup>
</xref>
</contrib>
<aff id="aff1"><label>‡</label>
State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China</aff>
<aff id="aff2"><label>§</label>
Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan 430070, China</aff>
<aff id="aff3"><label>¶</label>
Department of Veterinary and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, Minnesota 55108</aff>
<aff id="aff4"><label>‖</label>
Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602</aff>
<aff id="aff5"><label>**</label>
College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China</aff>
</contrib-group>
<author-notes><corresp id="cor1"><label>1</label>
To whom correspondence should be addressed:
<addr-line>State Key Laboratory of Agricultural Microbiology, College of
Veterinary Medicine, Huazhong Agricultural University, 1 Shi-zi-shan St.,
Wuhan 430070, China.</addr-line>
Tel.: <phone>86-18071438015</phone>
; Fax:
<fax>86-27-87280480</fax>
; E-mail:
<email>penggq@mail.hzau.edu.cn</email>
.</corresp>
<fn fn-type="edited-by"><p>Edited by Charles E. Samuel</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><day>13</day>
<month>9</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="epub"><day>26</day>
<month>7</month>
<year>2019</year>
</pub-date>
<pub-date pub-type="pmc-release"><day>26</day>
<month>7</month>
<year>2019</year>
</pub-date>
<pmc-comment> PMC Release delay is 0 months and 0 days and was based on the . </pmc-comment>
<volume>294</volume>
<issue>37</issue>
<fpage>13606</fpage>
<lpage>13618</lpage>
<history><date date-type="received"><day>7</day>
<month>6</month>
<year>2019</year>
</date>
<date date-type="rev-recd"><day>24</day>
<month>7</month>
<year>2019</year>
</date>
</history>
<permissions><copyright-statement>© 2019 Shen et al.</copyright-statement>
<copyright-year>2019</copyright-year>
<copyright-holder>Shen et al.</copyright-holder>
<license><license-p>Published under exclusive license by The American Society for
Biochemistry and Molecular Biology, Inc.</license-p>
<license-p>This article is made available via the PMC Open Access Subset for
unrestricted re-use and analyses in any form or by any means with
acknowledgement of the original source. These permissions are granted for
the duration of the COVID-19 pandemic or until permissions are revoked in
writing. Upon expiration of these permissions, PMC is granted a perpetual
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with existing copyright protections.</license-p>
</license>
</permissions>
<self-uri content-type="pdf" xlink:href="zbc03719013606.pdf"></self-uri>
<abstract><p>Coronaviruses are enveloped, single-stranded RNA viruses that are distributed
worldwide. They include transmissible gastroenteritis virus (TGEV), porcine
epidemic diarrhea virus (PEDV), and the human coronaviruses severe acute
respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome
coronavirus (MERS-CoV), many of which seriously endanger human health and
well-being. Only alphacoronaviruses and betacoronaviruses harbor nonstructural
protein 1 (nsp1), which performs multiple functions in inhibiting antiviral host
responses. The role of the C terminus of betacoronavirus nsp1 in virulence has
been characterized, but the location of the alphacoronavirus nsp1 region that is
important for virulence remains unclear. Here, using TGEV nsp1 as a model to
explore the function of this protein in alphacoronaviruses, we demonstrate that
alphacoronavirus nsp1 inhibits host gene expression. Solving the crystal
structure of full-length TGEV at 1.85-Å resolution and conducting several
biochemical analyses, we observed that a specific motif (amino acids
91–95) of alphacoronavirus nsp1 is a conserved region that inhibits host
protein synthesis. Using a reverse-genetics system based on CRISPR/Cas9
technology to construct a recombinant TGEV in which this specific nsp1 motif was
altered, we found that this mutation does not affect virus replication in cell
culture but significantly reduces TGEV pathogenicity in pigs. Taken together,
our findings suggest that alphacoronavirus nsp1 is an essential virulence
determinant, providing a potential paradigm for the development of a new
attenuated vaccine based on modified nsp1.</p>
</abstract>
<kwd-group><kwd>protein motif</kwd>
<kwd>pathogenesis</kwd>
<kwd>crystal structure</kwd>
<kwd>virulence factor</kwd>
<kwd>virus</kwd>
<kwd>coronavirus</kwd>
<kwd>host gene expression</kwd>
<kwd>immune evasion</kwd>
<kwd>nonstructural protein 1 (nsp1)</kwd>
<kwd>transmissible gastroenteritis virus (TGEV)</kwd>
</kwd-group>
<funding-group><award-group id="award1"><funding-source><institution-wrap><institution>National Natural Science Foundation of China (NSF)
</institution>
<institution-id institution-id-type="open-funder-registry">10.13039/501100001809</institution-id>
</institution-wrap>
</funding-source>
<award-id>31873020</award-id>
<award-id>31722056</award-id>
<principal-award-recipient><name><surname>Peng</surname>
<given-names>Guiqing</given-names>
</name>
</principal-award-recipient>
</award-group>
<award-group id="award2"><funding-source>National Key Research and Development Plan of
China</funding-source>
<award-id>2018YFD0500102</award-id>
<principal-award-recipient><name><surname>Peng</surname>
<given-names>Guiqing</given-names>
</name>
</principal-award-recipient>
</award-group>
<award-group id="award3"><funding-source>Huazhong Agricultural University Scientific and Technological
Self-Innovation Foundation</funding-source>
<award-id>2662017PY028</award-id>
<principal-award-recipient><name><surname>Peng</surname>
<given-names>Guiqing</given-names>
</name>
</principal-award-recipient>
</award-group>
</funding-group>
</article-meta>
</front>
<body><sec sec-type="intro"><title>Introduction</title>
<p>Coronaviruses (CoVs)<xref ref-type="fn" rid="FN1"><sup>2</sup>
</xref>
belong to a
large family of enveloped, single-stranded RNA viruses that are serious threats to
public health and have caused considerable damage (<xref rid="B1" ref-type="bibr">1</xref>
<xref ref-type="bibr" rid="B2">–</xref>
<xref rid="B5" ref-type="bibr">5</xref>
). Depending on serotype, CoVs can be generally
divided into <italic>Alpha</italic>
-, <italic>Beta</italic>
-,
<italic>Gamma</italic>
-, and <italic>Deltacoronavirus</italic>
(α-CoV,
β-CoV, γ-CoV, and δ-CoV, respectively) genera (<xref rid="B2" ref-type="bibr">2</xref>
). The α-CoVs include transmissible
gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), human
coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), and feline
infectious peritonitis virus (FIPV) (<xref rid="B1" ref-type="bibr">1</xref>
). The
β-CoVs include murine hepatitis virus (MHV), SARS-CoV, and MERS-CoV (<xref rid="B1" ref-type="bibr">1</xref>
).</p>
<p>CoV nonstructural protein 1 (nsp1) is the N-terminal component of the pp1a
polyprotein. Only α-CoVs and β-CoVs encode nsp1, whereas γ-CoVs
and δ-CoVs lack this protein (<xref rid="B6" ref-type="bibr">6</xref>
). Nsp1
sequences identified using standard search tools, such as BLAST, are divergent among
different CoVs. The sizes of α-CoV nsp1 and β-CoV nsp1 differ; the
α-CoVs encode nsp1 proteins of ∼9 kDa, which are substantially smaller
than the ∼20-kDa nsp1 proteins of β-CoVs. Although the sequence
homologies among TGEV, PEDV, and SARS-CoV nsp1 proteins are low, the core structures
share a relatively conserved domain (<xref rid="B7" ref-type="bibr">7</xref>
<xref ref-type="bibr" rid="B8">–</xref>
<xref rid="B9" ref-type="bibr">9</xref>
). This high structural similarity may explain why CoV nsp1 has the
conserved biological function of inhibiting host gene expression. However, the
critical region of β-CoV nsp1 required to inhibit protein synthesis is
different from that of α-CoV nsp1 (<xref rid="B9" ref-type="bibr">9</xref>
<xref ref-type="bibr" rid="B10">–</xref>
<xref rid="B12" ref-type="bibr">12</xref>
). This difference could imply that the
detailed mechanisms through which CoV nsp1 proteins inhibit host gene expression are
unpredictable. To date, the ability of β-CoV nsp1 to inhibit host gene
expression has been deeply studied. SARS-CoV nsp1 not only prevents mRNA translation
but also promotes its degradation by binding to the 40S ribosomal subunit (<xref rid="B13" ref-type="bibr">13</xref>
). Furthermore, MERS-CoV nsp1 selectively
targets mRNA synthesized in the host cell nucleus for degradation and thus inhibits
translation in host cells (<xref rid="B14" ref-type="bibr">14</xref>
). TGEV and PEDV
nsp1 proteins cannot bind the 40S ribosomal subunit to inhibit the translation of
host mRNA; however, HCoV-229E and HCoV-NL63 nsp1 proteins might bind the 40S
ribosomal subunit to affect host mRNA stability (<xref rid="B9" ref-type="bibr">9</xref>
, <xref rid="B15" ref-type="bibr">15</xref>
, <xref rid="B16" ref-type="bibr">16</xref>
).</p>
<p>At present, the specific pathway by which α-CoV nsp1 inhibits host gene
expression is unknown. However, the function of CoV nsp1 in viral immune system
evasion has been well-characterized in cell culture. For example, SARS-CoV nsp1
suppresses interferon (IFN) expression and host antiviral signaling pathways in
infected cells (<xref rid="B10" ref-type="bibr">10</xref>
, <xref rid="B17" ref-type="bibr">17</xref>
). Furthermore, MHV nsp1 efficiently interferes with the type
I IFN system (<xref rid="B11" ref-type="bibr">11</xref>
), whereas PEDV nsp1 mediates
cAMP-response element-binding protein (CREB)-binding protein (CBP) and NF-κB
degradation to inhibit type II IFN responses (<xref rid="B12" ref-type="bibr">12</xref>
, <xref rid="B18" ref-type="bibr">18</xref>
). Thus, nsp1 is considered
a possible major virulence factor for CoVs. Moreover, the contribution of nsp1 to
CoV pathogenesis has been directly demonstrated for MHV and SARS-CoV (<xref rid="B11" ref-type="bibr">11</xref>
, <xref rid="B17" ref-type="bibr">17</xref>
).
However, little direct evidence has indicated that α-CoV nsp1 is a virulence
factor. Here, we present the crystal structure of full-length TGEV nsp1. Further
structural and biochemical analyses indicated that a motif (amino acids
91–95) is important for the inhibition of host gene expression by α-CoV
nsp1. In addition, we demonstrate that the loss of nsp1-induced inhibition of host
protein synthesis does not affect the replication of TGEV but can significantly
reduce its virulence in piglets. This research improves our understanding of why
α-CoV nsp1 is necessary for virulence and may aid in the development of a new
attenuated vaccine.</p>
</sec>
<sec sec-type="results"><title>Results</title>
<sec><title>Transient α-CoV nsp1 expression affects cellular gene
expression</title>
<p>Using <italic>Renilla</italic>
luciferase (Rluc) assays, we assessed several
representative α-CoV nsp1 proteins for their ability to interfere with
host-cell gene expression. Human embryonic kidney (HEK-293T) cells were
cotransfected with plasmids expressing the nsp1 protein and pRL-SV40 plasmids
expressing SV40 promoter–driven Rluc. The results showed that TGEV, FIPV,
HCoV-229E, HCoV-NL63, and PEDV nsp1 significantly reduced luciferase reporter
gene expression. Furthermore, subsequent Western blot analysis using equal
amounts of intracellular proteins confirmed this finding (<xref ref-type="fig" rid="F1">Fig. 1</xref>
<italic>A</italic>
).</p>
<fig id="F1" orientation="portrait" position="float"><label>Figure 1.</label>
<caption><p><bold>α-CoV nsp1 inhibits protein synthesis.</bold>
<italic>A</italic>
, α-CoV nsp1 significantly inhibited the
expression of reporter genes in HEK-293T cells. The expression of nsp1
and GAPDH was detected by Western blot analysis using an anti-HA
antibody and an anti-GAPDH antibody, respectively
(<italic>top</italic>
). <italic>B</italic>
, HEK-293T cells were
cotransfected with pRL-SV40 and WT plasmids. At 12, 24, and 48 h
post-transfection, the cells were lysed and subjected to real-time
quantitative PCR analysis. TGEV nsp1 significantly inhibited the
synthesis of Rluc mRNA; SARS nsp1 was used as the positive control.
<italic>C</italic>
, cells were transfected with different doses of
the TGEV nsp1 plasmid (0–2.0 μg) for 24 h. The cells were
pulsed with 3 μ<sc>m</sc>
puromycin for 1 h at 37 °C and then
subjected to Western blot analysis (<italic>left</italic>
). The
grayscale values of the protein bands were analyzed by ImageJ
(<italic>right</italic>
). <italic>D</italic>
, cells were pulsed with
3 μ<sc>m</sc>
puromycin for 1 h after 0, 12, 24, and 36 h of
transfection and then subjected to Western blot analysis
(<italic>left</italic>
). The corresponding grayscale values of the
protein bands were analyzed by ImageJ (<italic>right</italic>
). The
<italic>error bars</italic>
show the S.D. of the results from three
independent experiments. *, <italic>p</italic>
< 0.05, significant;
**, <italic>p</italic>
< 0.01, highly significant; ***,
<italic>p</italic>
< 0.001, extremely significant.</p>
</caption>
<graphic xlink:href="zbc0381911180001"></graphic>
</fig>
<p>Then, we used TGEV nsp1 as a model to study the detailed function of α-CoV
nsp1. Using the SARS-CoV nsp1 protein as a positive control (<xref rid="B13" ref-type="bibr">13</xref>
), we examined Rluc mRNA in HEK-293T
cells using real-time quantitative PCR and found that TGEV nsp1 inhibited Rluc
mRNA synthesis at different time points (<xref ref-type="fig" rid="F1">Fig.
1</xref>
<italic>B</italic>
). To further explore whether TGEV nsp1 could
broadly inhibit gene expression in HEK-293T cells, we examined host proteins
using a ribopuromycylation assay. The broad-spectrum inhibitory activity of TGEV
nsp1 on host protein synthesis depended on both concentration and time (<xref ref-type="fig" rid="F1">Fig. 1</xref>
, <italic>C</italic>
and
<italic>D</italic>
).</p>
</sec>
<sec><title>Identification of the critical region of TGEV nsp1 required for inhibition of
protein synthesis</title>
<p>Although the structure of a truncated TGEV nsp1 protein (Protein Data Bank (PDB)
code <ext-link ext-link-type="pdb" xlink:href="3ZBD">3ZBD</ext-link>
) has been reported (<xref rid="B8" ref-type="bibr">8</xref>
), we wondered whether the C terminus could affect the
overall structure. We performed initial crystallization trials for the
full-length TGEV nsp1 (residues Met-1 to Arg-109). Fortunately, we were able to
determine the crystal structure of full-length TGEV nsp1 using the molecular
replacement method and refined it to 1.85-Å resolution in space group
P2<sub>1</sub>
. The details of the phasing and refinement steps are given in
<xref rid="T1" ref-type="table">Table 1</xref>
. Although the crystal
resolution was very high, the specific conformation of the C terminus remained
unclear. This was probably because the C-terminal loop was flexible, for which
it is difficult to obtain accurate information. Structural characterization
demonstrated that the structure of full-length TGEV nsp1 displayed eight
β-sheets and two α-helices (<xref ref-type="fig" rid="F2">Fig.
2</xref>
<italic>A</italic>
), and the full-length protein shared a common
skeleton with the truncated protein. The similarity of the three-dimensional
conformations indicated that the C terminus had little influence on the overall
TGEV nsp1 structure.</p>
<table-wrap id="T1" orientation="portrait" position="float"><label>Table 1</label>
<caption><p><bold>Data collection and refinement statistics</bold>
</p>
<p>The highest-resolution values are indicated in parentheses.
<italic>R</italic>
<sub>merge</sub>
=
∑∑|<italic>I</italic>
<sub>h</sub>
−
〈<italic>I</italic>
〉|/∑∑<italic>I</italic>
<sub>h</sub>
where <italic>I</italic>
<sub>h</sub>
is the intensity measurement of
reflection <italic>h</italic>
and 〈<italic>I</italic>
〉 is
the average intensity from multiple observations.
<italic>R</italic>
<sub>work</sub>
=
∑‖<italic>F<sub>o</sub>
</italic>
| −
|<italic>F<sub>c</sub>
</italic>
‖/∑|<italic>F<sub>o</sub>
</italic>
|
where <italic>F<sub>o</sub>
</italic>
and <italic>F<sub>c</sub>
</italic>
are the observed and calculated structure factors, respectively.
<italic>R</italic>
<sub>free</sub>
is equivalent to
<italic>R</italic>
<sub>work</sub>
, but 5% of the measured
reflections were excluded from the refinement and set aside for
cross-validation.</p>
</caption>
<table frame="hsides" rules="groups"><thead valign="bottom"><tr><th rowspan="1" colspan="1"></th>
<th align="center" rowspan="1" colspan="1">TGEV nsp1</th>
<th align="center" rowspan="1" colspan="1">TGEV
nsp1(91–95sg)</th>
</tr>
</thead>
<tbody valign="top"><tr><td align="left" rowspan="1" colspan="1"><bold>Data
collection</bold>
</td>
<td rowspan="1" colspan="1"></td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Space group</td>
<td align="left" rowspan="1" colspan="1">P2<sub>1</sub>
</td>
<td align="left" rowspan="1" colspan="1">P2<sub>1</sub>
</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Cell parameters
<italic>a</italic>
, <italic>b</italic>
, <italic>c</italic>
(Å)</td>
<td align="left" rowspan="1" colspan="1">36.16, 67.01, 90.10</td>
<td align="left" rowspan="1" colspan="1">33.44, 59.24, 57.17</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> α, β, γ
(°)</td>
<td align="left" rowspan="1" colspan="1">90.00, 93.98, 90.00</td>
<td align="left" rowspan="1" colspan="1">90.00, 97.54, 90.00</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Wavelength</td>
<td align="left" rowspan="1" colspan="1">1.0</td>
<td align="left" rowspan="1" colspan="1">0.97918</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Resolution range
(Å)</td>
<td align="left" rowspan="1" colspan="1">34.31-1.85</td>
<td align="left" rowspan="1" colspan="1">40.95-1.98</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Completeness (%)</td>
<td align="left" rowspan="1" colspan="1">96.9 (96.9)</td>
<td align="left" rowspan="1" colspan="1">97.6 (97.6)</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic>
<sub>merge</sub>
(last shell)</td>
<td align="left" rowspan="1" colspan="1">0.056 (0.476)</td>
<td align="left" rowspan="1" colspan="1">0.025 (0.403)</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>I</italic>
/σ
(last shell)</td>
<td align="left" rowspan="1" colspan="1">35.12 (4.43)</td>
<td align="left" rowspan="1" colspan="1">42.35 (9.42)</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Redundancy (last shell)</td>
<td align="left" rowspan="1" colspan="1">4.0 (3.9)</td>
<td align="left" rowspan="1" colspan="1">3.9 (3.8)</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"><bold>Refinement</bold>
</td>
<td rowspan="1" colspan="1"></td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Resolution (Å)</td>
<td align="left" rowspan="1" colspan="1">34.31-1.80</td>
<td align="left" rowspan="1" colspan="1">40.95-1.97</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> <italic>R</italic>
<sub>work</sub>
/<italic>R</italic>
<sub>free</sub>
</td>
<td align="left" rowspan="1" colspan="1">0.179/0.232</td>
<td align="left" rowspan="1" colspan="1">0.191/0.237</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of reflections</td>
<td align="left" rowspan="1" colspan="1">35,753</td>
<td align="left" rowspan="1" colspan="1">15,284</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of protein atoms</td>
<td align="left" rowspan="1" colspan="1">3,490</td>
<td align="left" rowspan="1" colspan="1">1,684</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of solvent atoms</td>
<td align="left" rowspan="1" colspan="1">430</td>
<td align="left" rowspan="1" colspan="1">103</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> No. of ions/ligands</td>
<td align="left" rowspan="1" colspan="1">0</td>
<td align="left" rowspan="1" colspan="1">0</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Mean B factor
(Å<sup>2</sup>
)</td>
<td align="left" rowspan="1" colspan="1">20.94</td>
<td align="left" rowspan="1" colspan="1">26.55</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> r.m.s.d.</td>
<td rowspan="1" colspan="1"></td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Bond
length (Å)</td>
<td align="left" rowspan="1" colspan="1">0.007</td>
<td align="left" rowspan="1" colspan="1">0.007</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Bond
angle (°)</td>
<td align="left" rowspan="1" colspan="1">0.864</td>
<td align="left" rowspan="1" colspan="1">0.784</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Ramachandran plot</td>
<td rowspan="1" colspan="1"></td>
<td rowspan="1" colspan="1"></td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Favored
(%)</td>
<td align="left" rowspan="1" colspan="1">98.34</td>
<td align="left" rowspan="1" colspan="1">97.58</td>
</tr>
<tr><td align="left" rowspan="1" colspan="1"> Allowed,
outlier (%)</td>
<td align="left" rowspan="1" colspan="1">1.66, 0.00</td>
<td align="left" rowspan="1" colspan="1">2.42, 0.00</td>
</tr>
</tbody>
</table>
</table-wrap>
<fig id="F2" orientation="portrait" position="float"><label>Figure 2.</label>
<caption><p><bold>Structure of full-length TGEV nsp1 and identification of the region
that inhibits protein synthesis.</bold>
<italic>A</italic>
, crystal structure of TGEV nsp1. The structure of
nsp1 is shown as a cartoon; α-helices are shown in
<italic>red</italic>
, and β-sheets are shown in
<italic>yellow. B</italic>
and <italic>C</italic>
, based on the
structure of TGEV nsp1, plasmids with mutated loop regions were
constructed. The recombinant plasmids were as follows:
nsp1(1–3sg), nsp1(11–15sg), nsp1(19–22sg),
nsp1(37–40sg), nsp1(46–48sg), nsp1(55–60sg),
nsp1(66–69sg), nsp1(76–84sg), nsp1(91–95sg), and
nsp1(105–109sg). HEK-293T cells were transfected with the control
plasmid (PCAGGS) or nsp1 plasmids. At 24 h, the cells were lysed and
subjected to Rluc assays and Western blot analysis. The <italic>error
bars</italic>
show the S.D. of the results from three independent
experiments. The <italic>asterisks</italic>
indicate statistical
significance calculated using Student's <italic>t</italic>
test. ***,
<italic>p</italic>
< 0.001.</p>
</caption>
<graphic xlink:href="zbc0381911180002"></graphic>
</fig>
<p>To identify which region in TGEV nsp1 was essential for its inhibitory activity,
we used methods that have been previously used to study important functional
regions in PEDV nsp1 (<xref rid="B9" ref-type="bibr">9</xref>
). Based on the
crystal structure of TGEV nsp1, we designed the following series of plasmids in
which random coils were replaced with flexible Ser-Gly-Ser-Gly (sg) linkers:
nsp1(1–3sg), nsp1(11–15sg), nsp1(19–22sg),
nsp1(37–40sg), nsp1(46–48sg), nsp1(52–60sg),
nsp1(66–69sg), nsp1(76–84sg), nsp1(91–95sg), and
nsp1(105–109sg) (<xref ref-type="fig" rid="F2">Fig. 2</xref>
,
<italic>B</italic>
and <italic>C</italic>
). The Rluc assay showed that the
loops comprising amino acids 37–40 and 91–95 were the most
important regions for the ability of TGEV nsp1 to inhibit reporter gene
expression (<xref ref-type="fig" rid="F2">Fig. 2</xref>
<italic>C</italic>
).
Subsequently, Western blot analysis indicated that nsp1(91–95sg)
expression was significantly higher than nsp1 expression (<xref ref-type="fig" rid="F2">Fig. 2</xref>
<italic>C</italic>
), again demonstrating that nsp1
could inhibit its own expression (<xref rid="B9" ref-type="bibr">9</xref>
, <xref rid="B15" ref-type="bibr">15</xref>
). We examined Rluc mRNA expression in
HEK-293T cells by real-time quantitative PCR and found that TGEV
nsp1(91–95sg) and TGEV nsp1(37–40sg) did not inhibit Rluc mRNA
synthesis at different time points (<xref ref-type="fig" rid="F3">Fig.
3</xref>
<italic>A</italic>
). We conducted the ribopuromycylation assay, and
the results showed that TGEV nsp1(37–40sg) significantly reduced the
inhibition of host gene expression (<xref ref-type="fig" rid="F3">Fig.
3</xref>
<italic>B</italic>
). Because the TGEV nsp1(37–40sg) protein
was found to be insoluble in the <italic>Escherichia coli</italic>
supernatant,
the motif (amino acids 37–40) was not analyzed further. Based on the
above experiments, we focused on TGEV nsp1(91–95sg). Finally, the
ribopuromycylation assay demonstrated that the mutant protein no longer
inhibited host protein synthesis in a dose- or time-dependent manner (<xref ref-type="fig" rid="F3">Fig. 3</xref>
, <italic>C</italic>
and
<italic>D</italic>
). These data indicate that the motif comprising amino
acids 91–95 is critical for TGEV nsp1–induced suppression of host
gene expression.</p>
<fig id="F3" orientation="portrait" position="float"><label>Figure 3.</label>
<caption><p><bold>The motif comprising amino acids 91–95 is a critical region
for TGEV nsp1-induced inhibition of host protein synthesis.</bold>
<italic>A</italic>
, HEK-293T cells were cotransfected with pRL-SV40
encoding the Rluc reporter gene downstream of the SV40 promoter and one
of the following plasmids: PCAGGS, PCAGGS-TGEV-nsp1-HA,
PCAGGS-TGEV-nsp1(37–40sg)-HA, and
PCAGGS-TGEV-nsp1(91–95sg)-HA, which encode no gene, TGEV nsp1,
TGEV nsp1(37–40sg), and TGEV nsp1(91–95sg), respectively.
At 12, 24, and 48 h post-transfection, the cells were lysed and
subjected to real-time quantitative PCR analysis. The values of TGEV
nsp1, TGEV nsp1(37–40sg), and TGEV nsp1(91–95sg) were
normalized to those of the untreated empty vector (PCAGGS) control,
which were set to 1. <italic>B</italic>
, HEK-293T cells were transfected
with different doses of the TGEV nsp1(37–40sg) plasmid
(0–2.0 μg) for 24 h. The cells were pulsed with 3
μ<sc>m</sc>
puromycin for 1 h at 37 °C and then subjected
to Western blot analysis (<italic>left</italic>
). The grayscale values
of the protein bands were analyzed by ImageJ (<italic>right</italic>
).
<italic>C</italic>
, cells were transfected with different doses of
the TGEV nsp1(91–95sg) plasmid (0–2.0 μg) for 24 h.
The cells were pulsed with 3 μ<sc>m</sc>
puromycin for 1 h at 37
°C and then subjected to Western blot analysis
(<italic>left</italic>
). The grayscale values of the protein bands
were analyzed by ImageJ (<italic>right</italic>
). <italic>D</italic>
,
the cells were pulsed with 3 μ<sc>m</sc>
puromycin for 1 h after
transfection for 0, 12, 24, and 36 h (<italic>left</italic>
) and then
subjected to Western blot analysis (<italic>right</italic>
). The
grayscale values of the protein bands were analyzed by ImageJ. Data are
represented as mean ± S.D., <italic>n</italic>
= 3. **,
<italic>p</italic>
< 0.01; <italic>ns</italic>
, not
significant.</p>
</caption>
<graphic xlink:href="zbc0381911180003"></graphic>
</fig>
</sec>
<sec><title>Structural similarity of WT and mutant TGEV nsp1</title>
<p>To explore whether a loss of protein activity by TGEV nsp1(91–95sg) was
due to structural changes, we constructed a TGEV nsp1(91–95sg) plasmid.
Expression and purification were performed as described for the full-length
construct. This new construct yielded crystals under different crystallization
conditions than those of the full-length protein, and native data were collected
to 1.98 Å. The details of the phasing and refinement steps are given in
<xref rid="T1" ref-type="table">Table 1</xref>
. A comparison of surface
electrostatics and shape between TGEV nsp1 and TGEV nsp1(91–95sg) led us
to speculate that the sg linker mutation from amino acids 91 to 95 did not
significantly change the overall structure of TGEV nsp1 (<xref ref-type="fig" rid="F4">Fig. 4</xref>
, <italic>A–F</italic>
).</p>
<fig id="F4" orientation="portrait" position="float"><label>Figure 4.</label>
<caption><p><bold>Structural comparisons of TGEV nsp1 and TGEV
nsp1(91–95sg).</bold>
<italic>A</italic>
, views from opposite sides of the electrostatic
surface of TGEV nsp1. <italic>B</italic>
, views from opposite sides of
the electrostatic surface of TGEV nsp1(91–95sg). Positive charges
are shown in <italic>blue</italic>
, and negative charges are shown in
<italic>red. C</italic>
, views from opposite sides of a cartoon
model of TGEV nsp1 with the motif (amino acids 91–95) marked in
<italic>black. D</italic>
, views from opposite sides of a cartoon
model of TGEV nsp1(91–95sg) with the motif (amino acids
91–95) marked in <italic>black</italic>
. TGEV nsp1 is shown in
<italic>yellow</italic>
, and TGEV nsp1(91–95sg) is shown in
<italic>red. E</italic>
, ribbon diagrams of the two structures.
<italic>F</italic>
, r.m.s.d. values between TGEV nsp1 and TGEV
nsp1(91–95sg). The r.m.s.d. values were calculated using
PDBeFold.</p>
</caption>
<graphic xlink:href="zbc0381911180004"></graphic>
</fig>
</sec>
<sec><title>The motif comprising amino acids 91–95 is important for the regulation
of host translation by α-CoV nsp1</title>
<p>We next determined whether this motif (amino acids 91–95) plays an
important role in inhibiting host protein synthesis in the context of other
α-CoV nsp1 proteins. To explore the corresponding motif in α-CoV
nsp1, we selected the previously mentioned representative sequences and aligned
them using ClustalW2 software. The α-CoV nsp1 amino acid sequence
alignment showed that the motif comprising amino acids 91–95 was a
relatively conserved domain (<xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>A</italic>
). Then, we constructed mutant nsp1 proteins from
FIPV, HCoV-229E, HCoV-NL63, and PEDV to explore whether the α-CoV nsp1
motif comprising amino acids 91–95 was an important region for the
suppression of host gene expression. Luciferase assays indicated that the
important function of this motif (amino acids 91–95) in inhibiting host
gene expression not only was exhibited by TGEV nsp1 but also could be extended
to nsp1 proteins from other α-CoVs (<xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>B</italic>
). Furthermore, Western blot analysis revealed
that the expression of the mutant proteins was higher than that of the WT
proteins, especially for TGEV nsp1 and HCoV-NL63 nsp1 (<xref ref-type="fig" rid="F5">Fig. 5</xref>
<italic>C</italic>
), consistent with the results of
the luciferase assays. In addition, we compared the Rluc values between the
mutant and the WT nsp1 proteins (<xref ref-type="fig" rid="F5">Fig.
5</xref>
<italic>D</italic>
). These data showed that the relevant motif
(amino acids 91–95) might be responsible for inhibiting host gene
expression, albeit to different degrees, among the α-CoV nsp1 proteins.
Consistent with our previous results (<xref rid="B9" ref-type="bibr">9</xref>
),
when we mutated the motif (amino acids 91–95) in PEDV nsp1, we again
observed partial recovery of the ability to suppress Rluc expression. Taken
together, these results indicated that the motif comprising amino acids
91–95 was an important conserved region for the regulation of host
translation by α-CoV nsp1.</p>
<fig id="F5" orientation="portrait" position="float"><label>Figure 5.</label>
<caption><p><bold>The motif comprising amino acids 91–95 is an important
region for α-CoV nsp1-induced inhibition of gene
expression.</bold>
<italic>A</italic>
, the following sequences from GenBank were used to
create the sequence alignment (given as the abbreviation and GenBank
accession number): TGEV, <ext-link ext-link-type="gen" xlink:href="HQ462571.1">HQ462571.1</ext-link>
; FIPV, <ext-link ext-link-type="gen" xlink:href="ADL71484.1">ADL71484.1</ext-link>
; PEDV, <ext-link ext-link-type="gen" xlink:href="AJP67455.1">AJP67455.1</ext-link>
; HCoV-229E, <ext-link ext-link-type="gen" xlink:href="CAA49377.1">CAA49377.1</ext-link>
; and HCoV-NL63, <ext-link ext-link-type="gen" xlink:href="AFV53147.1">AFV53147.1</ext-link>
. The residue numbers with reference to TGEV
nsp1 are labeled on <italic>top</italic>
of the <italic>panel</italic>
.
The regions comprising amino acids 91–95 are marked by
<italic>small black dots</italic>
in the α-CoV nsp1 proteins.
Residues conserved in most sequences are shown in <italic>red</italic>
and are <italic>boxed</italic>
with a <italic>white background</italic>
.
The sequences were aligned with ClustalW2, and the figure was prepared
with ESPript3.0. <italic>B</italic>
and <italic>C</italic>
, host protein
synthesis inhibited by “sg” replacement mutants of
α-CoV nsp1. HEK-293T cells were cotransfected with pRL-SV40 or
pRL-TK and one of the following plasmids: PCAGGS (control),
PCAGGS-TGEV-nsp1, PCAGGS-TGEV-nsp1(91–95sg), PCAGGS-FIPV-nsp1,
PCAGGS-FIPV-nsp1(91–95sg), PCAGGS-HCoV-229E-nsp1,
PCAGGS-HCoV-229E-nsp1(92–96sg), PCAGGS-HCoV-NL63-nsp1,
PCAGGS-HCoV-NL63-nsp1(92–96sg), PCAGGS-PEDV-nsp1, and
PCAGGS-PEDV-nsp1(92–96sg), which encode no protein, TGEV nsp1,
TGEV nsp1(91–95sg), FIPV nsp1, FIPV nsp1(91–95sg),
HCoV-229E nsp1, HCoV-229E nsp1(92–96sg), HCoV-NL63 nsp1, or
HCoV-NL63 nsp1(92–96sg), respectively. At 24 h post-transfection,
the cells were lysed and subjected to Rluc assays and Western blot
analysis. Data are represented as mean ± S.D., <italic>n</italic>
=
3. ***, <italic>p</italic>
< 0.001. <italic>D</italic>
, the -fold
change in Rluc activity in the mutant protein relative to the
corresponding WT protein is shown.</p>
</caption>
<graphic xlink:href="zbc0381911180005"></graphic>
</fig>
</sec>
<sec><title>Construction and recovery of a TGEV mutant virus</title>
<p>Although α-CoV nsp1 expression is known to suppress host gene expression,
its biological functions in viral replication have been largely unexplored. To
evaluate the role of TGEV nsp1 in inhibiting host gene expression during viral
replication, we used our reverse-genetics system to construct a recombinant TGEV
encoding a mutant nsp1 protein in which ORF3 was replaced by green fluorescent
protein (GFP) (<xref rid="B19" ref-type="bibr">19</xref>
). Based on the
previously described results, we replaced the important motif (amino acids
91–95) in the nsp1-coding sequence. In the resulting mutant virus, the
replicase gene start codon, translational reading frame, and residues required
for proteolytic release of nsp1 from the replicase polyprotein were maintained
(<xref ref-type="fig" rid="F6">Fig. 6</xref>
<italic>A</italic>
). The mutant
virus was successfully amplified by transfecting corresponding bacterial
artificial chromosome (BAC) plasmids into HEK-293T cells. Then, we collected the
amplified virus 24 h after transfection to infect TGEV-susceptible cells.
Characteristic green fluorescence consistent with the parental virus phenotype
was observed 36 h after infection (<xref ref-type="fig" rid="F6">Fig.
6</xref>
<italic>B</italic>
). The recombinant virus, which was subsequently
named TGEV(91–95sg), was verified by sequencing the corresponding
replaced regions. To assess the stability of TGEV(91–95sg), we analyzed
the nsp1-coding region by RT-PCR sequencing after seven passages in PK15 cells,
and no nucleotide changes were detected. Furthermore, Western blot analysis
confirmed that the virus successfully infected PK15 and swine testicle (ST)
cells (<xref ref-type="fig" rid="F6">Fig. 6</xref>
<italic>C</italic>
).</p>
<fig id="F6" orientation="portrait" position="float"><label>Figure 6.</label>
<caption><p><bold>Effects of the recombinant virus at the cellular level.</bold>
<italic>A</italic>
, DNA-based reverse-genetics BAC system for TGEV and
TGEV(91–95sg). The relative positions of the mutated genes used
for molecular cloning are indicated. <italic>B</italic>
and
<italic>C</italic>
, the recombinant virus was successfully used to
infect susceptible PK15 and ST cells. TGEV-BAC and
TGEV(91–95sg)-BAC were transfected into HEK-293T cells with
Lipofectamine 3000, and then the virus was inoculated into PK15 and ST
cells 24 h later. Green fluorescence was observed after 36 h. The
expression of TGEV N and TGEV nsp1 proteins in cells was also detected
by Western blot analysis using anti-TGEV N and anti-TGEV nsp1
antibodies, respectively. GAPDH served as the loading control.
<italic>D</italic>
and <italic>E</italic>
, multistep growth curves
for TGEV and TGEV(91–95sg) in PK15 and ST cells at a multiplicity
of infection of 0.1. Original magnification × 50 (<italic>scale
bars</italic>
400 μm). The virus titers at different time
points, as indicated, were determined by an end-point dilution assay.
Data are represented as mean ± S.D., <italic>n</italic>
= 3.</p>
</caption>
<graphic xlink:href="zbc0381911180006"></graphic>
</fig>
</sec>
<sec><title>Growth properties of TGEV and TGEV(91–95sg)</title>
<p>Following the successful recovery of TGEV(91–95sg), we next evaluated the
role of nsp1 in inhibiting host gene expression during viral replication. The
growth properties of TGEV(91–95sg) in various cell lines were measured by
performing a multistep growth assay with a viral multiplicity of infection of
0.1. The results showed that both viruses reached a plateau at ∼48 h
postinfection in PK15 and ST cells. Furthermore, the viral growth and peak
titers of TGEV(91–95sg) in susceptible cells were indistinguishable from
those of the WT virus, indicating that the <italic>nsp1</italic>
gene was not
critical for viral growth in cell culture (<xref ref-type="fig" rid="F6">Fig.
6</xref>
, <italic>D</italic>
and <italic>E</italic>
). To confirm this
conclusion, we also tested the nsp1 expression level during viral infection and
found that it was indistinguishable between the mutant and WT viruses (<xref ref-type="fig" rid="F6">Fig. 6</xref>
<italic>C</italic>
).</p>
</sec>
<sec><title>Pathogenicity of TGEV and TGEV(91–95sg)</title>
<p>To test the pathogenicity of TGEV(91–95sg), an animal experiment was
carried out. Piglets that had not been breastfed at birth were randomly divided
into two groups with five piglets in each group; in addition, a mock-infected
control group was formed that contained three piglets. The piglets were orally
inoculated at a dose of 1 × 10<sup>6</sup>
50% tissue culture infective
dose (TCID<sub>50</sub>
) with the respective chimeric virus or mock-infected
with Dulbecco's modified Eagle's medium (DMEM). The animal experiments showed
that replacement of the selected motif (amino acids 91–95) reduced the
pathogenic properties of TGEV. Furthermore, in the TGEV group, all five piglets
exhibited obvious dehydration and weight loss. Severe diarrhea began at 48 h
postinfection, and all piglets died within 96 h, indicating the acquisition of
lethal characteristics (<xref ref-type="fig" rid="F7">Fig.
7</xref>
<italic>C</italic>
). Interestingly, the clinical signs were
significantly less severe in the TGEV(91–95sg) group than in the TGEV
group (<xref ref-type="fig" rid="F7">Fig. 7</xref>
,
<italic>A–C</italic>
). To further assess the attenuation of
TGEV(91–95sg), we performed post-mortem examinations and hematoxylin and
eosin (H&E) staining. In the TGEV group, the stomach was obviously inflated
and had undigested flocculate. Moreover, the gastric fundus mucosa was
congested, flushed, and bleeding. The stomach wall and small intestinal wall
became thin, which was especially true for the jejunum, and animals presented
with intestinal dilation with an abundance of yellow liquid. In the
TGEV(91–95sg) group, the stomach was slightly inflated, and the gastric
fundus mucosa was not bleeding. The small intestinal wall was slightly thinner
than that in the mock group. Overall, the damage to the small intestine and
stomach in the TGEV group was significantly worse than that in the
TGEV(91–95sg) group (<xref ref-type="fig" rid="F7">Fig.
7</xref>
<italic>D</italic>
). Moreover, H&E staining revealed extensive
fine damage to different segments of the intestine, characterized by the
fragmentation and shedding of intestinal villi. The results showed that the
TGEV(91–95sg) group had only mild lesions in both the intestine and
stomach compared with the TGEV group (<xref ref-type="fig" rid="F7">Fig.
7</xref>
<italic>E</italic>
).</p>
<fig id="F7" orientation="portrait" position="float"><label>Figure 7.</label>
<caption><p><bold>Pathogenicity analysis of TGEV and TGEV(91–95sg).</bold>
<italic>A</italic>
, piglets were inoculated with the viruses. The
average weight gain of the piglets was recorded on the day of death or
euthanasia as the final time point. In the TGEV group, one piglet died
by the 3rd day, and the other piglets died by the 4th day. In the
TGEV(91–95sg) and mock groups, the piglets were euthanized on the
8th day. Data are represented as mean ± S.D., <italic>n</italic>
=
3–5. <italic>B</italic>
, clinical mental state scores of the
piglets in the different groups. The following criteria were used for
evaluation: 0, normal; 1, mild lethargy (slow to move; head down); 2,
moderate lethargy (stands but tends to lie down); 3, heavier lethargy
(lies down; occasionally stands); and 4, severe lethargy (recumbent;
moribund). Data are represented as mean ± S.D., <italic>n</italic>
= 3–5. <italic>C</italic>
, survival rates of the piglets in each
group. Survival curves for the piglets infected with the recovered
viruses in each group are shown. <italic>D</italic>
, gross lesions in
piglets inoculated with the recombinant viruses. The intestinal lesions
were examined on the day of death or after euthanasia at the final time
points. The necropsy images show transparent intestines observed in
piglets inoculated with TGEV and TGEV(91–95sg) but not in
mock-inoculated piglets. <italic>E</italic>
, histopathological
examination of the intestines from the recombinant TGEV-infected
piglets. Different segments, including the duodenum, jejunum, ileum,
large intestine, and stomach, were taken from each group and then
processed for H&E staining. Representative images are shown.
Original magnification × 100 (<italic>scale bars</italic>
200
μm).</p>
</caption>
<graphic xlink:href="zbc0381911180007"></graphic>
</fig>
<p>Next, we investigated how nsp1 contributed to the virulence of TGEV by analyzing
the viral infection efficiency as reflected by virus shedding (<xref ref-type="fig" rid="F8">Fig. 8</xref>
<italic>A</italic>
) and viral tissue
distribution (<xref ref-type="fig" rid="F8">Fig. 8</xref>
<italic>B</italic>
).
Fecal samples were obtained with fecal swabs, and virus shedding was tested by
regular PCR. The shedding of these groups was measured on days 1–7 and
was terminated because of the quick deaths of the infected piglets. At 7 days
postinoculation, all surviving piglets were euthanized to reduce the stress of
the other piglets. The results showed that the challenged piglets had viruses in
their feces. In contrast, no detectable virus shedding was found in the
mock-infected pigs. Furthermore, we performed sequencing of the corresponding
replaced regions to confirm the stability of the infective virus.
Immunohistochemistry showed that the virus was present in susceptible intestinal
tissues, consistent with the clinical results. Collectively, these data
demonstrate that TGEV(91–95sg) is strongly attenuated <italic>in
vivo</italic>
, and we conclude that the <italic>nsp1</italic>
gene is
necessary for virulence.</p>
<fig id="F8" orientation="portrait" position="float"><label>Figure 8.</label>
<caption><p><bold>Virus shedding in feces and immunohistochemistry of the intestine
and stomach.</bold>
<italic>A</italic>
, daily virus shedding in feces from the different
groups was measured by RT-PCR detection of viral genomes in fecal swabs.
The length of the target fragment is ∼1800 bp.
<italic>B</italic>
, immunohistochemistry of different tissue sections,
including the stomach, duodenum, jejunum, ileum, and large intestine,
from the piglets. The <italic>black arrowheads</italic>
point to the
virus in the tissue. Original magnification × 100 (<italic>scale
bars</italic>
200 μm).</p>
</caption>
<graphic xlink:href="zbc0381911180008"></graphic>
</fig>
</sec>
</sec>
<sec sec-type="discussion"><title>Discussion</title>
<p>Many viral proteins suppress host gene expression and modify host cell environments
to promote virus-specific translation (<xref rid="B20" ref-type="bibr">20</xref>
,
<xref rid="B21" ref-type="bibr">21</xref>
). As a result of such processes, these
proteins are often considered important virulence factors that contribute to the
design of live attenuated vaccines. For example, nonstructural protein 1 (NS1) of
the influenza virus plays a crucial role in replication and pathogenesis by
suppressing apoptosis-associated specklike protein ubiquitination; thus, vaccination
with a live attenuated H5N1 influenza vaccine lacking NS1 is safe (<xref rid="B22" ref-type="bibr">22</xref>
<xref ref-type="bibr" rid="B23">–</xref>
<xref rid="B24" ref-type="bibr">24</xref>
). The virion host
shutoff (vhs) protein is the key regulator of the induced early host shutoff
response, and herpes simplex viruses with vhs deleted have been proposed as live
attenuated vaccines (<xref rid="B25" ref-type="bibr">25</xref>
, <xref rid="B26" ref-type="bibr">26</xref>
). Human metapneumovirus protein M2-1
phosphorylation plays important regulatory roles in RNA synthesis, replication, and
pathogenesis, and inhibition of M2-1 phosphorylation may serve as a novel approach
for development of live attenuated vaccines (<xref rid="B27" ref-type="bibr">27</xref>
).</p>
<p>This phenomenon is also widely found in CoVs. Envelope proteins affect virulence, and
an attenuated virus with envelope gene deletion is a promising vaccine candidate
(<xref rid="B28" ref-type="bibr">28</xref>
<xref ref-type="bibr" rid="B29">–</xref>
<xref rid="B34" ref-type="bibr">34</xref>
). Although many viral proteins can induce host translational shutoff,
nsp1 was the first CoV gene 1 protein recognized to play an important role in
inhibiting host gene expression to regulate viral replication. In β-CoVs,
deletion of the functional region of nsp1 will be critical for developing attenuated
vaccines (<xref rid="B11" ref-type="bibr">11</xref>
, <xref rid="B17" ref-type="bibr">17</xref>
, <xref rid="B35" ref-type="bibr">35</xref>
). However, the role of
nsp1 in α-CoV pathogenesis is unknown. In our study, we replaced the important
motif in the TGEV nsp1-coding sequence using our reverse genetics technology. Then,
we successfully recovered the mutant virus. Subsequently, the viral growth and peak
titers of the mutant virus in susceptible cells were found to be indistinguishable
from those of the WT virus, indicating that the <italic>nsp1</italic>
gene was not a
critical factor driving viral growth in cell culture (<xref ref-type="fig" rid="F6">Fig. 6</xref>
, <italic>D</italic>
and <italic>E</italic>
). To confirm this
conclusion, we also tested the nsp1 expression level during viral infection and
found that it was indistinguishable between the mutant and WT viruses (<xref ref-type="fig" rid="F6">Fig. 6</xref>
<italic>C</italic>
). Furthermore, the
animal experiments revealed that the determinant region within TGEV nsp1 was
critical for viral virulence based on gain-of-function studies. Taken together, the
results of this study demonstrate, for the first time, that loss of α-CoV
nsp1–induced inhibition of host gene expression does not affect viral
replication but significantly reduces virulence.</p>
<p>Among the four CoV genera, only α-CoV and β-CoV encode nsp1. Previous
studies have indicated that the mechanisms by which nsp1 suppresses host gene
expression may differ among CoV species (<xref rid="B13" ref-type="bibr">13</xref>
<xref ref-type="bibr" rid="B14">–</xref>
<xref rid="B15" ref-type="bibr">15</xref>
, <xref rid="B36" ref-type="bibr">36</xref>
). The mechanisms
of nsp1-induced host gene suppression have been well-characterized for β-CoVs.
Nsp1 proteins of bat CoVs have been shown to bind viral RNA and to suppress host
translation (<xref rid="B37" ref-type="bibr">37</xref>
). SARS-CoV nsp1 uses a
two-pronged strategy to inhibit host gene expression by first interacting with the
40S ribosomal subunit and then inactivating translation (<xref rid="B13" ref-type="bibr">13</xref>
). MERS-CoV nsp1 selectively targets transcribed mRNA to
inhibit host gene expression (<xref rid="B14" ref-type="bibr">14</xref>
). MHV nsp1
interferes efficiently with the type I IFN system to enhance virulence (<xref rid="B11" ref-type="bibr">11</xref>
). Nonetheless, structural analysis indicates
that CoV nsp1s have a common origin (<xref rid="B7" ref-type="bibr">7</xref>
<xref ref-type="bibr" rid="B8">–</xref>
<xref rid="B9" ref-type="bibr">9</xref>
)
and that the nsp1 proteins of α-CoVs and β-CoVs share a biological
function to inhibit host gene expression. Furthermore, previous studies have
revealed that the C terminus of MHV and SARS-CoV nsp1 is an important functional
region for inhibiting host protein synthesis (<xref rid="B10" ref-type="bibr">10</xref>
, <xref rid="B11" ref-type="bibr">11</xref>
). However, the conserved
important domain underlying the α-CoV nsp1–induced inhibition of host
gene expression is unclear. Although we have reported that the loops at amino acid
positions 67–71, 78–85, and 103–110 generate a stable
functional region (<xref rid="B9" ref-type="bibr">9</xref>
), this functional domain
is not present in the TGEV nsp1 protein (<xref rid="B12" ref-type="bibr">12</xref>
).
To identify the common functional domain in α-CoV nsp1, we selected TGEV nsp1
as a model. Although the structure of a truncated TGEV nsp1 (PDB code <ext-link ext-link-type="pdb" xlink:href="3ZBD">3ZBD</ext-link>
) has been reported (<xref rid="B8" ref-type="bibr">8</xref>
),
we wondered whether the C terminus could affect the overall structure. Subsequently,
we determined the structure of full-length TGEV nsp1. Structural characterization
revealed that full-length TGEV nsp1 shared a common skeleton with truncated nsp1.
These similar conformations suggested that the C terminus had no influence on the
overall structure of TGEV nsp1. Although the 37–40 mutant protein
significantly reduced the inhibition of host gene expression, the mutant virus with
nsp1(37–40sg) was not successful. Although a previous study has also reported
that 5′-UTR of coronavirus can effect viral replication (<xref rid="B38" ref-type="bibr">38</xref>
), we are not sure whether the rescue failure
of this mutant virus is due to the loss of nsp1 activity or the effect of
5′-UTR. Through structural analysis and biochemical experiments, we verified
that the motif comprising amino acids 91–95 was necessary for the inhibition
of host gene expression by nsp1. Interestingly, this motif could be expanded to
other representative α-CoV nsp1 proteins (<xref ref-type="fig" rid="F5">Fig.
5</xref>
). In our study, we found that the motif comprising amino acids
91–95 is a conserved region in α-CoVs, which is conducive to
understanding the mechanism of α-CoV nsp1 inhibition of host gene
expression.</p>
<p>In summary, using a reverse-genetics platform, we obtained the first evidence that
TGEV nsp1 does not affect replication ability but significantly reduces
pathogenicity. We also found that the motif comprising amino acids 91–95 in
α-CoV nsp1 is a common functional region involved in the inhibition of host
gene expression. Overall, our results add detail to the molecular mechanism of
α-CoV virulence, which might contribute to the design of a novel attenuated
α-CoV vaccine based on nsp1 modifications.</p>
</sec>
<sec sec-type="methods"><title>Experimental procedures</title>
<sec><title>Cells and viruses</title>
<p>HEK-293T and ST cells (ATCC) were maintained at 37 °C with 5% CO<sub>2</sub>
in Gibco DMEM (high glucose, 4.5 g/liter) containing 10% fetal bovine serum.
PK15 cells (ATCC) were maintained in Gibco DMEM (low glucose, 1 g/liter)
supplemented with 10% fetal bovine serum. The pBAC-TGEV-GFP plasmid containing a
full-length infectious cDNA clone, which was derived from the highly virulent
TGEV strain WH-1 (GenBank<sup>TM</sup>
accession number <ext-link ext-link-type="gen" xlink:href="HQ462571">HQ462571</ext-link>
), has been described previously
(<xref rid="B19" ref-type="bibr">19</xref>
). To acquire the TGEV recombinant
virus, the plasmid was first transfected into HEK-293T cells, and then the virus
was recovered to infect virus-susceptible cells.</p>
</sec>
<sec><title>Plasmid construction</title>
<p>For expression in <italic>E. coli</italic>
, the full-length gene sequence of TGEV
nsp1 (GenBank accession number <ext-link ext-link-type="gen" xlink:href="HQ462571.1">HQ462571.1</ext-link>
) was cloned into a pET-42b(+) vector with an
N-terminal His<sub>6</sub>
tag via PCR amplification. The forward and reverse
primers contained the NdeI and XhoI restriction sites, respectively. The mutant
TGEV nsp1 sequence was inserted into pET-42b using the same method.</p>
<p>For expression in eukaryotic cells, WT nsp1 flanked with an N-terminal
hemagglutinin (HA) tag was cloned into the PCAGGS vector using the EcoRI and
XhoI restriction sites. To determine the functional region of TGEV nsp1, we used
a method reported previously (<xref rid="B9" ref-type="bibr">9</xref>
). The
following plasmids in which a random coil was replaced with a flexible sg linker
were engineered : nsp1(1–3sg), nsp1(11–15sg),
nsp1(19–22sg), nsp1(37–40sg), nsp1(46–48sg),
nsp1(55–60sg), nsp1(66–69sg), nsp1(76–84sg),
nsp1(91–95sg), and nsp1(105–109sg). All of the recombinant
expression plasmids were sequenced, and no unexpected mutations occurred.</p>
<p>To construct the recombinant virus containing mutant nsp1, a method previously
reported by our laboratory was used (<xref rid="B19" ref-type="bibr">19</xref>
).
The sequences of the relevant primers are shown in <xref rid="T2" ref-type="table">Table 2</xref>
. Each corresponding mutant virus was efficiently
constructed using CRISPR/Cas9 technology. Briefly, two specific restriction
sites encompassing the TGEV nsp1 sequence were selected. Then, we synthesized
two types of single-stranded DNA forward primers (sgnsp1F(91–95sg) and
sgnsp1R(91–95sg)) and a constant reverse primer (sgRNA) corresponding to
the two restriction sites. After performing annealing PCR using the forward and
reverse primers, the purified short DNA fragments from the PCR products were
transcribed using T7 RNA polymerase. The transcribed products corresponding to
the two sites were incubated with the nuclease Cas9 to digest the pTGEV-GFP BAC
plasmid <italic>in vitro</italic>
, and the digestion yielded a linearized BAC
and an ∼1.8-kb DNA fragment that included the TGEV nsp1 sequence.
Specifically, pTGEV-GFP BAC was digested in a 50-μl reaction mixture with
5 μg of pTGEV-GFP BAC, 5 μl of Cas9 (New England Biolabs), 10
μg of sgRNA, and 5 μl of Nuclease Reaction Buffer (New England
Biolabs) at 37 °C overnight.</p>
<table-wrap id="T2" orientation="portrait" position="float"><label>Table 2</label>
<caption><p><bold>Sequences of the primers used for CRISPR/Cas9</bold>
</p>
<p>sgRNA is a common bottom oligo for transcription template DNA. The T7
promoter sequence is presented in green, and the guide sequence for the
targeting of transcription template DNA is presented in red. The blue
sequence regions of sgnsp1F(91–95sg)/sgnsp1R(91–95sg) are
the regions that overlap with the sgRNA sequence.</p>
</caption>
<graphic xlink:href="zbc038191118t002"></graphic>
</table-wrap>
</sec>
<sec><title>Protein expression and purification</title>
<p>For protein expression, the recombinant plasmid was transformed into <italic>E.
coli</italic>
Trans BL21 (DE3) cells, which were grown at 37 °C in
lysogeny broth (LB) medium containing 50 μg/ml kanamycin until the optical
density at 600 nm (OD<sub>600</sub>
) reached 0.6–0.8. Then, 1 m<sc>m</sc>
isopropyl β-<sc>d</sc>
-thiogalactopyranoside was added to induce cell
growth for 8 h at 27 °C. Purification was performed using a method reported
previously (<xref rid="B9" ref-type="bibr">9</xref>
). The target protein was
further purified by a Superdex 200 (GE Healthcare) column with elution buffer
(20 m<sc>m</sc>
Tris-HCl and 200 m<sc>m</sc>
NaCl, pH 7.4). For crystallization,
the purified protein was concentrated to ∼10 mg/ml.</p>
</sec>
<sec><title>Crystallization, data collection, and structure determination</title>
<p>TGEV nsp1 and TGEV nsp1(91–95sg) were crystallized via the sitting-drop
vapor-diffusion method at 20 °C. Optimal crystals were obtained under the
following conditions through screening and optimization: 0.5 <sc>m</sc>
nickel
chloride and 4% PEG3350 for TGEV nsp1 and 0.2 <sc>m</sc>
ammonium phosphate
dibasic and 23% PEG3350 for TGEV nsp1(91–95sg). Single crystals were
first washed with 5, 10, 15 and 30% ethylene glycol (v/v) as a cryoprotectant
and then flash frozen in liquid nitrogen. All data collection steps were
performed on beamline BL17U at the Shanghai Synchrotron Radiation Facility
(SSRF) using a MAR 225 CCD detector (MAR Research). All of the diffraction
images were integrated, merged, and scaled using HKL-3000 software (<xref rid="B39" ref-type="bibr">39</xref>
). The structures were solved by
molecular replacement with PHASER (<xref rid="B40" ref-type="bibr">40</xref>
)
using the truncated structure of TGEV nsp1 (PDB code <ext-link ext-link-type="pdb" xlink:href="3ZBD">3ZBD</ext-link>
) as a starting model. Manual model
building was performed using Coot (<xref rid="B41" ref-type="bibr">41</xref>
),
and the structures were refined with Phenix (<xref rid="B42" ref-type="bibr">42</xref>
). The refinement statistics are shown in <xref rid="T1" ref-type="table">Table 1</xref>
. All of the structural figures were drawn using
PyMOL (<xref rid="B43" ref-type="bibr">43</xref>
). The root mean square
deviation (r.m.s.d.) was analyzed using PDBeFold (<ext-link ext-link-type="uri" xlink:href="https://www.ebi.ac.uk/msd-srv/ssm/">https://www.ebi.ac.uk/msd-srv/ssm/</ext-link>
).<xref ref-type="fn" rid="FN2"><sup>3</sup>
</xref>
The amino acid sequences of the CoV nsp1
proteins were aligned using the ClustalW2 software program (<xref rid="B44" ref-type="bibr">44</xref>
) and visualized with the ESPript 3
server (<ext-link ext-link-type="uri" xlink:href="http://espript.ibcp.fr/ESPript/ESPript/">http://espript.ibcp.fr/ESPript/ESPript/</ext-link>
)<sup>3</sup>
(<xref rid="B46" ref-type="bibr">46</xref>
).</p>
</sec>
<sec><title>Reporter assay, ribopuromycylation assay, and Western blot analysis</title>
<p>The functional plasmid and reporter gene plasmids were transfected into HEK-293T
cells using Lipofectamine 3000 (Thermo Fisher) according to the manufacturer's
instructions. At 24 h post-transfection, cell lysates were prepared and
subjected to an Rluc reporter activity assay (Promega). A ribopuromycylation
assay was performed as described previously (<xref rid="B9" ref-type="bibr">9</xref>
, <xref rid="B45" ref-type="bibr">45</xref>
). Briefly, cultured
HEK-293T cells were transfected with different doses of the different plasmids.
At various time points post-transfection, the cells were pulse-labeled with 3
μ<sc>m</sc>
puromycin and then incubated for an additional hour at 37
°C with 5% CO<sub>2</sub>
.</p>
<p>To determine the success of plasmid transfection or virus infection in the cells,
cell samples were collected for treatment. For treatment, the cells were first
gently washed twice with precooled PBS and then centrifuged at 4000 rpm for 5
min. The supernatant was discarded, radioimmune precipitation assay lysis buffer
(Beyotime) was added, and the samples were incubated with rotation at 4 °C
for 20 min. The extracts were prepared in SDS-PAGE sample buffer. Protein
expression was analyzed via Western blotting. The proteins were visualized using
an anti-HA antibody (Ab; Signalway Antibody (SAB)), an anti-puromycin Ab
(Millipore), an anti-TGEV N Ab (provided by the laboratory), and an anti-TGEV
nsp1 Ab (provided by the laboratory). Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) expression was detected with an anti-GAPDH monoclonal Ab (mAb;
Proteintech) to confirm equal protein loading. The corresponding grayscale value
of each protein band was analyzed with ImageJ.</p>
</sec>
<sec><title>Quantitative analysis</title>
<p>Quantitative analysis of mRNA was performed as described previously (<xref rid="B9" ref-type="bibr">9</xref>
). Briefly, RNA was extracted from the
tissue or cell samples, and a reverse transcription kit (Toyobo) was used to
quantitatively reverse transcribe 100 ng of RNA in a 20-μl system. The
reverse transcription program was performed according to the instructions of the
reverse transcription kit (Toyobo). The target mRNA and the endogenous control
RNA (18S rRNA) were amplified with a TaqMan One-Step Real-time PCR Master Mix
Reagent kit (Bio-Rad). Following the manufacturer's instructions, PCR was
performed on an ABI PRISM 7000 real-time thermocycler (Applied Biosystems,
Foster City, CA). The amount of target mRNA was normalized to the amount of
endogenous 18S rRNA.</p>
</sec>
<sec><title>Protein structure accession numbers and statistical analysis</title>
<p>The coordinates and structural characteristics of TGEV nsp1 and TGEV
nsp1(91–95sg) were submitted to the Research Collaboratory for Structural
Bioinformatics (RCSB) under PDB accession numbers <ext-link ext-link-type="pdb" xlink:href="6IVC">6IVC</ext-link>
and <ext-link ext-link-type="pdb" xlink:href="6IVD">6IVD</ext-link>
, respectively. Unpaired Student's
<italic>t</italic>
test and one-way analysis of variance with Bonferroni
correction (Prism version 6.01, GraphPad Software) were used to analyze
differences between groups. * indicates a significant difference
(<italic>p</italic>
< 0.05), ** indicates a highly significant difference
(<italic>p</italic>
< 0.01), and *** indicates an extremely significant
difference (<italic>p</italic>
< 0.001).</p>
</sec>
<sec><title>Animal experiments</title>
<p>Thirteen 1-day-old piglets from a TGEV-free sow were randomly divided into three
groups and fed fresh liquid milk diluted in warm water every 3 h. All piglets
were confirmed to be free of TGEV, PEDV, porcine deltacoronavirus, and rotavirus
by RT-PCR analysis of piglet feces before viral challenge. The piglet weights
were measured and recorded at the beginning of the challenge. The piglet
challenge group was inoculated orally with chimeric viral particles at a dose of
1 × 10<sup>6</sup>
TCID<sub>50</sub>
or mock-inoculated with 500 μl
of DMEM. At 7 days postinoculation, all surviving piglets were euthanized to
reduce the stress of the other piglets. Before necropsy, the weight of every
piglet was recorded. At necropsy, four sections of the intestine (duodenum,
jejunum, ileum, and large intestine) and the stomach were collected, fixed in
10% formalin for histopathological examination, and stained with H&E.</p>
</sec>
<sec><title>Ethics statement</title>
<p>The animal experiments were performed according to protocols approved by the
Scientific Ethics Committee of Huazhong Agricultural University (permit number
HZAUSW-2018-009). Animal care and maintenance procedures were in compliance with
the recommendations by the Regulations for the Administration of Affairs
Concerning Experimental Animals provided by the Ministry of Science and
Technology of China.</p>
</sec>
</sec>
<sec><title>Author contributions</title>
<p>Z. S. data curation; Z. S. software; Z. S. and G. W. formal analysis; Z. S.
validation; Z. S. visualization; Z. S. writing-original draft; Z. S., G. W., Y. Y.,
and J. S. project administration; G. W. methodology; L. F., S. X., Z. F. F., and G.
P. resources; F. L. and G. P. writing-review and editing; G. P. supervision; G. P.
funding acquisition.</p>
</sec>
</body>
<back><fn-group><fn fn-type="supported-by"><p>This work was supported by National Natural Science Foundation of China Grants
31873020 and 31722056, National Key Research and Development Plan of China Grant
2018YFD0500102, and Huazhong Agricultural University Scientific and
Technological Self-Innovation Foundation Program 2662017PY028. <named-content content-type="COI-statement">The authors declare that they have no conflicts
of interest with the contents of this article</named-content>
.</p>
</fn>
<fn fn-type="other"><p>The atomic coordinates and structure factors (codes <ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=6IVC">6IVC</ext-link>
and <ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/pdb/explore/explore.do?structureId=6IVD">6IVD</ext-link>
) have been deposited in the Protein Data Bank
(<ext-link ext-link-type="uri" xlink:href="http://www.pdb.org/">http://wwpdb.org/</ext-link>
).</p>
</fn>
<fn fn-type="other" id="FN2"><label>3</label>
<p>Please note that the JBC is not responsible for the long-term archiving and
maintenance of this site or any other third party hosted site.</p>
</fn>
</fn-group>
<fn-group content-type="abbreviations"><fn id="FN1"><label>2</label>
<p>The abbreviations used are: <def-list><def-item><term id="G1">CoV</term>
<def><p>coronavirus</p>
</def>
</def-item>
<def-item><term id="G2">HCoV</term>
<def><p>human coronavirus</p>
</def>
</def-item>
<def-item><term id="G3">FIPV</term>
<def><p>feline infectious peritonitis virus</p>
</def>
</def-item>
<def-item><term id="G4">MHV</term>
<def><p>murine hepatitis virus</p>
</def>
</def-item>
<def-item><term id="G5">TGEV</term>
<def><p>transmissible gastroenteritis virus</p>
</def>
</def-item>
<def-item><term id="G6">PEDV</term>
<def><p>porcine epidemic diarrhea virus</p>
</def>
</def-item>
<def-item><term id="G7">SARS-CoV</term>
<def><p>severe acute respiratory syndrome coronavirus</p>
</def>
</def-item>
<def-item><term id="G8">MERS-CoV</term>
<def><p>Middle East respiratory syndrome coronavirus</p>
</def>
</def-item>
<def-item><term id="G9">nsp1</term>
<def><p>nonstructural protein 1</p>
</def>
</def-item>
<def-item><term id="G10">IFN</term>
<def><p>interferon</p>
</def>
</def-item>
<def-item><term id="G11">Rluc</term>
<def><p><italic>Renilla</italic>
luciferase</p>
</def>
</def-item>
<def-item><term id="G12">PDB</term>
<def><p>Protein Data Bank</p>
</def>
</def-item>
<def-item><term id="G13">sg</term>
<def><p>Ser-Gly-Ser-Gly</p>
</def>
</def-item>
<def-item><term id="G14">BAC</term>
<def><p>bacterial artificial chromosome</p>
</def>
</def-item>
<def-item><term id="G15">ST</term>
<def><p>swine testicle</p>
</def>
</def-item>
<def-item><term id="G16">TCID<sub>50</sub>
</term>
<def><p>50% tissue culture infective dose</p>
</def>
</def-item>
<def-item><term id="G17">DMEM</term>
<def><p>Dulbecco's modified Eagle's medium</p>
</def>
</def-item>
<def-item><term id="G18">r.m.s.d.</term>
<def><p>root mean square deviation</p>
</def>
</def-item>
<def-item><term id="G19">H&E</term>
<def><p>hematoxylin and eosin</p>
</def>
</def-item>
<def-item><term id="G20">NS1</term>
<def><p>nonstructural protein 1</p>
</def>
</def-item>
<def-item><term id="G21">vhs</term>
<def><p>virion host shutoff</p>
</def>
</def-item>
<def-item><term id="G22">HA</term>
<def><p>hemagglutinin</p>
</def>
</def-item>
<def-item><term id="G23">sgRNA</term>
<def><p>single guide RNA</p>
</def>
</def-item>
<def-item><term id="G24">Ab</term>
<def><p>antibody</p>
</def>
</def-item>
<def-item><term id="G25">GAPDH</term>
<def><p>glyceraldehyde-3-phosphate dehydrogenase.</p>
</def>
</def-item>
</def-list>
</p>
</fn>
</fn-group>
<ack><title>Acknowledgment</title>
<p>We thank the staff at the Shanghai Synchrotron Radiation Facility (SSRF) BL17U1
beamline for assistance with the X-ray data collection.</p>
</ack>
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