MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells
Identifieur interne : 000A05 ( Pmc/Corpus ); précédent : 000A04; suivant : 000A06MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells
Auteurs : Bibekanand Mallick ; Zhumur Ghosh ; Jayprokas ChakrabartiSource :
- PLoS ONE [ 1932-6203 ] ; 2009.
Abstract
Severe acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA)–mediated host–virus interactions in bronchoalveolar stem cells (BASCs) at the onset of infection by correlating the “BASC–microRNome” with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA–mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-), a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this investigation reveals another mode of exploitation of cellular miRNA machinery by virus to their own advantage.
Url:
DOI: 10.1371/journal.pone.0007837
PubMed: 19915717
PubMed Central: 2773932
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PMC:2773932Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells</title><author><name sortKey="Mallick, Bibekanand" sort="Mallick, Bibekanand" uniqKey="Mallick B" first="Bibekanand" last="Mallick">Bibekanand Mallick</name><affiliation><nlm:aff id="aff1"><addr-line>Computational Biology Group, Indian Association for the Cultivation of Science, Calcutta, India</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ghosh, Zhumur" sort="Ghosh, Zhumur" uniqKey="Ghosh Z" first="Zhumur" last="Ghosh">Zhumur Ghosh</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Medicine and Department of Radiology, Stanford University School of Medicine, Stanford, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chakrabarti, Jayprokas" sort="Chakrabarti, Jayprokas" uniqKey="Chakrabarti J" first="Jayprokas" last="Chakrabarti">Jayprokas Chakrabarti</name><affiliation><nlm:aff id="aff1"><addr-line>Computational Biology Group, Indian Association for the Cultivation of Science, Calcutta, India</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19915717</idno><idno type="pmc">2773932</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2773932</idno><idno type="RBID">PMC:2773932</idno><idno type="doi">10.1371/journal.pone.0007837</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000A05</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A05</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells</title><author><name sortKey="Mallick, Bibekanand" sort="Mallick, Bibekanand" uniqKey="Mallick B" first="Bibekanand" last="Mallick">Bibekanand Mallick</name><affiliation><nlm:aff id="aff1"><addr-line>Computational Biology Group, Indian Association for the Cultivation of Science, Calcutta, India</addr-line></nlm:aff></affiliation></author><author><name sortKey="Ghosh, Zhumur" sort="Ghosh, Zhumur" uniqKey="Ghosh Z" first="Zhumur" last="Ghosh">Zhumur Ghosh</name><affiliation><nlm:aff id="aff2"><addr-line>Department of Medicine and Department of Radiology, Stanford University School of Medicine, Stanford, California, United States of America</addr-line></nlm:aff></affiliation></author><author><name sortKey="Chakrabarti, Jayprokas" sort="Chakrabarti, Jayprokas" uniqKey="Chakrabarti J" first="Jayprokas" last="Chakrabarti">Jayprokas Chakrabarti</name><affiliation><nlm:aff id="aff1"><addr-line>Computational Biology Group, Indian Association for the Cultivation of Science, Calcutta, India</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</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>Severe acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA)–mediated host–virus interactions in bronchoalveolar stem cells (BASCs) at the onset of infection by correlating the “BASC–microRNome” with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA–mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-), a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19915717</article-id><article-id pub-id-type="pmc">2773932</article-id><article-id pub-id-type="publisher-id">09-PONE-RA-12288R1</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0007837</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline"><subject>Computational Biology/Genomics</subject><subject>Computational Biology/Transcriptional Regulation</subject><subject>Genetics and Genomics/Gene Expression</subject></subj-group></article-categories><title-group><article-title>MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells</article-title><alt-title alt-title-type="running-head">miRNA Regulation in BASCs</alt-title></title-group><contrib-group><contrib contrib-type="author" equal-contrib="yes"><name><surname>Mallick</surname><given-names>Bibekanand</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" equal-contrib="yes"><name><surname>Ghosh</surname><given-names>Zhumur</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author"><name><surname>Chakrabarti</surname><given-names>Jayprokas</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Computational Biology Group, Indian Association for the Cultivation of Science, Calcutta, India</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Department of Medicine and Department of Radiology, Stanford University School of Medicine, Stanford, California, United States of America</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Jordan</surname><given-names>I. King</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">Georgia Institute of Technology, United States of America</aff><author-notes><corresp id="cor1">* E-mail: <email>zhumurg@stanford.edu</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: BM ZG. Performed the experiments: BM ZG. Analyzed the data: BM ZG. Contributed reagents/materials/analysis tools: JC. Wrote the paper: BM ZG.</p></fn></author-notes><pub-date pub-type="collection"><year>2009</year></pub-date><pub-date pub-type="epub"><day>13</day><month>11</month><year>2009</year></pub-date><volume>4</volume><issue>11</issue><elocation-id>e7837</elocation-id><history><date date-type="received"><day>17</day><month>8</month><year>2009</year></date><date date-type="accepted"><day>12</day><month>10</month><year>2009</year></date></history><permissions><copyright-statement>Mallick et al.</copyright-statement><copyright-year>2009</copyright-year><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</license-p></license></permissions><abstract><p>Severe acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA)–mediated host–virus interactions in bronchoalveolar stem cells (BASCs) at the onset of infection by correlating the “BASC–microRNome” with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA–mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-), a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this investigation reveals another mode of exploitation of cellular miRNA machinery by virus to their own advantage.</p></abstract><counts><page-count count="12"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>Severe Acute Respiratory Syndrome (SARS) is a new fulminant atypical pneumonia which emerged as a regional and global threat in 2002–2003 with a high mortality rate resulting from acute lung failure <xref rid="pone.0007837-Donnelly1" ref-type="bibr">[1]</xref>. The disease causing agent has been identified as a novel coronavirus termed as SARS-associated coronavirus (SARS-CoV) <xref rid="pone.0007837-Ksiazek1" ref-type="bibr">[2]</xref>, <xref rid="pone.0007837-Poutanen1" ref-type="bibr">[3]</xref>. The SARS-CoV is an enveloped virus containing a single stranded, positive-sense RNA genome which encodes 14 putative open reading frames encoding 28 potential proteins <xref rid="pone.0007837-Marra1" ref-type="bibr">[4]</xref>, <xref rid="pone.0007837-Rota1" ref-type="bibr">[5]</xref>. These include four structural proteins, spike (S) glycoprotein, matrix (M) protein, small envelope (E) protein, and nucleocapsid (N) protein <xref rid="pone.0007837-Marra1" ref-type="bibr">[4]</xref>. These proteins have various roles in aiding the virus to enter the host and spread infection. While the incidence of new cases of SARS waned in 2003–2004, many aspects of SARS disease pathogenesis and host-pathogen interactions remain unsolved. Limited pathologic studies reveal that the major site of SARS-CoV infection and morbidity is the respiratory tract. The target organ of SARS is mainly lungs <xref rid="pone.0007837-Gu1" ref-type="bibr">[6]</xref>. Among the various animal models that have been used to study the pathogenesis of SARS-CoV infection, the monkey model mimics the clinical course of SARS to a certain degree <xref rid="pone.0007837-Kuiken1" ref-type="bibr">[7]</xref>. The cellular tropism of SARS-CoV in mouse lung has also been investigated by Ling <italic>et al</italic>., 2006 <xref rid="pone.0007837-Ling1" ref-type="bibr">[8]</xref>. But still today, very little is known regarding the mode of SARS-CoV interaction with host cells at the onset of infection in the lungs at a molecular level, and also the cell types in which the primary viral infection and replication occurs.</p><p>The lung is an extremely complex, conditionally renewing organ which contains anatomically and functionally distinct epithelial stem cell populations which reside in distinct anatomical locations. The basal cells <xref rid="pone.0007837-Hong1" ref-type="bibr">[9]</xref>, Clara cells <xref rid="pone.0007837-Evans1" ref-type="bibr">[10]</xref> and type-II pneumocytes <xref rid="pone.0007837-Fehrenbach1" ref-type="bibr">[11]</xref> are the candidate stem/progenitor cells which can repair the injured lungs and contribute to local needs in times of tissue damage. Recently, Kim <italic>et al</italic>. have isolated a regional pulmonary stem cell population termed as bronchoalveolar stem cells (BASCs) residing at the bronchoalveolar duct junction of adult lungs <xref rid="pone.0007837-Kim1" ref-type="bibr">[12]</xref>. These have been identified and characterized as CD34<sup>+</sup> Sca-1<sup>+</sup> CD45<sup>-</sup> PE-CAM<sup>−</sup> cells expressing both cytoplasmic Clara cell secretion protein (CCSP) and prosurfactant protein-C proteins, which are markers for Clara cells and type-II pneumocytes respectively <xref rid="pone.0007837-Kim1" ref-type="bibr">[12]</xref>. We have searched for the evidence to establish BASC as the prime target cells of SARS infection initiation and replication.</p><p>To date, miRNA-mediated RNA interference is reported to be an essential tool to understand the regulatory pathways at molecular level that underlie infection biology <xref rid="pone.0007837-Lu1" ref-type="bibr">[13]</xref>, <xref rid="pone.0007837-Ghosh1" ref-type="bibr">[14]</xref>. They not only participate in executive decisions but also perform much of the grunt work to micromanage protein output. Inspite of being such potential regulatory elements, miRNAs have so far missed attention in the pathogenesis of SARS-CoV infection.</p><p>MiRNAs are small (19–25 nucleotides) endogenous noncoding RNAs that have been shown to influence the abundance and translational efficiency of cognate mRNAs <xref rid="pone.0007837-Bartel1" ref-type="bibr">[15]</xref>, <xref rid="pone.0007837-Ghosh2" ref-type="bibr">[16]</xref>. Viruses, which typically employ many components of the host gene expression machinery, also encode miRNAs. On the contrary, miRNA-biogenesis pathway poses some serious problem for RNA viruses and a group of DNA viruses (poxviruses) to encode miRNAs <xref rid="pone.0007837-Ghosh1" ref-type="bibr">[14]</xref>. SARS-CoV being an RNA virus is assumed not to encode its own miRNAs. But in due course of evolution, the virus might have developed certain highly sophisticated molecular mechanisms to exploit the cellular biosynthetic machinery of host cells and elude the cellular defense mechanisms. It is most likely that in certain situations SARS-CoV uses the cellular miRNA transcripts in order to foster their own agenda.</p><p>Given the proposed involvement of BASCs in SARS disease <xref rid="pone.0007837-Ling1" ref-type="bibr">[8]</xref>, elucidation of the miRNA-mediated regulatory mechanisms responsible for making BASCs the prime targets of SARS infection might provide new avenues to explore the underlying mechanisms of SARS infection.</p><sec id="s1a"><title>Onset of SARS infection</title><p>The entry of SARS-CoV into cells is mediated through interaction between spike (S) glycoprotein of the virus and angiotensin-converting enzyme 2 (ACE2), the primary receptor of SARS-CoV on the host cell <xref rid="pone.0007837-Li1" ref-type="bibr">[17]</xref>. ACE2 is a homolog of angiotensin-converting enzyme (ACE), which plays an important role in the renin–angiotensin system for blood pressure homeostasis. Recent studies of Li <italic>et al</italic>. <xref rid="pone.0007837-Li2" ref-type="bibr">[18]</xref> have shown a protective effect of ACE2 against experimental lung fibrosis through its ability to degrade local tissue angiotensin II (ANG II) in response to bleomycin. Furthermore, in the pathogenesis of lung fibrosis, they have proposed the involvement of the down regulation of ACE2 as an integral component of the sequence of events leading to lung collagen deposition. The molecular mechanisms responsible for the loss of ACE2 in lung fibrosis are currently under investigation. A typical clinical feature associated with SARS is also pulmonary fibrosis and associated lung failure whose underlying mechanism still remains elusive <xref rid="pone.0007837-Zhao1" ref-type="bibr">[19]</xref>. Investigations leading to the underlying molecular mechanisms responsible for the loss of ACE2 in pulmonary fibrosis might provide a clue to the reason behind fibrosis associated lung injury in SARS.</p><p>Several reports suggest that there is a close interaction between SARS-CoV and respiratory epithelia which play an important role in the genesis of SARS <xref rid="pone.0007837-Yu1" ref-type="bibr">[20]</xref>. Jia <italic>et al</italic>. <xref rid="pone.0007837-Jia1" ref-type="bibr">[21]</xref> have investigated interactions between SARS-CoV and human airway epithelia and indicated that the state of cell differentiation and ACE2 expression levels are important determinants of the susceptibility of human airway epithelia to infection. To date, the molecular switches regulating the state of cellular differentiation of an infected cell and ACE2 expression have not been studied properly. In this work, we have proposed the role of miRNA in modulating the expression of ACE2 along with the stage specific cellular differentiation at the site of infection initiation. Further we have tried to focus on the role of SARS-CoV which dictates these events for its successful transmission and replication.</p><p>The POU-homeodomain transcription factor Oct-4 (Pou5f1) plays a central role in self-renewal, pluripotency, and lineage commitment. Alterations in Oct-4 expression promote differentiation and leads to the specification of ectodermal, endodermal or mesodermal primitive progenitors <xref rid="pone.0007837-Campbell1" ref-type="bibr">[22]</xref>. A small number of Oct-4-expressing cells have been observed at the bronchoalveolar junction of the neonatal lung <xref rid="pone.0007837-Ling1" ref-type="bibr">[8]</xref>. We have investigated miRNA-mediated modulatory role on Oct-4+ BASCs and have correlated their contribution to SARS infection.</p><p>Our analysis establishes a complex interplay between miRNAs, BASCs and certain essential factors related to the disease which might provide a better understanding of SARS pathogenesis. The multifarious relationships shared by genes related to the disease-system pathway, modulatory effects of miRNAs within the prime target cells of infection in response to indefinite cues impose the need of an interaction map. This study develops a complex interaction map between disease-related factors, miRNAs and BASCs in SARS disease pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Further, our investigation towards investigating the therapeutic potential of miRNA-mediated RNAi as an effective antiviral agent against SARS might unravel some of the efficient therapeutic measures against the deadly disease in future.</p></sec></sec><sec id="s2"><title>Results and Discussion</title><p>We have investigated the cellular tropism of SARS-CoV (<xref ref-type="table" rid="pone-0007837-t001">Table 1</xref>) in the BASCs present in lungs and attempted to establish the role of BASC-miRNAs towards understanding the pathogenesis of SARS in lung fibrosis. We describe the role of BASCs as the target cells of SARS infection and show how a repertoire of BASC miRNAs act as molecular switches to contribute to this host-pathogen interaction.</p><table-wrap id="pone-0007837-t001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.t001</object-id><label>Table 1</label><caption><title>SARS-CoV strains considered in our study.</title></caption><alternatives><graphic id="pone-0007837-t001-1" xlink:href="pone.0007837.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Name of the Strains</td><td align="left" rowspan="1" colspan="1">Accession no.</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Urbani</td><td align="left" rowspan="1" colspan="1">AY278741</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus ZJ01</td><td align="left" rowspan="1" colspan="1">AY286320</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS Coronavirus CDC#200301157</td><td align="left" rowspan="1" colspan="1">AY714217</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Taiwan TC1</td><td align="left" rowspan="1" colspan="1">AY338174</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Taiwan TC3</td><td align="left" rowspan="1" colspan="1">AY348314</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus ZJ0301 from China</td><td align="left" rowspan="1" colspan="1">DQ182595</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TOR2</td><td align="left" rowspan="1" colspan="1">AY274119</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus GD01</td><td align="left" rowspan="1" colspan="1">AY278489</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus FRA</td><td align="left" rowspan="1" colspan="1">AY310120</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Shanhgai LY</td><td align="left" rowspan="1" colspan="1">AY322207</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus NS-1</td><td align="left" rowspan="1" colspan="1">AY508724</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus civet007</td><td align="left" rowspan="1" colspan="1">AY572034</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus civet010</td><td align="left" rowspan="1" colspan="1">AY572035</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus civet020</td><td align="left" rowspan="1" colspan="1">AY572038</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TJF</td><td align="left" rowspan="1" colspan="1">AY654624</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus B039</td><td align="left" rowspan="1" colspan="1">AY686864</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus A022</td><td align="left" rowspan="1" colspan="1">AY686863</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus WH20</td><td align="left" rowspan="1" colspan="1">AY772062</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TWH</td><td align="left" rowspan="1" colspan="1">AP006557</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TWJ</td><td align="left" rowspan="1" colspan="1">AP006558</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TWK</td><td align="left" rowspan="1" colspan="1">AP006559</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TWS</td><td align="left" rowspan="1" colspan="1">AP006560</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus TWY</td><td align="left" rowspan="1" colspan="1">AP006561</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Frankfurt 1</td><td align="left" rowspan="1" colspan="1">AY291315</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus HSR 1</td><td align="left" rowspan="1" colspan="1">AY323977</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Sino3-11</td><td align="left" rowspan="1" colspan="1">AY485278</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus Sino1-11</td><td align="left" rowspan="1" colspan="1">AY485277</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus ShanghaiQXC2</td><td align="left" rowspan="1" colspan="1">AY463060</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus ShanghaiQXC1</td><td align="left" rowspan="1" colspan="1">AY463059</td></tr><tr><td align="left" rowspan="1" colspan="1">SARS coronavirus LLJ-2004</td><td align="left" rowspan="1" colspan="1">AY595412</td></tr></tbody></table></alternatives></table-wrap><sec id="s2a"><title>Primary site of SARS infection</title><p>Which is the primary site of SARS infection and how do these corona virus interacts with those cells to enter the host and eventually invades the host immune system to spread itself - is a pertinent question which prevails and is a debated issue. In order to throw light on this very important question, we looked upon at the SARS<sup>+</sup> cell markers and BASC markers (specific to each of its developmental stages from multipotent state to fully differentiated state).</p><p>It has been reported that SARS-infected cells are unlikely to be pneumocytes (both type I or type II) or cytokeratin<sup>+</sup> epithelial cells and it is quite obvious since SARS-CoV cannot infect or replicate in a fully differentiated cell <xref rid="pone.0007837-Chen1" ref-type="bibr">[23]</xref>. Further, SARS<sup>+</sup> cells are distinct from cells expressing the macrophage/monocyte specific marker CD68. But SARS<sup>+</sup> cells are found to express the functional receptor ACE2 as well as the stem/progenitor cell marker CD34 and Oct-4 <xref rid="pone.0007837-Chen1" ref-type="bibr">[23]</xref>. Chen <italic>et al</italic> have also shown that SARS<sup>+</sup> cells are CD45<sup>−</sup>. Hence it has been confirmed that the SARS<sup>+</sup> cells in the infected lung were a subset of putative stem/progenitor cells expressing CD34, Oct-4 and ACE2. Further, it is unlikely for SARS<sup>+</sup> cells to produce differentiated cell markers. The presence of SARS<sup>+</sup> cells within the multiple cell types at the bronchiolar lining layers makes it difficult to isolate SARS<sup>+</sup> cells. Some of the SARS<sup>+</sup> cells remains overlapped with adjacent cytokeratin<sup>+</sup> cells which might be mistakenly interpreted as colocalized cells. In such a situation BASC (Sca-1<sup>+</sup> CD34<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup>) in terminal bronchioles located exclusively at the broncho-alveolar duct junction (BADJ) stands a good selection to be investigated as the prime target cells of SARS-CoV infection.</p></sec><sec id="s2b"><title>Characterizing BASC at BADJ as the prime target cell of SARS infection initiation</title><p>A percentage of the Sca-1<sup>+</sup> CD4<sup>−</sup> Pecam<sup>−</sup> cell populations at BADJ are CD34<sup>+</sup>. This Sca-1<sup>+</sup> CD34<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup> population is enriched for BASCs <xref rid="pone.0007837-Kim1" ref-type="bibr">[12]</xref> became evident from the fact that it contained no ciliated cells. In addition to clonal colony formation, these cells exhibited extensive self-renewal in culture. They also had a greater capacity for differentiation compared to other lung epithelial cells. Sca-1<sup>+</sup> CD34<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup> BASC cultures further confirmed the multilineage differentiation capacity of BASCs. Within seven to ten days they show up positive for differentiated cell markers as they get differentiated into Clara-like cells (CCA<sup>+</sup> SP-C<sup>−</sup>), AT2-like cells (SP-C<sup>+</sup> CCA) and AT1-like cells (AQ5<sup>+</sup>).</p><p>Within the Sca-1<sup>+</sup> CD4<sup>−</sup> Pecam<sup>−</sup> cells the other percentage of cells showed CD34<sup>−</sup> and contained ciliated cells. The Oct-4<sup>+</sup> colony cells which are Sca-1<sup>+</sup> CD34<sup>−</sup> SSEA-1<sup>+</sup> cytokeratin<sup>+</sup> were shown to be succeptible to SARS-CoV infection by Ling <italic>et al</italic>. <xref rid="pone.0007837-Ling1" ref-type="bibr">[8]</xref>. These might be a subset of Sca-1<sup>+</sup> CD4<sup>−</sup> Pecam<sup>−</sup> CD34<sup>−</sup> population (<xref ref-type="fig" rid="pone-0007837-g001">Figure 1</xref>). Later studies have shown that cytokeratin<sup>+</sup> cells do not express the functional receptor ACE2 <xref rid="pone.0007837-Chen1" ref-type="bibr">[23]</xref>. Further, SARS<sup>+</sup> cells do not express cytokeratin and are CD34<sup>+</sup> SSEA-1<sup>−</sup>. Phagocytosis is a common mode of virus entry within surrounding cells in SARS infection <xref rid="pone.0007837-Yilla1" ref-type="bibr">[24]</xref>. Hence, presence of SARS antigen within the subset of Oct-4<sup>+</sup> colony cells which are Sca-1<sup>+</sup> CD34<sup>−</sup> SSEA-1<sup>+</sup> cytokeratin<sup>+</sup> is most likely due to phagocytosis. Thereby, it is evident that these Oct-4 cells (Sca-1<sup>+</sup> CD34<sup>−</sup> SSEA-1<sup>+</sup> cytokeratin<sup>+</sup>) are not the prime target cells for SARS-CoV infection.</p><fig id="pone-0007837-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.g001</object-id><label>Figure 1</label><caption><title>Lower respiratory tract focusing on bronchoalveolar duct junction (BADJ) of the lung.</title><p>Venn diagram represents an enlarged section of the BADJ showing up the different subsets of the distinct cellular population (Oct-4<sup>+</sup> ACE2<sup>+</sup>) in this region of the lung. A subset of this cellular population is enriched in BASCs identified as Sca-1<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup> CD34<sup>+</sup>, the other subset being Sca-1<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup> CD34<sup>−</sup>. On differentiation Sca-1<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup> CD34<sup>+</sup> cells show positive for CCA and SP-C; Sca-1<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup> CD34<sup>−</sup> cells show positive for CCA, SP-C, and ciliated cells.</p></caption><graphic xlink:href="pone.0007837.g001"></graphic></fig><p>On the contrary, the BASCs (Sca-1<sup>+</sup> CD34<sup>+</sup> CD45<sup>−</sup> Pecam<sup>−</sup>) must be another subset of these Oct-4<sup>+</sup> ACE2<sup>+</sup> epithelial colony cells (<xref ref-type="fig" rid="pone-0007837-g001">Figure 1</xref>) which provides a more favorable environment for SARS-CoV entry and replication. Absence of ciliated cells and CD34<sup>+</sup> makes them the most probable targets of SARS-CoV at the onset of infection.</p></sec><sec id="s2c"><title>miRNAs as modulators of BASC differentiation</title><p>Since SARS-CoV cannot infect or replicate in a fully differentiated cell, it is indeed essential for the virus to control the differentiation stages in an infected BASC cells so that they do not reach a fully differentiated state until the virus has undergone successful transmission and replication within this primary site of infection. Hence, it is important to monitor the expression levels of the different developmental stage specific markers of SARS infected BASC and also that of the corresponding regulatory switches that control their level of expression.</p><p>MiRNAs are widespread agents of post-transcriptional gene silencing and have been strongly linked with stem cells <xref rid="pone.0007837-Shcherbata1" ref-type="bibr">[25]</xref>, <xref rid="pone.0007837-Zhang1" ref-type="bibr">[26]</xref>. They exhibit a high degree of stage- and tissue-specificity, and therefore it is important to scan the profiling data for those miRNAs that operate during the narrow windows of development of a BASC in order to understand their regulatory impact on the expression levels of essential marker genes and transcription factors within the cell. In order to model the regulatory mechanisms within a SARS<sup>+</sup> cell, we have tried to understand the underlying regulatory mechanisms within a normal BASC cells mediated by the molecular switches- the miRNAs.</p><p>It has been established that the virus exploits the host-miRNA milieu to foster their own agenda of evading the host immune system, thereby establish a consistent infection and continue replication.</p></sec><sec id="s2d"><title>mh–miRNAs targeting host factors</title><p>We undertook an intensive transcriptome-wide search for candidate mh-miRNAs (BASC-miRNAs which are homologous between mouse-human whole genomes) (<xref ref-type="table" rid="pone-0007837-t002">Table 2</xref>) targeting the developmental stage specific transcriptional factors and marker genes of BASCs. <xref ref-type="table" rid="pone-0007837-t003">Table 3</xref> shows the list of the miRNAs targeting the potential stage specific factors of BASCs. Further, we have also elucidated the mh-miRNAs targeting ACE2. The target factors have further been mapped with the expression levels of their corresponding miRNAs within BASCs which shows their integrated correlation contributing towards each developmental stage of BASCs in a healthy individual (<xref ref-type="fig" rid="pone-0007837-g002">Figure 2</xref>). SARS-CoV definitely interferes with these miRNA mediated regulation to dictate the cell fate and continue its replication. Hence as a future advancement of this work, it will be worthy designing benchwork to detect the correlation of these miRNA-mRNA expressions within a SARS-CoV infected cell.</p><fig id="pone-0007837-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.g002</object-id><label>Figure 2</label><caption><title>Fold change of the differentially expressed host miRNAs in BASC targeting host and virulent viral factors <xref rid="pone.0007837-Qian1" ref-type="bibr">[61]</xref>.</title></caption><graphic xlink:href="pone.0007837.g002"></graphic></fig><table-wrap id="pone-0007837-t002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.t002</object-id><label>Table 2</label><caption><title>Significantly expressed mh–miRNAs in bronchio-alveolar stem cells.</title></caption><alternatives><graphic id="pone-0007837-t002-2" xlink:href="pone.0007837.t002"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">miRNAs</td><td align="left" rowspan="1" colspan="1">Mouse Accession No.</td><td align="left" rowspan="1" colspan="1">Human Accession No.</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">miR-142-3p</td><td align="left" rowspan="1" colspan="1">MIMAT0000155</td><td align="left" rowspan="1" colspan="1">MIMAT0000434</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-19a</td><td align="left" rowspan="1" colspan="1">MIMAT0000651</td><td align="left" rowspan="1" colspan="1">MIMAT0000073</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-144</td><td align="left" rowspan="1" colspan="1">MIMAT0000156</td><td align="left" rowspan="1" colspan="1">MIMAT0000436</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-374</td><td align="left" rowspan="1" colspan="1">MIMAT0003727</td><td align="left" rowspan="1" colspan="1">MIMAT0004955</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-7a</td><td align="left" rowspan="1" colspan="1">MIMAT0000677</td><td align="left" rowspan="1" colspan="1">MIMAT0000252</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-186</td><td align="left" rowspan="1" colspan="1">MIMAT0000215</td><td align="left" rowspan="1" colspan="1">MIMAT0000456</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-15b*</td><td align="left" rowspan="1" colspan="1">MIMAT0004521</td><td align="left" rowspan="1" colspan="1">MIMAT0004586</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-340-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0004651</td><td align="left" rowspan="1" colspan="1">MIMAT0004692</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-223</td><td align="left" rowspan="1" colspan="1">MIMAT0000665</td><td align="left" rowspan="1" colspan="1">MIMAT0000280</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-451</td><td align="left" rowspan="1" colspan="1">MIMAT0001632</td><td align="left" rowspan="1" colspan="1">MIMAT0001631</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-106a</td><td align="left" rowspan="1" colspan="1">MIMAT0000385</td><td align="left" rowspan="1" colspan="1">MIMAT0000103</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-140</td><td align="left" rowspan="1" colspan="1">MIMAT0000151</td><td align="left" rowspan="1" colspan="1">MIMAT0000431</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-421</td><td align="left" rowspan="1" colspan="1">MIMAT0004869</td><td align="left" rowspan="1" colspan="1">MIMAT0003339</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-142-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0000154</td><td align="left" rowspan="1" colspan="1">MIMAT0000433</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-20b</td><td align="left" rowspan="1" colspan="1">MIMAT0003187</td><td align="left" rowspan="1" colspan="1">MIMAT0001413</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-98</td><td align="left" rowspan="1" colspan="1">MIMAT0000545</td><td align="left" rowspan="1" colspan="1">MIMAT0000096</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-92a</td><td align="left" rowspan="1" colspan="1">MIMAT0000539</td><td align="left" rowspan="1" colspan="1">MIMAT0000092</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-15b</td><td align="left" rowspan="1" colspan="1">MIMAT0000124</td><td align="left" rowspan="1" colspan="1">MIMAT0000417</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-146b</td><td align="left" rowspan="1" colspan="1">MIMAT0003475</td><td align="left" rowspan="1" colspan="1">MIMAT0002809</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-20a</td><td align="left" rowspan="1" colspan="1">MIMAT0000529</td><td align="left" rowspan="1" colspan="1">MIMAT0000075</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-301a</td><td align="left" rowspan="1" colspan="1">MIMAT0000379</td><td align="left" rowspan="1" colspan="1">MIMAT0000688</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-148b</td><td align="left" rowspan="1" colspan="1">MIMAT0000580</td><td align="left" rowspan="1" colspan="1">MIMAT0000759</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-15a</td><td align="left" rowspan="1" colspan="1">MIMAT0000526</td><td align="left" rowspan="1" colspan="1">MIMAT0000068</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-218</td><td align="left" rowspan="1" colspan="1">MIMAT0000663</td><td align="left" rowspan="1" colspan="1">MIMAT0000275</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-17</td><td align="left" rowspan="1" colspan="1">MIMAT0000649</td><td align="left" rowspan="1" colspan="1">MIMAT0000070</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-106b</td><td align="left" rowspan="1" colspan="1">MIMAT0000386</td><td align="left" rowspan="1" colspan="1">MIMAT0000680</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-25</td><td align="left" rowspan="1" colspan="1">MIMAT0000652</td><td align="left" rowspan="1" colspan="1">MIMAT0000081</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-486</td><td align="left" rowspan="1" colspan="1">MIMAT0003130</td><td align="left" rowspan="1" colspan="1">MIMAT0002177</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-193</td><td align="left" rowspan="1" colspan="1">MIMAT0000223</td><td align="left" rowspan="1" colspan="1">MIMAT0000459</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-151-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0004536</td><td align="left" rowspan="1" colspan="1">MIMAT0004697</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-93</td><td align="left" rowspan="1" colspan="1">MIMAT0000540</td><td align="left" rowspan="1" colspan="1">MIMAT0000093</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-101b</td><td align="left" rowspan="1" colspan="1">MIMAT0000616</td><td align="left" rowspan="1" colspan="1">MIMAT0000099</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-127</td><td align="left" rowspan="1" colspan="1">MIMAT0000139</td><td align="left" rowspan="1" colspan="1">MIMAT0000446</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-483</td><td align="left" rowspan="1" colspan="1">MIMAT0004782</td><td align="left" rowspan="1" colspan="1">MIMAT0004761</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-574-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0004893</td><td align="left" rowspan="1" colspan="1">MIMAT0004795</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-21</td><td align="left" rowspan="1" colspan="1">MIMAT0000530</td><td align="left" rowspan="1" colspan="1">MIMAT0000076</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-200b</td><td align="left" rowspan="1" colspan="1">MIMAT0000233</td><td align="left" rowspan="1" colspan="1">MIMAT0000318</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-342-3p</td><td align="left" rowspan="1" colspan="1">MIMAT0000590</td><td align="left" rowspan="1" colspan="1">MIMAT0000753</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-151-3p</td><td align="left" rowspan="1" colspan="1">MIMAT0000161</td><td align="left" rowspan="1" colspan="1">MIMAT0000757</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-429</td><td align="left" rowspan="1" colspan="1">MIMAT0001537</td><td align="left" rowspan="1" colspan="1">MIMAT0001536</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-335-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0000766</td><td align="left" rowspan="1" colspan="1">MIMAT0000765</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-16</td><td align="left" rowspan="1" colspan="1">MIMAT0000527</td><td align="left" rowspan="1" colspan="1">MIMAT0000069</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-181d</td><td align="left" rowspan="1" colspan="1">MIMAT0004324</td><td align="left" rowspan="1" colspan="1">MIMAT0002821</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-99b</td><td align="left" rowspan="1" colspan="1">MIMAT0000132</td><td align="left" rowspan="1" colspan="1">MIMAT0000689</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-146a</td><td align="left" rowspan="1" colspan="1">MIMAT0000158</td><td align="left" rowspan="1" colspan="1">MIMAT0000449</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-331-3p</td><td align="left" rowspan="1" colspan="1">MIMAT0000571</td><td align="left" rowspan="1" colspan="1">MIMAT0000760</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-574-3p</td><td align="left" rowspan="1" colspan="1">MIMAT0004894</td><td align="left" rowspan="1" colspan="1">MIMAT0003239</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-10a</td><td align="left" rowspan="1" colspan="1">MIMAT0000648</td><td align="left" rowspan="1" colspan="1">MIMAT0000253</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-200a</td><td align="left" rowspan="1" colspan="1">MIMAT0000519</td><td align="left" rowspan="1" colspan="1">MIMAT0000682</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-423-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0004825</td><td align="left" rowspan="1" colspan="1">MIMAT0004748</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-17*</td><td align="left" rowspan="1" colspan="1">MIMAT0000650</td><td align="left" rowspan="1" colspan="1">MIMAT0000071</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-671-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0003731</td><td align="left" rowspan="1" colspan="1">MIMAT0003880</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-126-3p</td><td align="left" rowspan="1" colspan="1">MIMAT0000138</td><td align="left" rowspan="1" colspan="1">MIMAT0000445</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-125a-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0000135</td><td align="left" rowspan="1" colspan="1">MIMAT0000443</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-92b</td><td align="left" rowspan="1" colspan="1">MIMAT0004899</td><td align="left" rowspan="1" colspan="1">MIMAT0003218</td></tr><tr><td align="left" rowspan="1" colspan="1">let-7d*</td><td align="left" rowspan="1" colspan="1">MIMAT0000384</td><td align="left" rowspan="1" colspan="1">MIMAT0004484</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-27a</td><td align="left" rowspan="1" colspan="1">MIMAT0000537</td><td align="left" rowspan="1" colspan="1">MIMAT0000084</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-214</td><td align="left" rowspan="1" colspan="1">MIMAT0000661</td><td align="left" rowspan="1" colspan="1">MIMAT0000271</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-150</td><td align="left" rowspan="1" colspan="1">MIMAT0000160</td><td align="left" rowspan="1" colspan="1">MIMAT0000451</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-652</td><td align="left" rowspan="1" colspan="1">MIMAT0003711</td><td align="left" rowspan="1" colspan="1">MIMAT0003322</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-210</td><td align="left" rowspan="1" colspan="1">MIMAT0000658</td><td align="left" rowspan="1" colspan="1">MIMAT0000267</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-18a</td><td align="left" rowspan="1" colspan="1">MIMAT0000528</td><td align="left" rowspan="1" colspan="1">MIMAT0000072</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-361</td><td align="left" rowspan="1" colspan="1">MIMAT0000704</td><td align="left" rowspan="1" colspan="1">MIMAT0000703</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-152</td><td align="left" rowspan="1" colspan="1">MIMAT0000162</td><td align="left" rowspan="1" colspan="1">MIMAT0000438</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-24</td><td align="left" rowspan="1" colspan="1">MIMAT0000219</td><td align="left" rowspan="1" colspan="1">MIMAT0000080</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-148a</td><td align="left" rowspan="1" colspan="1">MIMAT0000516</td><td align="left" rowspan="1" colspan="1">MIMAT0000243</td></tr><tr><td align="left" rowspan="1" colspan="1">let-7g</td><td align="left" rowspan="1" colspan="1">MIMAT0000121</td><td align="left" rowspan="1" colspan="1">MIMAT0000414</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-19b</td><td align="left" rowspan="1" colspan="1">MIMAT0000513</td><td align="left" rowspan="1" colspan="1">MIMAT0000074</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-130b</td><td align="left" rowspan="1" colspan="1">MIMAT0000387</td><td align="left" rowspan="1" colspan="1">MIMAT0000691</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-128</td><td align="left" rowspan="1" colspan="1">MIMAT0000140</td><td align="left" rowspan="1" colspan="1">MIMAT0000424</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-181b</td><td align="left" rowspan="1" colspan="1">MIMAT0000673</td><td align="left" rowspan="1" colspan="1">MIMAT0000257</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-29b</td><td align="left" rowspan="1" colspan="1">MIMAT0000127</td><td align="left" rowspan="1" colspan="1">MIMAT0000100</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-455</td><td align="left" rowspan="1" colspan="1">MIMAT0003742</td><td align="left" rowspan="1" colspan="1">MIMAT0004784</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-221</td><td align="left" rowspan="1" colspan="1">MIMAT0000669</td><td align="left" rowspan="1" colspan="1">MIMAT0000278</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-30c</td><td align="left" rowspan="1" colspan="1">MIMAT0000514</td><td align="left" rowspan="1" colspan="1">MIMAT0000244</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-27b</td><td align="left" rowspan="1" colspan="1">MIMAT0000126</td><td align="left" rowspan="1" colspan="1">MIMAT0000419</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-30a</td><td align="left" rowspan="1" colspan="1">MIMAT0000128</td><td align="left" rowspan="1" colspan="1">MIMAT0000087</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-320</td><td align="left" rowspan="1" colspan="1">MIMAT0000666</td><td align="left" rowspan="1" colspan="1">MIMAT0000510</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-23b</td><td align="left" rowspan="1" colspan="1">MIMAT0000125</td><td align="left" rowspan="1" colspan="1">MIMAT0000418</td></tr><tr><td align="left" rowspan="1" colspan="1">let-7d</td><td align="left" rowspan="1" colspan="1">MIMAT0000383</td><td align="left" rowspan="1" colspan="1">MIMAT0000065</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-23a</td><td align="left" rowspan="1" colspan="1">MIMAT0000532</td><td align="left" rowspan="1" colspan="1">MIMAT0000078</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-125b-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0000136</td><td align="left" rowspan="1" colspan="1">MIMAT0000423</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-30d</td><td align="left" rowspan="1" colspan="1">MIMAT0000515</td><td align="left" rowspan="1" colspan="1">MIMAT0000245</td></tr><tr><td align="left" rowspan="1" colspan="1">let-7a</td><td align="left" rowspan="1" colspan="1">MIMAT0000521</td><td align="left" rowspan="1" colspan="1">MIMAT0000062</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-199a-5p</td><td align="left" rowspan="1" colspan="1">MIMAT0000229</td><td align="left" rowspan="1" colspan="1">MIMAT0000231</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-26a</td><td align="left" rowspan="1" colspan="1">MIMAT0000533</td><td align="left" rowspan="1" colspan="1">MIMAT0000082</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-191</td><td align="left" rowspan="1" colspan="1">MIMAT0000221</td><td align="left" rowspan="1" colspan="1">MIMAT0000440</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-30e</td><td align="left" rowspan="1" colspan="1">MIMAT0000248</td><td align="left" rowspan="1" colspan="1">MIMAT0000692</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-30b</td><td align="left" rowspan="1" colspan="1">MIMAT0000130</td><td align="left" rowspan="1" colspan="1">MIMAT0000420</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-26b</td><td align="left" rowspan="1" colspan="1">MIMAT0000534</td><td align="left" rowspan="1" colspan="1">MIMAT0000083</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-181a</td><td align="left" rowspan="1" colspan="1">MIMAT0000210</td><td align="left" rowspan="1" colspan="1">MIMAT0000256</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-143</td><td align="left" rowspan="1" colspan="1">MIMAT0000247</td><td align="left" rowspan="1" colspan="1">MIMAT0000435</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-195</td><td align="left" rowspan="1" colspan="1">MIMAT0000225</td><td align="left" rowspan="1" colspan="1">MIMAT0000461</td></tr><tr><td align="left" rowspan="1" colspan="1">miR-145</td><td align="left" rowspan="1" colspan="1">MIMAT0000157</td><td align="left" rowspan="1" colspan="1">MIMAT0000437</td></tr><tr><td align="left" rowspan="1" colspan="1">let-7c</td><td align="left" rowspan="1" colspan="1">MIMAT0000523</td><td align="left" rowspan="1" colspan="1">MIMAT0000064</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt101"><label></label><p>mh-miRNAs- mouse miRNAs having complete homology with human miRNAs.</p></fn></table-wrap-foot></table-wrap><table-wrap id="pone-0007837-t003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.t003</object-id><label>Table 3</label><caption><title>Predicted miRNA–mRNA pairs within the host system.</title></caption><alternatives><graphic id="pone-0007837-t003-3" xlink:href="pone.0007837.t003"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Target Genes</td><td align="left" rowspan="1" colspan="1">mh-miRNAs</td><td align="left" rowspan="1" colspan="1">Functions</td></tr></thead><tbody><tr><td colspan="3" align="left" rowspan="1"><bold>Class A</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">SCA1 (ATXN1)</td><td align="left" rowspan="1" colspan="1">miR-101miR-19bmiR-200amiR-106amiR-93miR-150</td><td align="left" rowspan="1" colspan="1">Stem Cell Antigen-1 is identified as a potential marker for BASCs located at the BADJ <xref rid="pone.0007837-Kim1" ref-type="bibr">[12]</xref>, <xref rid="pone.0007837-McQualter1" ref-type="bibr">[27]</xref>.</td></tr><tr><td align="left" rowspan="1" colspan="1">OCT4 (POU5F1)</td><td align="left" rowspan="1" colspan="1">miR-146b-5p</td><td align="left" rowspan="1" colspan="1">Octamer-binding transcription factor 4+ (Oct-4) is a stem cell marker protein. It acts as a master switch in differentiation by regulating cells that have pluripotent potential. Recently, it has been found to be expressed in pulmonary cells which are a target for SARS infection <xref rid="pone.0007837-Ling1" ref-type="bibr">[8]</xref>, <xref rid="pone.0007837-Chen1" ref-type="bibr">[23]</xref>.</td></tr><tr><td align="left" rowspan="1" colspan="1">CD34</td><td align="left" rowspan="1" colspan="1">let-7glet-7dlet-7c</td><td align="left" rowspan="1" colspan="1">These are membrane-bound stem cell markers for BASCs. This is also expressed in SARS-infected lung cells <xref rid="pone.0007837-Chen1" ref-type="bibr">[23]</xref>, <xref rid="pone.0007837-Martin1" ref-type="bibr">[28]</xref>.</td></tr><tr><td colspan="3" align="left" rowspan="1"><bold>Class B</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">SP-C (SFTPC)</td><td align="left" rowspan="1" colspan="1">miR-331-3p</td><td align="left" rowspan="1" colspan="1">Marker protein of alveolar type-2(AT2-like) cells which is a subset of the differentiated BASC population. Hence is quiescent in normal lung but proliferate in response to bronchiolar and alveolar epithelial injury <xref rid="pone.0007837-Eramo1" ref-type="bibr">[29]</xref>, <xref rid="pone.0007837-Kim2" ref-type="bibr">[30]</xref>.</td></tr><tr><td align="left" rowspan="1" colspan="1">CCSP-2</td><td align="left" rowspan="1" colspan="1">miR-181amiR-497</td><td align="left" rowspan="1" colspan="1">This is the Clara cell marker protein. These are also quiescent in normal lung and proliferate in response to bronchiolar and alveolar epithelial injury <xref rid="pone.0007837-Kim2" ref-type="bibr">[30]</xref>.</td></tr><tr><td align="left" rowspan="1" colspan="1">Aquaporin5</td><td align="left" rowspan="1" colspan="1">miR-331-3pmiR-497</td><td align="left" rowspan="1" colspan="1">Marker protein of AT1-like cells which is another subset of the differentiated BASC population. Aquaporin5 plays a critical role in the maintenance of normal lung water homeostasis <xref rid="pone.0007837-Kim2" ref-type="bibr">[30]</xref>, <xref rid="pone.0007837-Nielsen1" ref-type="bibr">[31]</xref>.</td></tr><tr><td colspan="3" align="left" rowspan="1"><bold>Class C</bold></td></tr><tr><td align="left" rowspan="1" colspan="1">ACE2</td><td align="left" rowspan="1" colspan="1">miR-186miR-93miR-200b</td><td align="left" rowspan="1" colspan="1">Angiotensin-converting enzyme 2 (ACE2), the primary receptor of SARS-CoV on the host cell. It is a negative regulator of the rennin-angiotensin system (RAS) in the setting of acute lung injury and in response to pulmonary infection with the SARS-CoV <xref rid="pone.0007837-Li1" ref-type="bibr">[17]</xref>, <xref rid="pone.0007837-Oudit1" ref-type="bibr">[32]</xref>.</td></tr><tr><td align="left" rowspan="1" colspan="1">CCR1</td><td align="left" rowspan="1" colspan="1">miR-223</td><td align="left" rowspan="1" colspan="1">Inflammatory chemokine receptor for CCL3 and CCL5 & responsible for lung fibrosis <xref rid="pone.0007837-Glass1" ref-type="bibr">[33]</xref>, <xref rid="pone.0007837-Tokuda1" ref-type="bibr">[34]</xref>.</td></tr></tbody></table></alternatives><table-wrap-foot><fn id="nt102"><label></label><p>We have predicted the differentially expressed mh–miRNAs which operate within the narrow windows of development of BASCs by targeting a set of host encoded mRNAs.</p></fn><fn id="nt103"><label></label><p><bold>Class A</bold> specifies the host mRNAs corresponding to BASC marker proteins which designates its undifferentiated state as well as the state of proliferation and/initiation of differentiation.</p></fn><fn id="nt104"><label></label><p><bold>Class B</bold> specifies the host mRNAs corresponding to BASC marker proteins as it reaches a fully differentiated state.</p></fn><fn id="nt105"><label></label><p><bold>Class C</bold> specifies the host mRNAs corresponding to the receptor proteins which participates in SARS-CoV pathogenesis.</p></fn><fn id="nt106"><label></label><p>These miRNA–mRNA pairs specific to BASC are important to understand host-virus interaction network.</p></fn></table-wrap-foot></table-wrap></sec><sec id="s2e"><title>Host miRNA targets in viral genome</title><p>We have searched for the host miRNA targets within the virulent genes of SARS-CoV. <xref ref-type="table" rid="pone-0007837-t004">Table 4</xref> includes the BASC-miRNA candidates targeting the most important virulent proteins responsible for viral infection. Interestingly, miRNAs-17*, -574-5p and -214 targets all the four viral virulent proteins viz. S, N, M, E and orf1a. miR-148a has its target in ORF1a, E, S and M. miR-223 and miR-98 has an exclusive correlation with their targets within the 3′ UTRs of N and S respectively (<xref ref-type="fig" rid="pone-0007837-g002">Figure 2</xref>).</p><table-wrap id="pone-0007837-t004" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.t004</object-id><label>Table 4</label><caption><title>Host miRNAs targeting virulent protein coding mRNAs of SARS-CoV.</title></caption><alternatives><graphic id="pone-0007837-t004-4" xlink:href="pone.0007837.t004"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Target genes</td><td align="left" rowspan="1" colspan="1">mh-miRNAs</td><td align="left" rowspan="1" colspan="1">Functions of target genes</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Spike (S)</td><td align="left" rowspan="1" colspan="1">miR-214miR-574-5pmiR-17*miR-148amiR-98</td><td align="left" rowspan="1" colspan="1">Responsible for viral attachment and entry into host cells by interacting with ACE2 <xref rid="pone.0007837-Rota1" ref-type="bibr">[5]</xref>, <xref rid="pone.0007837-Gallagher1" ref-type="bibr">[35]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">Envelop (E)</td><td align="left" rowspan="1" colspan="1">miR-574-5pmiR-214miR-17*miR-148a</td><td align="left" rowspan="1" colspan="1">E is responsible for virion envelope morphogenesis and acts as a viroporin, inducing the formation of hydrophilic pores in cellular membranes <xref rid="pone.0007837-Wilson1" ref-type="bibr">[36]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">Membrane (M)</td><td align="left" rowspan="1" colspan="1">miR-574-5pmiR-214miR-17*miR-148a</td><td align="left" rowspan="1" colspan="1">Induction of apoptosis <xref rid="pone.0007837-Chana1" ref-type="bibr">[37]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">Nucleocapsid (N)</td><td align="left" rowspan="1" colspan="1">miR-574-5pmiR-214miR-17*miR-223</td><td align="left" rowspan="1" colspan="1">Promotes tissue fibrosis <xref rid="pone.0007837-Zhao1" ref-type="bibr">[19]</xref>N protein inhibit cell cycle progression <xref rid="pone.0007837-Milan1" ref-type="bibr">[38]</xref>, <xref rid="pone.0007837-Yan1" ref-type="bibr">[39]</xref></td></tr><tr><td align="left" rowspan="1" colspan="1">ORF1a</td><td align="left" rowspan="1" colspan="1">miR-574-5pmiR-214miR-17*miR-148a</td><td align="left" rowspan="1" colspan="1">Encodes viral replicase proteins <xref rid="pone.0007837-Snijder1" ref-type="bibr">[40]</xref>, <xref rid="pone.0007837-Prentice1" ref-type="bibr">[41]</xref></td></tr></tbody></table></alternatives></table-wrap><p>Normally presence of such host miRNA targets in SARS-CoV genome indicates that the corresponding miRNAs are a part of the host's innate antiviral defense. On the contrary, the RNA virus co-opt host-miRNAs to suppress their own replication to evade immune elimination and establish a strong infection. It has been found that human infecting single-stranded RNA viruses are enriched for target sequences of human miRNAs <xref rid="pone.0007837-Watanabe1" ref-type="bibr">[42]</xref>. Or else rapidly evolving RNA viruses might evolve targets that match host miRNAs in order to increase their host specificity. It is very likely that the rapidly evolving virus take advantage of slowly evolving host miRNAs to increase their survival in the host, analogous to the manner in which viruses take advantage of other cellular processes. Co-option of host miRNAs by SARS-CoV to suppress their own replication will allow it to be in a latent state and escape the host immune system at the initial time point of transmission and infection. Once successful transmission within the host system takes place, antiviral miRNA target sequences in the virus can be expected to mutate rapidly to maximize mismatches and thus minimize the impact of ‘antiviral’ miRNAs. Rapid mutation in SARS-CoV is supported by the fact that SARS-CoV lack proof reading activity in their polymerases. RNA-dependent polymerase in SARS-CoV misincorporates 8.26 bases per million <xref rid="pone.0007837-Ye1" ref-type="bibr">[43]</xref>.</p><p>While investigating the seed sequence conservation of the viral-specific host miRNAs (<xref ref-type="fig" rid="pone-0007837-g003">Figure 3</xref>) we found that there is a strong bias towards G, Y(pYrimidine) and R (PuRine) bases at the 3<sup>rd</sup>, 4<sup>th</sup> and 5<sup>th</sup> position respectively of the seed region. This implies that a single mutation within the complementary regions of this conserved 3-mer motif within the viral targets will disrupt the miRNA seed-target match and will enable the virus to escape the antiviral effect of host-miRNAs after successful transmission within the host. Such host-miRNA sequence conservation helps the virus to mutate the target regions with more precision and at ease so as to escape the effect of all the 4 miRNAs together.</p><fig id="pone-0007837-g003" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.g003</object-id><label>Figure 3</label><caption><title>Sequence alignment between the common viral specific host miRNA mature sequences from 5′ to 3′ end.</title><p>The seed region (position 2 to 7) and the conserved positions (3<sup>rd</sup>, 4<sup>th</sup>, and 5<sup>th</sup>) within the seed region are highlighted within boxes.</p></caption><graphic xlink:href="pone.0007837.g003"></graphic></fig><p>The miRNA expression profiles of three miRNAs-17*, -574-5p and -214 targeting S, N, Orf1a, M and E shows that they are upregulated within uninfected BASCs. Presumably at the onset of SARS infection the expression of these targets is suppressed which is likely to control viral replication and consequently help the virus to evade the host immune system. But the targets for downregulated miR-148a within ORF1a, M, S and E might escape repression and maintain replication at a lower rate. Further, the exclusive miRNAs viz. miR-223 and -98 for N and S protein respectively are highly downregulated (8.7 fold and 5.6 fold respectively) in BASCs. N and S proteins are important for viral transmission in host. This provides a clue towards the fact that the S protein and N protein (having targets corresponding to these miRNAs) of SARS-CoV takes advantage of this and escapes miRNA-mediated repression which is a rescue to the virus for effective transmission at the initial stage of viral infection.</p></sec><sec id="s2f"><title>Key miRNA–mRNA pairs correlating with virus entry, replication, and host–virus interaction</title><sec id="s2f1"><title>ACE2-S interaction</title><p>It has been shown that proinflammatory and fibrogenic cytokine pathways are activated within the first 24–48 hr following pulmonary insult resulting from SARS-CoV infection <xref rid="pone.0007837-Nicholls1" ref-type="bibr">[44]</xref>. High initial levels of these activities are associated with persistent pulmonary damage and increased risks of subsequent pulmonary fibrosis and poor outcome in diffuse alveolar damage. Further, it has been reported that ACE2 mRNA, protein and enzymatic activity is severely decreased in pulmonary fibrosis resulting from SARS-CoV infection <xref rid="pone.0007837-Li1" ref-type="bibr">[17]</xref>, <xref rid="pone.0007837-Li2" ref-type="bibr">[18]</xref>. Studies have also been performed which support that differential regulation of host cell mRNA transcription and protein synthesis occurs in a SARS-CoV infected cells <xref rid="pone.0007837-Xiao1" ref-type="bibr">[45]</xref>.</p><p>SARS-CoV S protein is a multifunctional protein which plays pivotal roles in the biology and pathogenesis of SARS-CoV. It has been shown that S protein mediates viral infection by binding to cellular receptor ACE2 and thus induces membrane fusion. SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression <xref rid="pone.0007837-Kuba1" ref-type="bibr">[46]</xref>. Further a recent work of Imai <italic>et al</italic>. showed that on injecting SARS-CoV Spike into mice worsens acute lung failure in vivo, which can be attenuated by blocking the renin–angiotensin pathway. This suggests that the S-protein mediated activation of the pulmonary RAS influences the pathogenesis of ALI/ARDS and SARS <xref rid="pone.0007837-Imai1" ref-type="bibr">[47]</xref>. But the molecular mechanism by which S protein down regulates ACE2 in lung cells following SARS injury is not well addressed till to date.</p><p>In our work we observed that the miRNAs- miR-186,-93 and -200b targeting ACE2 are found to be highly down regulated in normal BASCs thereby showing that they do not interfere with the expression level of ACE2 and thus prevents lung fibrogenesis by limiting the local accumulation of the profibrotic peptide ANG II in an uninfected state. Considering the expression of S-protein, we find that its target corresponding to highly downregulated miR-98 probably escapes miRNA-mediated host RNAi defense at the onset of infection and interferes with ACE2 expression. <xref ref-type="fig" rid="pone-0007837-g004">Figure 4</xref> shows how the virus takes advantage of the microRNA-mediated complex interplay between the host and viral factors within BASCs. The question still remains regarding the molecular mechanism by which this S-protein represses the expression of ACE2.</p><fig id="pone-0007837-g004" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.g004</object-id><label>Figure 4</label><caption><title>Modelled miRNA–mediated host–virus interaction within BASC in SARS-CoV pathogenesis.</title><p>Host miRNAs are central to viral pathogenesis. This figure illustrates how the host miRNA and SARS-CoV interactions can explain the features of SARS-CoV pathogenesis such as (A) entry of the virus in an undifferentiated BASC, (B) causing acute infection during the proliferative stage and onset of BASC differentiation, and (C) complete clearance of virus in a fully differentiated BASC.</p></caption><graphic xlink:href="pone.0007837.g004"></graphic></fig><p>S-proteins have been shown to act as transcription factor activating the promoters of GRP94/78 genes inducing ER stress in a SARS-CoV infected cell <xref rid="pone.0007837-Jin1" ref-type="bibr">[48]</xref>. The role of S protein as a transcription factor has obliged us to think that they might play a role to activate the miRNA promoters of miR-186,-93 and -200b targeting ACE2 to enhance their expression resulting in a reduced expression of ACE2 in an infected host. Whether this decrease in ACE2 expression is a result of miRNA mediated gene silencing controlled by S-protein will be an interesting and compelling question for further study.</p></sec><sec id="s2f2"><title>Oct-4-N interaction</title><p>Oct-4 plays a crucial role towards regulating the chromatin structure in a state consistent with self-renewal and later facilitating the expression of genes that keeps the cell poised to respond to cues that lead to differentiation. Hence this is one of the essential factors which control the state of differentiation in BASCs. Furthermore, it has a significant role in apoptosis. miR-146b-5p is seen to target Oct-4 in normal BASCs. The downregulated expression of this miRNA explains for the uninterrupted expression of Oct-4 resulting its normal function in an uninfected cell. We have harnessed the high possibility of a complicated crosstalk between the ability of N-protein to control the expression of Oct-4 in an infected cell.</p><p>N protein is known to be the most abundantly expressed protein of SARS-CoV. The most unique and significant property of it is revealed in its ability to act as a sequence specific DNA binding factor. It has been reported to bind NFkB response element of COX2 promoter and enhance COX2 gene expression <xref rid="pone.0007837-Yan2" ref-type="bibr">[49]</xref> which effectively is beneficial for its existence within the host. This reveals the power of the N-protein to manipulate the entire gene expression programme of the infected cell. The common miRNAs, miR-214,-574-5p and -17* having targets in N protein are upregulated in BASCs. But miR-223, the unique miRNA targeting N-protein is highly downregulated. This signifies that the N-protein targets corresponding to miR-223 is going to escape miRNA-mediated downregulation at the onset of infection (See <xref ref-type="fig" rid="pone-0007837-g004">Figure 4</xref>). Further it seems that N-protein plays a role in interfering with the expression modulation of Oct-4 by binding to the promoter of miR-146-5p and controls the different stages of BASC differentiation as well as prevents apoptosis of SARS infected cells.</p></sec></sec><sec id="s2g"><title>Immunity-related issues—induction of inflammatory chemokines in SARS-CoV–infected cells might be an advantage for the virus to replicate</title><p>All respiratory virus disease results from two concurrent pathological components: ongoing virus replication and the resulting inflammatory response. Even if antivirals clearly inhibit virus replication, the biochemical and cellular inflammatory responses to the initial infection related events continue despite diminished virus titer <xref rid="pone.0007837-Bonville1" ref-type="bibr">[50]</xref>. While acute inflammatory responses are generally beneficial in nature and have been shown to limit virus replication in situ, prolonged, uncontrolled inflammation has been recognized as a significant component contributing to the pathological sequelae to the morbidity and mortality of severe acute respiratory syndrome coronavirus infection <xref rid="pone.0007837-Nicholls1" ref-type="bibr">[44]</xref>. A histopathologic analysis of SARS-CoV infected lung in mice showed that there has been an induction of significant changes in the expression levels of the chemokine receptor CCR1 which acts as receptors for CCL3 and CCL5. The expression levels of these inflammatory chemokines were increased by viral infection of the lung <xref rid="pone.0007837-Glass1" ref-type="bibr">[33]</xref>. It has been shown that CCR1 play an essential role in the pathogenesis of pulmonary fibrosis in lung-injury models <xref rid="pone.0007837-Tokuda1" ref-type="bibr">[34]</xref>. Further, previous work suggests that antagonism of signaling via the chemokine receptor CCR1 is a potent strategy that attenuates leukocyte recruitment in response to severe respiratory virus infection <xref rid="pone.0007837-Bonville1" ref-type="bibr">[50]</xref>.</p><p>Although there has been a subsequent increase in inflammatory cytokines related to innate immune response in response to viral infection, there has been a lack of immunoregulatory cytokines (T or B lymphocytes) in the lung during infection which confirms the fact that the expression of viral replication proteins remains at a quiescent state so as to avoid viral clearance by the activation of immunoregulatory cytokines by adaptive immune responses at the onset of the infection. On the contrary, activation of inflammatory chemokines might be acting as a beneficial phenomenon for the replication of various viruses <xref rid="pone.0007837-Lee1" ref-type="bibr">[51]</xref>, <xref rid="pone.0007837-Chang1" ref-type="bibr">[52]</xref>.</p><p>Emerging studies suggest that miRNAs provide an added layer in orchestrating immune responses <xref rid="pone.0007837-Asirvatham1" ref-type="bibr">[53]</xref>. miRNAs function in shaping immunity by regulating the repertoire of genes expressed in immune cells and the magnitude and duration of responses to particular pathogens <xref rid="pone.0007837-Baltimore1" ref-type="bibr">[54]</xref>. Hence we investigated the underlying miRNA induced molecular mechanisms responsible for these changes in the lung and we found that miR-223 has its target in the 3′-UTR of CCR1. Previously, miR-223 was experimentally identified in chorioamnionitis-related inflammation and its predicted target genes include several genes involved in inflammation and immune responses. Further, miR-223 is known to promote granulocyte differentiation and is a part of a regulatory loop that involves C/EBP and NFI-A <xref rid="pone.0007837-Fazi1" ref-type="bibr">[55]</xref>.</p><p>Interestingly miR-223 has its target in N protein of SARS-CoV too and it is one of the miRNAs which probably helps the virus to escape the RNAi mediated silencing at the onset of infection. Or in another way we may say that the virus encodes the target of miR-223 to escape the RNAi mediated repression and at the same time takes advantage of the effect of induction of the inflammatory chemokine responses to accelerate lung fibrosis. But the controlled expression of target CCR1 in an uninfected cell inspite of the significantly low expression of its corresponding miR-223 in BASCs suggest that there must be a cascaded fine tuned interaction masterminded by the virus which is going on within the infected cell. It is known that N protein activates NF-KB in Vero E6 cells <xref rid="pone.0007837-Milan1" ref-type="bibr">[38]</xref>. CCR1 is one of the target genes of NF-KB which is activated at a later stage <xref rid="pone.0007837-Law1" ref-type="bibr">[56]</xref>. Hence, we propose that within BASCs, reduced expression of miR-223 helps N protein to enter the host cell. On successful entry, N protein might be using miR-223 once again to activate CCR1 via NF-KB at its replicative stage to enhance lung fibrosis and at the same time gets empowered to manipulate the entire gene expression of the host cell by controlling the expression of Oct-4. The exact mechanism of such miRNA-mediated events needs further investigation. Further, checking the expression levels of N-protein, CCR1 as well as miR-223 in an infected BASC will provide a more compact conclusion.</p></sec><sec id="s2h"><title>Conclusion</title><p>Based on these findings, we propose that BASCs (Sca-1<sup>+</sup> CD34<sup>+</sup> CD45<sup>−</sup> Pecam<sup>-</sup>) are a subset of Oct-4<sup>+</sup> ACE2<sup>+</sup> cells and are the chief targets of SARS-CoV infection. SARS-CoV enters the host; infects the BASCs of the lungs and controls its developmental stages via the molecular switches-miRNAs. Co-option of host miRNAs by viruses reveal their intelligent plan to control their replication in order to evade immune elimination until they undergoes successful transmission and establish a strong infection. Thereafter, they undergo rapid mutation to maximize the target-miRNA mismatches and enhance their replication before the cell reaches at a fully differentiated state. On successful replication, the virus infects resident, infiltrating, and circulating immune cells. The circulating immune cells carry the virus to other organs and causes damages to the immune cells of spleen, peripheral and central lymph nodes and other lymphoid tissues. The immune defense being weakened significantly, leads to rapid deterioration of the pneumonia. It is worth to test these cells for the expression of Oct-4 and ACE2 for further confirmatory conclusion.</p><p>The proposed host miRNA-dependent mechanism probably acts in concert with the host and viral factors for establishing a strong infection. Our proposal shifts away from the simplistic notion that host miRNAs with specific sequence complementarity to viruses are indicative of a bonafide innate antiviral immune mechanism to an established viral pathogen.</p><p>Although the immediate threat of SARS is over, we intend to continue with its therapeutic measures partly to guard for its possible return. Moreover, this will help us to transform our knowledge from SARS to other emerging viral diseases specially causing lung injuries which most certainly will be a deadly threat in future. Lot of attempts have been made and is still going on to design effective antiviral agents against this deadly disease <xref rid="pone.0007837-Cinati1" ref-type="bibr">[57]</xref>–<xref rid="pone.0007837-Li3" ref-type="bibr">[60]</xref>. While some of these approaches showed partial efficiency to combat SARS infection, the others need further clinical investigations to be used as a proper therapeutic reagent.</p><p>This era has witnessed an increase in the development of exogenous siRNA therapeutics against viruses. The potential therapeutic effect of modulating host-miRNA levels is also worth considering. This emerging picture of miRNA regulation in SARS-CoV infected individual will make the host-virus interaction far richer and more complex than the crisp linear pathways of the previous decade, with miRNAs participating in executive decisions at the interesting and vulnerable nodes in regulatory networks. By boosting the level of the host virus-specific miRNAs, it might be possible to turn what was a desirable outcome for the virus into a desirable outcome for the patient. Under the current hypothesis, we would predict that decreasing the level of host virus-specific miRNAs would result in promotion of viral replication, leading to immunological recognition and clearance of the virus by the host immune system. Further, increasing the level of miRNAs exclusive for the N and S protein might also block viral entry. However, manipulating the level of host miRNAs could have unintended consequences because the physiological functions of the miRNAs might be altered or viral pathology might be enhanced. Further silencing of miRNAs with antagomirs specifically within the infected cell could become another therapeutic strategy where miRNAs participate in disease aetiology. Nevertheless, these potential interventions merit further evaluation.</p></sec></sec><sec id="s3"><title>Materials and Methods</title><p>To identify the miRNAs involved in the cellular tropism of SARS-CoV in lungs, we undertook an intensive search for potential miRNA candidates in the BASCGAP (Broncho-alveolar stem cell genome anatomy project) sequence data. Our miRNA sequences were derived from the Sanger Institute miRBase release 12.0 (<ext-link ext-link-type="uri" xlink:href="http://microrna.sanger.ac.uk/sequences">http://microrna.sanger.ac.uk/sequences</ext-link>). The global miRNA expression profile of our BASCGAP source sequences were derived from mouse BASCs <xref rid="pone.0007837-Qian1" ref-type="bibr">[61]</xref>.</p><sec id="s3a"><title>miRNA—mouse-human (mh–miRNA)</title><p>Our search strategy was motivated by the need to apply pure-homology based selection of miRNAs so that the results represent a closer approach to mimic the molecular mechanisms that underlie disease pathway behaviors and response in a human host. Further the overall miRNA expression profile of mouse and human lungs are similar <xref rid="pone.0007837-Williams1" ref-type="bibr">[62]</xref>. From the BASCGAP sequence data of 116 miRNAs, we screened out the BASC-miRNAs lying at the intersection of mouse-human whole genomes. This filter is based on a homology search (SSEARCH), performed with miRBase (release 12.0) and is applied on the mature candidates from the BASCGAP sequence data. E-value cutoff considered for SSEARCH was <10.0. Mature miRNA sequence homology was one of the stringent parameters considered in our search. <xref ref-type="table" rid="pone-0007837-t002">Table 2</xref> represents the screened output set of 95 mature candidates termed as mh-miRNAs.</p></sec><sec id="s3b"><title>mRNA targets</title><p>We have considered the developmental stage specific marker proteins of BASCs, which includes the transcription factor Pou5f1; ACE2-the most important receptor of SARS-CoV (<xref ref-type="table" rid="pone-0007837-t003">Table 3</xref>). In addition to these, we have chosen the virulent protein coding genes, viz. Nucleocapsid (N), Spike(S), Envelope (E), Membrane (M) and Orf1a from 30 strains of SARS-CoV (<xref ref-type="table" rid="pone-0007837-t001">Table 1</xref> and <xref ref-type="table" rid="pone-0007837-t004">Table 4</xref>) for our investigation. Annotated UTR sequences of the target genes were retrieved from UTRdb <xref rid="pone.0007837-Mignone1" ref-type="bibr">[63]</xref>.</p></sec><sec id="s3c"><title>Target prediction</title><p>We adopted RNAhybrid <xref rid="pone.0007837-Rehmsmeier1" ref-type="bibr">[64]</xref>, that predicts the most favorable hybridization sites between miRNA and UTR regions and generates energy minimized duplex structures using the Dynamic Programming technique. The resultant miRNA-mRNA duplexes are consistent with the following structural and energy constraints at the first step of screening the targets: (i) Binding energy cutoff < = −20 kcal/mol <xref rid="pone.0007837-Rehmsmeier1" ref-type="bibr">[64]</xref>; (ii) complementarity of minimum seven bases to the 5′ end of the miRNA; (iii) minimization of GU base pair within the miRNA-target duplex, a maximum of only one GU base pair between 2 and 7 position of the miRNA is allowed; (iv) minimization of loop size throughout the duplex.</p><p>Although, currently available target prediction methods are diverse, both in approach and performance and all have room for further improvement, perfect seed pairing centered on nucleotides 2–7 is considered as the most important criteria for prediction reliability. Furthermore, in order to screen out the most favorable target in the case of miRNAs having multiple targets we incorporated the constraint of site efficacy (hierarchy of site efficacy follows- 8 mer>7 mer-m8>7 mer-A1>6 mer) following the work of Grimson <italic>et al.</italic> and Friedman <italic>et al</italic>. <xref rid="pone.0007837-Grimson1" ref-type="bibr">[65]</xref>, <xref rid="pone.0007837-Friedman1" ref-type="bibr">[66]</xref>. 3′ compensatory pairing of miRNA to its target have been considered to compensate for imperfect/weak seed matches <xref rid="pone.0007837-Bartel2" ref-type="bibr">[67]</xref>. We have further checked the effect of cooperativity as well as the UTR binding location.</p></sec><sec id="s3d"><title>miRNA–mRNA enrichment analysis</title><p>For <italic>bantam</italic> miRNAs, good minimum free energies (MFEs) can occur frequently by chance. The longer a putative target sequence, the better such random energies will be. Hence, statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies and a Poisson approximation of multiple binding sites.</p></sec><sec id="s3e"><title>Microarray data analysis in BASCs</title><p>miRNA expression profile of 2×10<sup>6</sup> mouse BASCs (CD45<sup>−</sup>CD31<sup>−</sup>CD34<sup>+</sup> Sca-1<sup>+</sup>) and 4×10<sup>6</sup> control cells(CD45<sup>−</sup>CD31<sup>−</sup>CD34<sup>−</sup>Sca-1<sup>−</sup>) are determined using miRNA microarray. The data considered for our analysis is obtained by first subtracting the background and then normalizing the signals using a LOWESS filter (locally weighted regression). Background is determined using a regression-based background mapping method <xref rid="pone.0007837-Bolstad1" ref-type="bibr">[68]</xref>. The miRNA dataset was filtered according to the standard procedure to exclude spots with minimum intensity and size. Further for the dual-sample experiments, the ratio of the two sets of detected signals (log2 transformed, balanced) are calculated. Thereafter, t-values are calculated for each miRNA between the test and the control groups, and P-values are computed from the theoretical t-distribution. miRNAs with P-values below a critical P-value (typically 0.01) are the differentially detected signals and were selected as our global miRNA expression profile data set (of mouse BASCs) in BASCGAP. The flowchart of methodology is provided in <xref ref-type="fig" rid="pone-0007837-g005">Figure 5</xref>.</p><fig id="pone-0007837-g005" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0007837.g005</object-id><label>Figure 5</label><caption><title>miRNA–mRNA prediction pipelines.</title><p>This flow chart summarizes the steps followed and yields from BASCGAP consortium sequences to predict the miRNA-mRNA pairs involved in SARS pathogenesis.</p></caption><graphic xlink:href="pone.0007837.g005"></graphic></fig></sec></sec></body><back><ack><p>We thank the reviewers for their useful comments and suggestions to improve the manuscript. We also thank our colleagues for critical reading of the manuscript.</p></ack><fn-group><fn fn-type="COI-statement"><p><bold>Competing Interests: </bold>The authors have declared that no competing interests exist.</p></fn><fn fn-type="financial-disclosure"><p><bold>Funding: </bold>This work is funded by Department of Science and Technology, Government of India. 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