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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein <xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Freundt, Eric C" sort="Freundt, Eric C" uniqKey="Freundt E" first="Eric C." last="Freundt">Eric C. Freundt</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Yu, Li" sort="Yu, Li" uniqKey="Yu L" first="Li" last="Yu">Li Yu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Park, Elizabeth" sort="Park, Elizabeth" uniqKey="Park E" first="Elizabeth" last="Park">Elizabeth Park</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lenardo, Michael J" sort="Lenardo, Michael J" uniqKey="Lenardo M" first="Michael J." last="Lenardo">Michael J. Lenardo</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Xu, Xiao Ning" sort="Xu, Xiao Ning" uniqKey="Xu X" first="Xiao-Ning" last="Xu">Xiao-Ning Xu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19403678</idno><idno type="pmc">2698541</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2698541</idno><idno type="RBID">PMC:2698541</idno><idno type="doi">10.1128/JVI.00367-09</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000672</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000672</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein <xref ref-type="fn" rid="fn1">▿</xref> </title><author><name sortKey="Freundt, Eric C" sort="Freundt, Eric C" uniqKey="Freundt E" first="Eric C." last="Freundt">Eric C. Freundt</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Yu, Li" sort="Yu, Li" uniqKey="Yu L" first="Li" last="Yu">Li Yu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Park, Elizabeth" sort="Park, Elizabeth" uniqKey="Park E" first="Elizabeth" last="Park">Elizabeth Park</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Lenardo, Michael J" sort="Lenardo, Michael J" uniqKey="Lenardo M" first="Michael J." last="Lenardo">Michael J. Lenardo</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author><author><name sortKey="Xu, Xiao Ning" sort="Xu, Xiao Ning" uniqKey="Xu X" first="Xiao-Ning" last="Xu">Xiao-Ning Xu</name><affiliation><nlm:aff id="aff1"></nlm:aff></affiliation></author></analytic><series><title level="j">Journal of Virology</title><idno type="ISSN">0022-538X</idno><idno type="eISSN">1098-5514</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Viruses such as hepatitis C and the severe acute respiratory syndrome coronavirus (SARS-CoV) encode proteins that are distributed between mitochondria and the nucleus, but little is known about the factors that control partitioning between these sites. SARS-CoV encodes a unique accessory gene called open reading frame (ORF) 3b that, like other unique accessory genes in SARS-CoV, likely contributes to viral pathogenicity. The ORF 3b protein is 154 amino acids and is predicted to express from the second ORF in subgenomic RNA3. In this report, we have characterized the molecular components that regulate intracellular localization of the ORF 3b protein. We demonstrate unique shuttling behavior of ORF 3b, whereby the protein initially accumulates in the nucleus and subsequently translocates to mitochondria. Following nuclear localization, ORF 3b traffics to the outer membrane of mitochondria via a predicted amphipathic α-helix. Additionally, ORF 3b contains a consensus nuclear export sequence, and we demonstrate that nuclear export and thus mitochondrial translocation are dependent on a leptomycin B-sensitive nuclear export mechanism. We further show that ORF 3b inhibits induction of type I interferon induced by retinoic acid-induced gene 1 and the mitochondrial antiviral signaling protein. Our observations provide insights into the cellular localization of ORF 3b that may enhance our understanding of the mechanisms by which ORF 3b contributes to SARS-CoV pathogenesis. The findings reported here reveal that for multilocalized proteins, consideration of the spatiotemporal distribution may be crucial for understanding viral protein behavior and function.</p></div></front></TEI><pmc article-type="research-article"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">J Virol</journal-id><journal-id journal-id-type="publisher-id">jvi</journal-id><journal-title>Journal of Virology</journal-title><issn pub-type="ppub">0022-538X</issn><issn pub-type="epub">1098-5514</issn><publisher><publisher-name>American Society for Microbiology (ASM)</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19403678</article-id><article-id pub-id-type="pmc">2698541</article-id><article-id pub-id-type="publisher-id">0367-09</article-id><article-id pub-id-type="doi">10.1128/JVI.00367-09</article-id><article-categories><subj-group subj-group-type="heading"><subject>Virus-Cell Interactions</subject></subj-group></article-categories><title-group><article-title>Molecular Determinants for Subcellular Localization of the Severe Acute Respiratory Syndrome Coronavirus Open Reading Frame 3b Protein <xref ref-type="fn" rid="fn1">▿</xref> </article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Freundt</surname><given-names>Eric C.</given-names></name><xref ref-type="aff" rid="aff1">1</xref><xref ref-type="aff" rid="aff1">2</xref></contrib><contrib contrib-type="author"><name><surname>Yu</surname><given-names>Li</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Park</surname><given-names>Elizabeth</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Lenardo</surname><given-names>Michael J.</given-names></name><xref ref-type="aff" rid="aff1">1</xref></contrib><contrib contrib-type="author"><name><surname>Xu</surname><given-names>Xiao-Ning</given-names></name><xref ref-type="aff" rid="aff1">2</xref><xref ref-type="corresp" rid="cor1">*</xref></contrib></contrib-group><aff id="aff1">Laboratory of Immunology, Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,<label>1</label> Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom<label>2</label></aff><author-notes><fn id="cor1"><label>*</label><p>Corresponding author. Mailing address: Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom. Phone: 44 1865 222401. Fax: 44 1865 222628. E-mail: <email>xiaoning.xu@imm.ox.ac.uk</email></p></fn></author-notes><pub-date pub-type="ppub"><month>7</month><year>2009</year></pub-date><pub-date pub-type="epub"><day>29</day><month>4</month><year>2009</year></pub-date><volume>83</volume><issue>13</issue><fpage>6631</fpage><lpage>6640</lpage><history><date date-type="received"><day>18</day><month>2</month><year>2009</year></date><date date-type="accepted"><day>16</day><month>4</month><year>2009</year></date></history><permissions><copyright-statement>Copyright © 2009, American Society for Microbiology</copyright-statement></permissions><self-uri xlink:title="pdf" xlink:href="zjv01309006631.pdf"></self-uri><abstract><p>Viruses such as hepatitis C and the severe acute respiratory syndrome coronavirus (SARS-CoV) encode proteins that are distributed between mitochondria and the nucleus, but little is known about the factors that control partitioning between these sites. SARS-CoV encodes a unique accessory gene called open reading frame (ORF) 3b that, like other unique accessory genes in SARS-CoV, likely contributes to viral pathogenicity. The ORF 3b protein is 154 amino acids and is predicted to express from the second ORF in subgenomic RNA3. In this report, we have characterized the molecular components that regulate intracellular localization of the ORF 3b protein. We demonstrate unique shuttling behavior of ORF 3b, whereby the protein initially accumulates in the nucleus and subsequently translocates to mitochondria. Following nuclear localization, ORF 3b traffics to the outer membrane of mitochondria via a predicted amphipathic α-helix. Additionally, ORF 3b contains a consensus nuclear export sequence, and we demonstrate that nuclear export and thus mitochondrial translocation are dependent on a leptomycin B-sensitive nuclear export mechanism. We further show that ORF 3b inhibits induction of type I interferon induced by retinoic acid-induced gene 1 and the mitochondrial antiviral signaling protein. Our observations provide insights into the cellular localization of ORF 3b that may enhance our understanding of the mechanisms by which ORF 3b contributes to SARS-CoV pathogenesis. The findings reported here reveal that for multilocalized proteins, consideration of the spatiotemporal distribution may be crucial for understanding viral protein behavior and function.</p></abstract></article-meta></front><floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label><caption><p>Confocal analysis of the intracellular localization of the SARS-CoV ORF 3b. (A) Vero cells were transfected with ORF 3b-EGFP, counterstained with MitoTracker (red) or Hoechst (blue) to identify mitochondria and nuclei, respectively, and observed by confocal microscopy. Cells were observed that displayed exclusively nuclear (a to d), nuclear and mitochondrial ORF 3b (e to h), and exclusively mitochondrial (i to l) localization of ORF 3b. (B) Time-lapse microscopy of a single live Vero cell expressing ORF 3b-EGFP starting at 24 hpt. Images were captured every hour for 5 h, as indicated at the bottom left of each image.</p></caption><graphic xlink:href="zjv0130920570001"></graphic></fig><fig position="float" id="f2"><label>FIG. 2.</label><caption><p>FRAP analysis of nuclear and mitochondrial ORF 3b-EGFP. Vero cells were transfected with ORF 3b-EGFP, and cells expressing nuclear ORF 3b (A) or mitochondrial ORF 3b (B) were selected for analysis. (A) ROIs are shown that were photobleached (green), unbleached in the nucleus (orange), and an unbleached in the background (purple). (B) The photobleached ROI (green), an unbleached ROI including mitochondria (purple), and an unbleached ROI of background fluorescence (orange) are depicted. The time of bleaching is indicated on graphs by black arrowheads. FRAP was repeated two additional times, and similar results were obtained.</p></caption><graphic xlink:href="zjv0130920570002"></graphic></fig><fig position="float" id="f3"><label>FIG. 3.</label><caption><p>Intracellular targeting of ORF 3b-EGFP truncations. (A) Schematic representation of C-terminal EGFP constructs used to determine the mitochondrial localization sequence. The ability of the constructs to localize to mitochondria (Mito) is indicated. (B) Vero cells were transfected with constructs encoding EGFP fusion proteins containing the full-length ORF 3b protein (1 to 154-EGFP) or truncations expressing residues 1 to 70 (1-70-EGFP) or 1 to 90 (1-90-EGFP), as indicated, and counterstained with MitoTracker (red) or Hoechst (blue) to identify mitochondria and nuclei, respectively, and observed by confocal microscopy at 24 hpt.</p></caption><graphic xlink:href="zjv0130920570003"></graphic></fig><fig position="float" id="f4"><label>FIG. 4.</label><caption><p>Characterization of the mitochondrial targeting sequence of the ORF 3b protein. (A) The sequence of amino acids 70 to 154 of ORF 3b is shown with secondary structural predictions (H, helix) made by JPRED. Numbers indicate confidence of prediction (0, low; 9, high). (B) Helical wheel depiction of a predicted amphipathic helix formed by amino acids 74 to 85. (C) Protease protection assay. A mitochondrial fraction isolated from cells expressing pF-ORF 3b-EGFP was treated with proteinase K as described in Materials and Methods and subjected to immunoblotting to detect ORF 3b (anti-Flag), the outer mitochondrial transporter Tom20, or the internal mitochondrial protein OxPhos-I. (D) Mutational analysis of predicted helix. HEK293T cells were transfected with the indicated constructs and imaged by confocal microscopy at 24 hpt. Larger groups of cells expressing the unmutated ORF 3b protein (wild-type [WT]) and the K78K86 mutant are shown.</p></caption><graphic xlink:href="zjv0130920570004"></graphic></fig><fig position="float" id="f5"><label>FIG. 5.</label><caption><p>Evidence for a functional nuclear export sequence in ORF 3b. (A) Protein sequence alignment of nuclear export motifs of proteins described to rely on CRM1-dependent nuclear export and ORF 3b (Φ is L, I, V, F, M; X is any amino acid). (B) Vero cells transfected with ORF 3b-EGFP were counted to determine the percentage displaying nuclear, mitochondrial, or both (intermediate) types of ORF 3b protein localization. Cells were scored at 8 hpt (<italic>n</italic> = 189 cells) and 16 hpt after incubation without (16 hr) (<italic>n</italic> = 186 cells) or with (16 hr+LMB) (<italic>n</italic> = 189 cells) 5 ng/ml LMB. Histograms show means ± standard deviations of three independent transfections. (C) Vero cells were transfected with ORF 3b-EGFP (3b) (a to c and g to i) or HIV Rev-EGFP (Rev) (d to f and j to l) and counterstained with Hoechst to demarcate nuclei. Transfected cells were incubated in the absence or presence of the specific inhibitor of CRM1-mediated export, LMB, as indicated.</p></caption><graphic xlink:href="zjv0130920570005"></graphic></fig><fig position="float" id="f6"><label>FIG. 6.</label><caption><p>SARS-CoV ORF 3b inhibits transcription factors for type I IFN signaling induced by overexpression of RIG-I-N or MAVS. Histograms of luciferase reporter activity from HEK293T cells cotransfected with Flag-myc ORF 3b (3b) or empty vector (EV) and an NF-κB reporter plasmid (A and C) or an IRF-3 reporter plasmid (B) and pRLTK and expression constructs for either pcDNA (−), MAVS, or RIG-I-N. Cells were lysed at 16 hpt, and both firefly and <italic>Renilla</italic> luciferase activities were measured. Luciferase activity was normalized to pRLTK signal and is depicted as RLU. (C) Cells were treated with 10 ng/ml human TNF-α (TNF) at 16 hpt for 6 h prior to analysis. Histograms representing RLU are shown ± standard deviations of three replicates. *, <italic>P</italic> = 0.0003; **, <italic>P</italic> < 0.0001 (unpaired Student's <italic>t</italic> test).</p></caption><graphic xlink:href="zjv0130920570006"></graphic></fig><table-wrap position="float" id="t1"><label>TABLE 1.</label><caption><p>Oligonucleotides used for cloning and site-directed mutagenesis</p></caption><table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Primer name<xref ref-type="table-fn" rid="t1fn1"><italic>a</italic></xref></th><th colspan="1" rowspan="1" align="center" valign="bottom">Primer sequence (5′-3′)</th></tr></thead><tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">3b FOR</td><td colspan="1" rowspan="1" align="left" valign="top">CCGGAATTCATGATGCCAACTACTTTGTTTGCTGG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">3b REV</td><td colspan="1" rowspan="1" align="left" valign="top">TCCCCCCGGGCACGTACCTGTTTCTTCCGAA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">1 FOR</td><td colspan="1" rowspan="1" align="left" valign="top">GGGAATTCCACCATGATGCCAACTACTTTGTTTGCTGG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">20 FOR</td><td colspan="1" rowspan="1" align="left" valign="top">GGGAATTCCACCATGACAGTGTCACAGATACAATTGT</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">70 Rev</td><td colspan="1" rowspan="1" align="left" valign="top">ACACCGGTGCAAACTTCGGTGAAATAGCCATGTAC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">90 REV</td><td colspan="1" rowspan="1" align="left" valign="top">ACACCGGTGCAGAATGTAGCATTTTCAATACCAGTG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">110 REV</td><td colspan="1" rowspan="1" align="left" valign="top">ACACCGGTGCAGCCGTCGATTGTGTGTATTTGCA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">130 REV</td><td colspan="1" rowspan="1" align="left" valign="top">ACACCGGTGCTAGTAGTCGTCGTCGGCTCATCA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L74A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">GAAGTTTACTACCAGCGCGAGTCTACACAAATTA</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L74A REV</td><td colspan="1" rowspan="1" align="left" valign="top">TAATTTGTGTAGACTCGCGCTGGTAGTAAACTTC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">K78A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">CCAGCTTGAGTCTACACGCATTACTACAGACACTGG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">K78A REV</td><td colspan="1" rowspan="1" align="left" valign="top">CCAGTGTCTGTAGTAATGCGTGTAGACTCAAGCTGG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L79A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">CTTGAGTCTACACAAAGCACTACAGACACTGGTATTG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L79A REV</td><td colspan="1" rowspan="1" align="left" valign="top">CAATACCAGTGTCTGTAGTGCTTTGTGTAGACTCAAG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L83A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">CAAATTACTACAGACAGCGGTATTGAAAATGCTACATTC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L83A REV</td><td colspan="1" rowspan="1" align="left" valign="top">GAATGTAGCATTTTCAATACCGCTGTCTGTAGTAATTTG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">V84A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">CAAATTACTACAGACACTGGCATTGAAAATGCTACATTC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">V84A REV</td><td colspan="1" rowspan="1" align="left" valign="top">GAATGTAGCATTTTCAATGCCAGTGTCTGTAGTAATTTG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">K86A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">ACAGACACTGGTATTGGCAATGCTACATTCTTCATC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">K86A REV</td><td colspan="1" rowspan="1" align="left" valign="top">GATGAAGAATGTAGCATTGCCAATACCAGTGTCTGT</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L88A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">CACTGGTATTGAAAATGGCACATTCTTCATCTTTAACAAGC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L88A REV</td><td colspan="1" rowspan="1" align="left" valign="top">GCTTGTTAAAGATGAAGAATGTGCCATTTTCAATACCAGTG</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L93A FOR</td><td colspan="1" rowspan="1" align="left" valign="top">ATGCTACATTCTTCATCTGCAACAAGCTTGTTAAAGAC</td></tr><tr><td colspan="1" rowspan="1" align="left" valign="top">L93A REV</td><td colspan="1" rowspan="1" align="left" valign="top">GTCTTTAACAAGCTTGTTGCAGATGAAGAATGTAGCAT</td></tr></tbody></table><table-wrap-foot><fn id="t1fn1"><label>a</label><p>FOR, forward; 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