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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Expression of SARS-coronavirus envelope protein in <italic>Escherichia coli</italic> cells alters membrane permeability</title><author><name sortKey="Liao, Y" sort="Liao, Y" uniqKey="Liao Y" first="Y." last="Liao">Y. Liao</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author><author><name sortKey="Lescar, J" sort="Lescar, J" uniqKey="Lescar J" first="J." last="Lescar">J. Lescar</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author><author><name sortKey="Tam, J P" sort="Tam, J P" uniqKey="Tam J" first="J. P." last="Tam">J. P. Tam</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author><author><name sortKey="Liu, D X" sort="Liu, D X" uniqKey="Liu D" first="D. X." last="Liu">D. X. Liu</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">15522242</idno><idno type="pmc">7092894</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7092894</idno><idno type="RBID">PMC:7092894</idno><idno type="doi">10.1016/j.bbrc.2004.10.050</idno><date when="2004">2004</date><idno type="wicri:Area/Pmc/Corpus">000E22</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000E22</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Expression of SARS-coronavirus envelope protein in <italic>Escherichia coli</italic> cells alters membrane permeability</title><author><name sortKey="Liao, Y" sort="Liao, Y" uniqKey="Liao Y" first="Y." last="Liao">Y. Liao</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author><author><name sortKey="Lescar, J" sort="Lescar, J" uniqKey="Lescar J" first="J." last="Lescar">J. Lescar</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author><author><name sortKey="Tam, J P" sort="Tam, J P" uniqKey="Tam J" first="J. P." last="Tam">J. P. Tam</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author><author><name sortKey="Liu, D X" sort="Liu, D X" uniqKey="Liu D" first="D. X." last="Liu">D. X. Liu</name><affiliation><nlm:aff id="aff1">School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation><affiliation><nlm:aff id="aff2">Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</nlm:aff></affiliation></author></analytic><series><title level="j">Biochemical and Biophysical Research Communications</title><idno type="ISSN">0006-291X</idno><idno type="eISSN">1090-2104</idno><imprint><date when="2004">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in <italic>Escherichia coli</italic>. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in <italic>E. coli</italic> cells.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Agirre, A" uniqKey="Agirre A">A. Agirre</name></author><author><name sortKey="Barco, A" uniqKey="Barco A">A. Barco</name></author><author><name sortKey="Carrasco, L" uniqKey="Carrasco L">L. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Biochem Biophys Res Commun</journal-id><journal-id journal-id-type="iso-abbrev">Biochem. Biophys. Res. Commun</journal-id><journal-title-group><journal-title>Biochemical and Biophysical Research Communications</journal-title></journal-title-group><issn pub-type="ppub">0006-291X</issn><issn pub-type="epub">1090-2104</issn><publisher><publisher-name>Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15522242</article-id><article-id pub-id-type="pmc">7092894</article-id><article-id pub-id-type="publisher-id">S0006-291X(04)02295-8</article-id><article-id pub-id-type="doi">10.1016/j.bbrc.2004.10.050</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Expression of SARS-coronavirus envelope protein in <italic>Escherichia coli</italic> cells alters membrane permeability</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Liao</surname><given-names>Y.</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Lescar</surname><given-names>J.</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Tam</surname><given-names>J.P.</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Liu</surname><given-names>D.X.</given-names></name><email>dxliu@imcb.a-star.edu.sg</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="aff2" ref-type="aff">b</xref><xref rid="cor1" ref-type="corresp">*</xref></contrib></contrib-group><aff id="aff1"><label>a</label>School of Biological Science, Nanyang Technological University, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</aff><aff id="aff2"><label>b</label>Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673, Singapore</aff><author-notes><corresp id="cor1"><label>*</label>Corresponding author. Fax: +65 67791117 <email>dxliu@imcb.a-star.edu.sg</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>30</day><month>10</month><year>2004</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><day>3</day><month>12</month><year>2004</year></pub-date><pub-date pub-type="epub"><day>30</day><month>10</month><year>2004</year></pub-date><volume>325</volume><issue>1</issue><fpage>374</fpage><lpage>380</lpage><history><date date-type="received"><day>27</day><month>9</month><year>2004</year></date></history><permissions><copyright-statement>Copyright © 2004 Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2004</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><p>To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in <italic>Escherichia coli</italic>. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in <italic>E. coli</italic> cells.</p></abstract><kwd-group><title>Keywords</title><kwd>Severe acute respiratory syndrome coronavirus</kwd><kwd>Envelope protein</kwd><kwd>Membrane permeability</kwd><kwd><italic>Escherichia coli</italic></kwd><kwd>E protein</kwd><kwd>Oligomerization</kwd><kwd>Viroporin</kwd></kwd-group></article-meta></front><body><p id="para.0010">A novel coronavirus was identified as the causative agent of the recent epidemic of severe acute respiratory syndrome (SARS) <xref rid="bib18" ref-type="bibr">[18]</xref>. Similar to other coronaviruses, SARS coronavirus (SARS-CoV) is an enveloped virus with a single strand, positive-sense RNA genome of 29.7 kb in length. Upon virus entry into cells, a 3′-coterminal nested set of 9 mRNAs is produced <xref rid="bib20" ref-type="bibr">[20]</xref>. The genome-length mRNA, mRNA1, expresses two overlapping replicase proteins in the form of polyproteins 1a and 1a/b. The polyproteins are subsequently processed into at least 16 putative nonstructural proteins (NSP1–NSP16) by virus-encoded proteinases <xref rid="bib20" ref-type="bibr">[20]</xref>. The mature proteins comprise proteinases, RNA-dependent RNA polymerase, and helicase, involved in the genomic and subgenomic RNA synthesis. The four structural proteins, arranged in the order 5′-S-E-M-N-3′, are encoded by subgenomic mRNA 2, 4, 5, and 9, respectively. In addition, eight putative nonstructural proteins, 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b, are encoded by mRNA3, 6, 7, 8, and 9, respectively <xref rid="bib20" ref-type="bibr">[20]</xref>. They are unique proteins of SARS-CoV and very little is known about the functions of these nonstructural proteins <xref rid="bib18" ref-type="bibr">[18]</xref>, <xref rid="bib20" ref-type="bibr">[20]</xref>.</p><p id="para.0015">Coronavirus infection of cultured cells causes typical cytopathic effects (CPEs), including rounding-up and fusion of the infected cells, detachment of cells from the culture dishes, cell lysis, and death <xref rid="bib13" ref-type="bibr">[13]</xref>. Among them, formation of giant syncytial cells is the hallmark of early CPE in cells infected with most coronaviruses. The prominent CPE in cells infected with SARS-CoV, however, is rounding up of the infected cells, cell detachment, and lysis <xref rid="bib9" ref-type="bibr">[9]</xref>. Typically, two types of membrane active proteins are able to modify membrane permeability, leading to lysis of virus-infected cells <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>. The first type of these membrane active proteins is the viral proteins with fusogenic activity. One example of such proteins is the hepatitis C virus (HCV) E1 protein <xref rid="bib5" ref-type="bibr">[5]</xref>. The second type is a group of small, hydrophobic proteins termed viroporins <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>.</p><p id="para.0020">Viroporins are small, highly hydrophobic viral proteins that could interact with cellular membranes and modify membrane permeability to ions or other small molecules. In this manner, viroporins disorganize the membrane, cause cell lysis, and facilitate the release of viral progeny. This group of viral proteins is usually expressed at late stages of the infection cycles, forming hydrophilic pores in the plasma membrane and inducing a general increase in permeability to ions and small molecules, but not to macromolecules <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>. Viroporins have been found in many viruses, such as HCV p7 protein <xref rid="bib8" ref-type="bibr">[8]</xref>, <xref rid="bib15" ref-type="bibr">[15]</xref>, human immunodeficiency virus type 1 (HIV-1) Vpu <xref rid="bib6" ref-type="bibr">[6]</xref>, influenza A virus M2 <xref rid="bib16" ref-type="bibr">[16]</xref>, hepatitis A virus 2B <xref rid="bib10" ref-type="bibr">[10]</xref>, Semliki Forest virus 6K <xref rid="bib19" ref-type="bibr">[19]</xref>, picornavirus 2B <xref rid="bib1" ref-type="bibr">[1]</xref>, <xref rid="bib2" ref-type="bibr">[2]</xref>, <xref rid="bib11" ref-type="bibr">[11]</xref>, and avian reovirus p10 protein <xref rid="bib3" ref-type="bibr">[3]</xref>.</p><p id="para.0025">In this study, we aimed to identify and characterize SARS-CoV proteins that may induce membrane permeability upon expression in bacterial cells. Among five proteins tested, the E protein can obviously enhance membrane permeability to <italic>o</italic>-nitrophenyl-β-<sc>d</sc>-galactopyranoside (ONPG), a β-galactosidase substrate, and hygromycin B (HB), an antibiotic which can inhibit protein synthesis. Analysis of the E protein expressed in bacterial and mammalian cells demonstrated that the protein may form homodimer and homotrimer, and site-directed mutagenesis studies revealed that two cysteine residues of the E protein located at amino acid positions 40 and 44 are essential for the formation of oligomers and for induction of membrane permeability in <italic>Escherichia coli</italic> cells. These results together with the predicted structural similarity with known membrane-permeable proteins of other viruses suggest that the E protein might be a putative viroporin.</p><sec id="section.0010"><title>Experimental procedures</title><p id="para.0030"><italic>Transient expression of SARS-CoV sequence in mammalian cells</italic>. HeLa cells were grown at 37 °C in 5% CO<sub>2</sub> and maintained in Glasgow's modified Eagle's medium supplemented with 10% fetal calf serum.</p><p id="para.0035">SARS-CoV sequences were placed under the control of a T7 promoter and transiently expressed in mammalian cells using a vaccinia virus-T7 expression system. Briefly, 60–80% confluent monolayers of HeLa cells grown on 35-mm dishes (Falcon) were infected with 10 plaque-forming units/cell of a recombinant vaccinia virus (vTF7-3) that expresses T7 RNA polymerase. Two hours later, cells were transfected with 5 μg of plasmid DNA mixed with transfection reagent (DOTAP) according to the instructions of the manufacturer (Roche). The transfection mixture was replaced with fresh culture medium 6 h post-transfection. Cells were harvested and stored at −80 °C.</p><p id="para.0040"><italic>Expression of proteins in E. coli</italic>. Plasmids were transformed into <italic>E. coli</italic> strain BL21 (DE3). A single colony was grown in LB medium overnight and then diluted 100-fold in LB medium or M9 medium supplemented with 0.2% glucose. When the absorbance of the cultures reached 0.6 at 600 nm, 1 mM IPTG was added to the medium to induce protein synthesis.</p><p id="para.0045">At indicated times post-induction, the cell density of bacterial cultures was determined by measuring the light scattering at 600 nm.</p><p id="para.0050"><italic>Western blot analysis</italic>. Total protein from bacterial or HeLa cells was lysed with 2× SDS loading buffer (with or without 200 mM DTT) plus 10 mM of iodoacetamide and subjected to SDS–PAGE. Proteins were transferred to PVDF membrane (Stratagene) and blocked overnight at 4 °C in blocking buffer (5% fat free milk powder in TBST buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween 20). The membrane was incubated with a 1:1000 diluted primary antibody in blocking buffer for 1 h at room temperature. After washing three times with TBST, the membrane was incubated with 1:2000 diluted anti-mouse IgG conjugated with horseradish peroxidase (DAKO) in blocking buffer for 1 h at room temperature. After washing for three times with TBST, the polypeptides were detected with a chemiluminescence detection kit (ECL, Amersham Biosciences) according to the instructions of the manufacturer.</p><p id="para.0055"><italic>β</italic>-<italic>Galactosidase and hygromycin B assays</italic>. To measure the entry of ONPG into bacterial cells, 1 ml of bacterial cultures was removed at indicated times post-induction. After centrifugation, cells were re-suspended in 1 ml of fresh M9 medium containing 2 mM ONPG, a β-galactosidase substrate. Cells were incubated for 10 min at 30 °C, and the reaction was stopped by addition of 0.4 ml of 1 M sodium carbonate. Samples were centrifuged, and the absorbance at 420 nm was measured to estimate the cleavage of ONPG.</p><p id="para.0060">To detect the entry of HB into bacterial cells, 1 mM of HB was added to the medium 50 min post-induction. After incubation for 30 min, 1 μCi of [<sup>35</sup>S]methionine per ml was added to the medium and incubated at 37 °C for 15 min. The bacterial cells were then harvested and subjected to SDS–PAGE. The proteins were detected by autoradiography.</p><p id="para.0065"><italic>Polymerase chain reaction and site-directed mutagenesis</italic>. Amplification of the respective template DNAs with appropriate primers was performed with pfu DNA polymerase (Strategene) under the standard buffer conditions with 2 mM MgCl<sub>2</sub>. The PCR conditions were 35 cycles of 94 °C for 45 s, 50–58 °C for 45 s, and 72 °C for 45 s to 2 min. The annealing temperature and extension time were subjected to adjustments according to the melting temperatures of the primers used and the lengths of the PCR fragments synthesized. Site-directed mutagenesis was carried out with two rounds of PCR and two pairs of primers.</p><p id="para.0070"><italic>Construction of plasmids</italic>. Plasmids pET24-E, pET24-3a, pET24-3b, pET24-6, pET24-7a, and pET24-HCVE1 were constructed by cloning an <italic>Nde</italic>I/<italic>Xho</italic>I-digested PCR fragment into <italic>Nde</italic>I/<italic>Xho</italic>I-digested pET24a vector. All the constructs have a His tag fused to the 3′ end of the genes. The two primers for SARS E protein are: 5′-CGGGATATCCCATATGTACTCATTCGTTTCGGAA-3′ and 5′-CGGAATTCTTACTCGAGGACCAGAAGATCAGGAACTCC-3′. The two primers for SARS 3a protein are: 5′-CGGGATATCCCATATGGATTTGTTTATGAGATTT-3′ and 5′-CGGAATTCTTACTCGAGCAAAGGC-ACGCTAGTAGTTCGT-3′. The two primers for SARS 3b protein are: 5′-CGGGATATCCCATATGATGCCAACTACTTTGTTT-3′ and 5′-CGGAATTCTTACTCGAGACGTACCTGTTTCTTCCGAA-A-3′. The two primers for SARS 6 protein are: 5′-CGGGATATCCCATATGTTTCATCTTGTTGACTTC-3′ and 5′-CGGAATTCTTACTCGAGTGGATAATCTAACTCCATAGG-3′. The two primers for SARS 7a protein are: 5′-CGGGATATCCCATATGAAAATTATTCTCTTCCTG-3′ and 5′-CGGAATTCTTACTCGAGTTCTGTCTTTCTCTTAATGGT-3′. The two primers for HCV E1 protein are: 5′-CGGGATATCCCATATGTACCAAGTGCGCAATTCCTCG-3′ and 5′-CCGGAATTCTTAGCGGCCGCCGCGTCGACGCCGGCAAATAG-3′.</p><p id="para.0075">Plasmid pFlagE was constructed by cloning an <italic>Eco</italic>RV- and <italic>Eco</italic>RI-digested PCR fragment into <italic>Eco</italic>RV- and <italic>Eco</italic>RI-digested pFlag. The Flag tag is fused to the N-terminal end of the E protein. The two primers used are: 5′-CGGGATATCCCATATGTATCATTCGTTTCGGAA-3′ and 5′-CGGAATTCTTACTCGAGGACCAGAAGATCAGGAACTCC-3′.</p></sec><sec id="section.0015"><title>Results and discussion</title><sec id="section.0020"><title>Retardation of bacterial growth by SARS-CoV E, 6, and 7a proteins</title><p id="para.0080">The effect of overexpression of several SARS-CoV proteins containing potential transmembrane domain on bacterial growth was initially tested. For this purpose, nucleotide sequences covering the SARS-CoV E, 3a, 3b, 6, and 7a proteins and HCV E1 protein, respectively, were cloned into pET24a vector, and were expressed in <italic>E. coli</italic> BL21 (DE3), a strain carrying integrated in the chromosome the T7 RNA polymerase gene under the control of a lac UV5 promoter that can be induced by IPTG. HCV E1 protein was chosen as a positive control, as it has been shown to be able to increase membrane permeability when overexpressed in <italic>E. coli</italic> cells <xref rid="bib5" ref-type="bibr">[5]</xref>. As shown in <xref rid="fig1" ref-type="fig">Fig. 1</xref>A, expression of 3a and 3b proteins rendered no obvious effects on the growth of bacteria, compared with bacterial cells carrying the empty plasmid pET24a. Certain degrees of retardation of bacterial growth were observed in cells expressing the E, 6, and 7a and HCV E1 proteins (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A).<fig id="fig1"><label>Fig. 1</label><caption><p>(A) Effects of expression of SARS-CoV proteins on bacterial growth. <italic>E. coli</italic> strain BL21 (DE3) cells carrying pET24a-E, pET24a-3a, pET24a-3b, pET24a-6, pET24a-7a, pET24a-HCVE1, and pET24a were induced with 1 mM IPTG. The cell densities were measured at 600 nm at indicated times post-induction. Cells carrying pET24a-HCVE1 were used as positive control, and cells carrying pET24a were used as negative control. (B) Analysis of the expression of SARS-CoV proteins. Bacterial cells carrying plasmid pET24a-E, pET24a-3a, pET24a-3b, pET24a-6, pET24a-7a, and pET24a-HCVE1 were induced with 1 mM IPTG. To detect the protein expression directly, 150 μg/ml of rifampicin was added 30 min post-induction. After incubation for 2 h, cells were labeled with [<sup>35</sup>S]methionine for 15 min. Electrophoresis of cell extracts was carried out in SDS–15% polyacrylamide gels (lanes 1–6). The expression of these proteins from unlabeled bacterial cells harvested at 2 h post-induction was analyzed by Western blotting using anti-His monoclonal antibody (lanes 7–12). Numbers on the left indicate molecular masses in kiloDaltons.</p></caption><graphic xlink:href="gr1"></graphic></fig></p><p id="para.0085">The expression of these proteins was then checked by labeling bacterial proteins with [<sup>35</sup>S]methionine in the presence of rifampicin. <xref rid="fig1" ref-type="fig">Fig. 1</xref>B shows the presence of protein bands with apparent molecular masses of 9.3 (lane 1), 32 (lane 2), 18.5 (lane 3), 14.8 (lane 5), and 21.7 kDa (lane 6), corresponding to the expected His-tagged E, 3a, 3b, and 7a and HCV E1 proteins, respectively. However, no band corresponding to the His-tagged protein 6 (8.5 kDa) was detected (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B, lane 4). To confirm the expression and identities of these proteins, Western blot analysis was conducted using anti-His antibody. The 9.3-kDa E protein (lane 7), 32-kDa 3a protein (lane 8), 14.8-kDa 7a protein (lane 11), and 21.7-kDa HCV E1 protein (lane 12) were detected, confirming the expression of these proteins. However, the antibody fails to react with the 3b protein (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B, lane 9). The reason for the failure to detect the His-tagged 3b protein is currently uncertain. Once again, no obvious band corresponding to protein 6 was detected (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B, lane 10), indicating the expression of this protein at a very low level in the bacterial cells. As significant inhibition of the growth of bacterial cells carrying this gene was observed (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A), it suggests that the protein may be highly toxic to bacteria. This protein was excluded in the subsequent studies. It was also noted that induction of the empty vector, pET24a, affects the growth of bacteria carrying the plasmid, compared with bacteria carrying pET24-3a and pET24-3b (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A). This may reflect the fact that overexpression of the multiple cloning site-His tag region in pET24a might affect bacterial growth.</p></sec><sec id="section.0025"><title>Modification of membrane permeability by the expression of E protein</title><p id="para.0090">To test if the observed inhibition of bacterial growth by the expression of E and 7a proteins is caused by the modification of membrane permeability, HB, an antibiotic which can inhibit host cell protein synthesis but is normally impermeable to cells within a short period of time, was added to the culture medium after induction of protein expression with IPTG. When the cell membrane permeability is altered, it can translocate into cells to block cellular protein translation. Cells were metabolically labeled with [<sup>35</sup>S]methionine for 15 min after addition of HB. Expression of SARS-CoV E protein allows the entry of HB to cells, as host protein synthesis was completely blocked (<xref rid="fig2" ref-type="fig">Fig. 2</xref>A, lanes 1 and 2). Similar inhibitory effect on host protein synthesis was observed in bacterial cells expressing the positive control protein, HCV E1 protein (<xref rid="fig2" ref-type="fig">Fig. 2</xref>A, lanes 5 and 6). No obvious effect on membrane permeability was observed in cells expressing 7a protein (<xref rid="fig2" ref-type="fig">Fig. 2</xref>A, lanes 3 and 4).<fig id="fig2"><label>Fig. 2</label><caption><p>Modification of membrane permeability by SARS-CoV E protein. (A) Entry of HB into bacterial cells. BL21 (DE3) cells transformed with pET24-E, pET24-7a, and pET24-HCVE1 were induced with 1 mM IPTG. At 1 h post induction, 2 mM HB was added. After incubation for 15 min, proteins were metabolically labeled with [<sup>35</sup>S]methionine for 15 min. Cell extracts were analyzed by SDS–15% PAGE. Cells carrying pET24-HCVE1 were used as positive control. Numbers on the left indicate molecular masses in kiloDaltons. (B) Entry of ONPG into bacterial cells. BL21 (DE3) cells carrying pET24-E, pET24-7a, pET24-HCVE1, and pET24a were induced with 1 mM IPTG. Two millimolar ONPG was added at the indicated times and incubated at 30 °C for 10 min. The β-galactosidase activity was determined by measuring the absorbance at 420 nm. Cells carrying pET24-HCVE1 were used as positive control and cells carrying pET24a were used as negative control.</p></caption><graphic xlink:href="gr2"></graphic></fig></p><p id="para.0095">To further confirm the observation that expression of SARS-CoV E protein can induce changes in membrane permeability, entry of ONPG into bacterial cells was analyzed. ONPG, a substrate of β-galactosidase, is normally excluded by the membrane of intact cells. Entry of ONPG into bacterial cells was easily monitored by measuring its conversion to a colored compound by the β-galactosidase activity present in bacterial cells. As shown in <xref rid="fig2" ref-type="fig">Fig. 2</xref>B, induction of the expression of E protein caused a clear increase in the entry of ONPG into cells. These results confirm that expression of SARS-CoV E protein could increase membrane permeability in bacterial cells.</p></sec><sec id="section.0030"><title>Oligomerization of E protein</title><p id="para.0100">We next tested if E protein could form oligomers by analysis of the E protein expressed in bacteria and mammalian cells on both reducing and non-reducing SDS–PAGE gels. After induction with IPTG, the bacterial cells were harvested and lysed with the protein loading buffer plus 10 mM of iodoacetamide to irreversibly block the free cysteinyl thiols to form disulfide bonds. Under such conditions, electrophoresis of the bacterially expressed E protein on reducing SDS–PAGE gel showed the detection of a 9.3 kDa band, representing the monomer of the E protein (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lane 2). In addition to the 9.3-kDa protein band, analysis of the same sample on non-reducing SDS–PAGE gel showed the detection of a protein band migrating at the position of 27 kDa (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lane 1), representing a putative homotrimer of the E protein.<fig id="fig3"><label>Fig. 3</label><caption><p>Oligomerization of SARS-CoV E protein. The His-tagged E protein expressed in bacteria (lanes 1 and 2) and the flag-tagged E protein expressed in HeLa cells were separated on SDS–15% polyacrylamide gels under reducing (lanes 2 and 4) and non-reducing (lanes 1 and 3) conditions. Polypeptides were transferred to PVDF membrane and probed with anti-His (lanes 1 and 2) and anti-Flag (lanes 3 and 4) monoclonal antibodies, respectively. After incubation with horseradish peroxidase-conjugated anti-mouse IgG, the E protein was detected by a chemiluminescence detection kit. Numbers on the left indicate molecular masses in kiloDaltons.</p></caption><graphic xlink:href="gr3"></graphic></fig></p><p id="para.0105">The E protein was then expressed in HeLa cells. To detect the protein expression, an 11-amino-acid Flag tag was fused to the N-terminus of the E protein. Analysis of the E protein expressed in HeLa cell on reducing SDS–PAGE gel showed the detection of the 9.3-kDa monomer (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lane 4). Both the 9.3-kDa monomer and the 27-kDa trimer were observed under non-reducing conditions (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lane 3).</p><p id="para.0110">Occasionally, a protein band migrating between the 9.3-kDa monomer and the 27-kDa trimer could be detected when the E protein expressed in both bacterial and mammalian cells was analyzed under non-reducing conditions (<xref rid="fig3" ref-type="fig">Fig. 3</xref>, lanes 1 and 3; and see <xref rid="fig4" ref-type="fig">Fig. 4</xref>B). It may represent dimerization of the E protein. These results suggest that the E protein may form homo-dimers and trimers in both bacterial and mammalian cells.<fig id="fig4"><label>Fig. 4</label><caption><p>Mutational analysis of the role of three cysteine residues of E protein in its modification of membrane permeability and oligomerization. (A) Entry of HB into bacterial cells expressing wild type and mutant E proteins. BL21 (DE3) cells transformed with pET24-E, pET24-M1, pET24-M2, pET24-M3, pET24-M4, and pET24-M5 were induced with 1 mM IPTG. At 1 h post-induction, 2 mM HB was added. After incubation for 15 min, proteins were metabolically labeled with [<sup>35</sup>S]methionine for 15 min. Cell extracts were analyzed by SDS–15% polyacrylamide gel. Numbers on the left indicate molecular mass in kiloDaltons. (B) Oligomerization of wild type and mutant E protein. The His-tagged wild type and mutant E proteins expressed in BL21 (DE3) were separated on SDS–15% polyacrylamide gels under non-reducing conditions. Polypeptides were transferred to PVDF membrane and probed with anti-His monoclonal antibody. After incubation with horseradish peroxidase-conjugated anti-mouse IgG, the E protein was detected by a chemiluminescence detection kit. Numbers on the left indicate molecular masses in kiloDaltons.</p></caption><graphic xlink:href="gr4"></graphic></fig></p></sec><sec id="section.0035"><title>The essential roles of two cysteine residues of E protein in its modification of membrane permeability and oligomerization in E. coli cells</title><p id="para.0115">Examination of the amino acid sequence of E protein showed that the protein contains three cysteine residues at amino acid positions 40 (C1), 43 (C2), and 44 (C3), respectively. To analyze the involvement of these cysteine residues in the oliogomerization of E protein, site-directed mutagenesis of these cysteine residues to alanine either individually or in combination of two or three was made to generate five mutants (M1–M5). Mutant M1 contains mutation at the C1 position, M2 at C2, and M3 at C3. M4 contains mutations at both the C1 and C2 positions, and M5 contains mutations at all three positions. These mutants were then expressed in <italic>E. coli</italic> BL21 (DE3). As can be seen in <xref rid="fig4" ref-type="fig">Fig. 4</xref>A, expression of wild type E protein and mutant M2 showed obvious induction of membrane permeability, as significant inhibition of protein synthesis by HB was observed (lanes 1 and 2; and lanes 5 and 6). Expression of mutants M1, M3, M4, and M5 showed no obvious increase of membrane permeability to HB (<xref rid="fig4" ref-type="fig">Fig. 4</xref>A, lanes 3, 4, and 7–12), indicating that these mutations may abolish the function of E protein in its induction of membrane permeability in bacterial cells. These results demonstrate that C1 and C3 residues are crucial for the function of E protein.</p><p id="para.0120">Analysis of the expression of wild type and mutant E protein was then carried out to see if the two cysteine residues are involved in the formation of the oligomers. As shown in <xref rid="fig4" ref-type="fig">Fig. 4</xref>B, both the 9.3-kDa monomer and the 27-kDa homotrimer were detected in bacterial cells expressing wild type E protein and M2 under non-reducing conditions (lanes 1 and 3). Only the 9.3-kDa monomer was detected in bacterial cells expressing M1, M3, M4, and M5 under the same non-reducing conditions (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B, lanes 2, 4, 5, and 6). As mentioned, a protein band of approximately 18 kDa migrating between the 9.3-kDa monomer and the 27-kDa homotrimer was detected in cells expressing both wild type E protein and M2 (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B, lanes 1 and 3). These results confirm that the two cysteine residues are involved in oligomerization of the E protein by formation of interchain disulfide bonds.</p><p id="para.0125">Cytolytic animal viruses encode small hydrophobic integral membrane proteins to modify host cell membrane permeability during their infection cycles. These proteins contain at least one transmembrane domain that interacts with and expands the lipid bilayer, and could form hydrophilic pores in the membrane by oligomerization, with hydrophilic residues presumably facing the aqueous lumen of the pore <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>. The hydrophilic channels would allow low molecular weight hydrophilic molecules across the membrane barrier, leading to the disruption of membrane potential, collapse of ionic gradients, and release of essential compounds from the cell. Alterations in ion concentration, such as enhancing the intracellular concentration of sodium ions, in the cytoplasm of virus-infected cells at the beginning of viral protein synthesis would favor virus replication. This would promote translation of viral versus cellular mRNAs, as translation of mRNAs from many cytolytic animal viruses is fairly resistant to high sodium concentrations. In contrast, high sodium concentrations are inhibitory to the translation of most cellular mRNAs <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>. Progressive membrane damage during viral replication cycles would also result in cell lysis, promote virus release, and facilitate virus spread to surrounding cells. Disruption of the function of viroporins would therefore abrogate viral infectivity, rendering this group of viral proteins suitable targets for the development of antiviral drugs.</p><p id="para.0130">Is the SARS-CoV E protein a viroporin? Data presented in this study demonstrated that expression of E protein in bacteria inhibits bacterial growth, increase the entry of ONPG and HB into bacterial cells. Furthermore, the protein could form homo-dimers and trimers by interchain disulfide bonds in both bacterial and mammalian cells. These observations support that SARS-CoV E protein might function as a viroporin. Further studies are required to address if similar modification of membrane permeability can be observed in other systems, including yeast and mammalian cells.</p><p id="para.0135">Coronavirus E protein is a minor virion component <xref rid="bib14" ref-type="bibr">[14]</xref>. It plays an essential role in virion assembly and morphogenesis <xref rid="bib12" ref-type="bibr">[12]</xref>. Computer-aided programs predict that SARS-CoV E protein may contain a putative α-helical transmembrane domain from amino acids 11–33. Similar to other viroporins, SARS-CoV E protein also contains some hydrophilic residues in the helical region. These functional and structural features resemble the influenza virus M2 protein, the best characterized ion-channel forming viroporin with four α-helices and a tetrameric quaternary structure <xref rid="bib17" ref-type="bibr">[17]</xref>. If the SARS-CoV E protein were proved to be able to form ion channels in cellular membranes, it would open a way to design anti-SARS therapeutic approaches.</p><p id="para.0140">Is induction of membrane permeability a general feature of coronavirus E protein? Although the E proteins from different coronaviruses share a low homology in their primary amino acid sequences, the general structural features consisting of an α-helical transmembrane domain are conserved. It suggests that the E protein may play very similar roles, including induction of host cell membrane permeability changes critical for the replication of coronaviruses. 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Gen. Virol.</source><year>2003</year></element-citation></ref></ref-list><ack><title>Acknowledgments</title><p>This work was supported by grants from Nanyang Technological University (SUG15/02) and from the Biomedical Research Council (BMRC 03/1/22/17/220), Agency for Science Technology and Research, Singapore.</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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