Proteolytic Processing and Deubiquitinating Activity of Papain-Like Proteases of Human Coronavirus NL63▿
Identifieur interne : 000715 ( Pmc/Curation ); précédent : 000714; suivant : 000716Proteolytic Processing and Deubiquitinating Activity of Papain-Like Proteases of Human Coronavirus NL63▿
Auteurs : Zhongbin Chen ; Yanhua Wang ; Kiira Ratia ; Andrew D. Mesecar ; Keith D. Wilkinson ; Susan C. BakerSource :
- Journal of Virology [ 0022-538X ] ; 2007.
Abstract
Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease
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DOI: 10.1128/JVI.02747-06
PubMed: 17392370
PubMed Central: 1900296
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<author><name sortKey="Chen, Zhongbin" sort="Chen, Zhongbin" uniqKey="Chen Z" first="Zhongbin" last="Chen">Zhongbin Chen</name>
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<author><name sortKey="Wang, Yanhua" sort="Wang, Yanhua" uniqKey="Wang Y" first="Yanhua" last="Wang">Yanhua Wang</name>
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<author><name sortKey="Ratia, Kiira" sort="Ratia, Kiira" uniqKey="Ratia K" first="Kiira" last="Ratia">Kiira Ratia</name>
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<author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name>
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<author><name sortKey="Wilkinson, Keith D" sort="Wilkinson, Keith D" uniqKey="Wilkinson K" first="Keith D." last="Wilkinson">Keith D. Wilkinson</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
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<author><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C." last="Baker">Susan C. Baker</name>
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<sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Proteolytic Processing and Deubiquitinating Activity of Papain-Like Proteases of Human Coronavirus NL63<xref ref-type="fn" rid="fn1">▿</xref>
</title>
<author><name sortKey="Chen, Zhongbin" sort="Chen, Zhongbin" uniqKey="Chen Z" first="Zhongbin" last="Chen">Zhongbin Chen</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wang, Yanhua" sort="Wang, Yanhua" uniqKey="Wang Y" first="Yanhua" last="Wang">Yanhua Wang</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Ratia, Kiira" sort="Ratia, Kiira" uniqKey="Ratia K" first="Kiira" last="Ratia">Kiira Ratia</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name>
<affiliation><nlm:aff id="aff1"></nlm:aff>
</affiliation>
</author>
<author><name sortKey="Wilkinson, Keith D" sort="Wilkinson, Keith D" uniqKey="Wilkinson K" first="Keith D." last="Wilkinson">Keith D. Wilkinson</name>
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<author><name sortKey="Baker, Susan C" sort="Baker, Susan C" uniqKey="Baker S" first="Susan C." last="Baker">Susan C. Baker</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="2007">2007</date>
</imprint>
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<front><div type="abstract" xml:lang="en"><p>Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease <italic>cis</italic>
-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.</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</publisher-name>
</publisher>
</journal-meta>
<article-meta><article-id pub-id-type="pmid">17392370</article-id>
<article-id pub-id-type="pmc">1900296</article-id>
<article-id pub-id-type="publisher-id">2747-06</article-id>
<article-id pub-id-type="doi">10.1128/JVI.02747-06</article-id>
<article-categories><subj-group subj-group-type="heading"><subject>Genome Replication and Regulation of Viral Gene Expression</subject>
</subj-group>
</article-categories>
<title-group><article-title>Proteolytic Processing and Deubiquitinating Activity of Papain-Like Proteases of Human Coronavirus NL63<xref ref-type="fn" rid="fn1">▿</xref>
</article-title>
</title-group>
<contrib-group><contrib contrib-type="author"><name><surname>Chen</surname>
<given-names>Zhongbin</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>Wang</surname>
<given-names>Yanhua</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Ratia</surname>
<given-names>Kiira</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Mesecar</surname>
<given-names>Andrew D.</given-names>
</name>
<xref ref-type="aff" rid="aff1">3</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Wilkinson</surname>
<given-names>Keith D.</given-names>
</name>
<xref ref-type="aff" rid="aff1">4</xref>
</contrib>
<contrib contrib-type="author"><name><surname>Baker</surname>
<given-names>Susan C.</given-names>
</name>
<xref ref-type="aff" rid="aff1">1</xref>
<xref ref-type="corresp" rid="cor1">*</xref>
</contrib>
</contrib-group>
<aff id="aff1">Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, Maywood, Illinois,<label>1</label>
Department of Biochemistry and Molecular Biology, Beijing Institute of Radiation Medicine, Beijing, China,<label>2</label>
Center for Pharmaceutical Biotechnology and Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, Illinois,<label>3</label>
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia<label>4</label>
</aff>
<author-notes><fn id="cor1"><label>*</label>
<p>Corresponding author. Mailing address: Department of Microbiology and Immunology, Loyola University Medical Center, 2160 South First Avenue, Bldg. 105, Rm. 3929, Maywood, IL 60153. Phone: (708) 216-6910. Fax: (708) 216-9574. E-mail: <email>sbaker1@lumc.edu</email>
</p>
</fn>
</author-notes>
<pub-date pub-type="ppub"><month>6</month>
<year>2007</year>
</pub-date>
<pub-date pub-type="epub"><day>28</day>
<month>3</month>
<year>2007</year>
</pub-date>
<volume>81</volume>
<issue>11</issue>
<fpage>6007</fpage>
<lpage>6018</lpage>
<history><date date-type="received"><day>13</day>
<month>12</month>
<year>2006</year>
</date>
<date date-type="accepted"><day>15</day>
<month>3</month>
<year>2007</year>
</date>
</history>
<copyright-statement>Copyright © 2007, American Society for Microbiology</copyright-statement>
<copyright-year>2007</copyright-year>
<self-uri xlink:title="pdf" xlink:href="zjv01107006007.pdf"></self-uri>
<abstract><p>Human coronavirus NL63 (HCoV-NL63), a common human respiratory pathogen, is associated with both upper and lower respiratory tract disease in children and adults. Currently, no antiviral drugs are available to treat CoV infections; thus, potential drug targets need to be identified and characterized. Here, we identify HCoV-NL63 replicase gene products and characterize two viral papain-like proteases (PLPs), PLP1 and PLP2, which process the viral replicase polyprotein. We generated polyclonal antisera directed against two of the predicted replicase nonstructural proteins (nsp3 and nsp4) and detected replicase proteins from HCoV-NL63-infected LLC-MK2 cells by immunofluorescence, immunoprecipitation, and Western blot assays. We found that HCoV-NL63 replicase products can be detected at 24 h postinfection and that these proteins accumulate in perinuclear sites, consistent with membrane-associated replication complexes. To determine which viral proteases are responsible for processing these products, we generated constructs representing the amino-terminal end of the HCoV-NL63 replicase gene and established protease <italic>cis</italic>
-cleavage assays. We found that PLP1 processes cleavage site 1 to release nsp1, whereas PLP2 is responsible for processing both cleavage sites 2 and 3 to release nsp2 and nsp3. We expressed and purified PLP2 and used a peptide-based assay to identify the cleavage sites recognized by this enzyme. Furthermore, by using K48-linked hexa-ubiquitin substrate and ubiquitin-vinylsulfone inhibitor specific for deubiquitinating enzymes (DUBs), we confirmed that, like severe acute respiratory syndrome (SARS) CoV PLpro, HCoV-NL63 PLP2 has DUB activity. The identification of the replicase products and characterization of HCoV-NL63 PLP DUB activity will facilitate comparative studies of CoV proteases and aid in the development of novel antiviral reagents directed against human pathogens such as HCoV-NL63 and SARS-CoV.</p>
</abstract>
</article-meta>
</front>
<floats-wrap><fig position="float" id="f1"><label>FIG. 1.</label>
<caption><p>Detection of replicase products from HCoV-NL63-infected cells. (A) Schematic diagram illustrating HCoV-NL63 ORFs, the predicted processing of replicase polyprotein to nsp's, and regions used to generate anti-R3 (R3) and anti-R4 (R4) sera. The PLP domain (PLP1 and PLP2) and picornavirus 3C-like protease domain (3CLpro) are indicated. (B) Indirect immunofluorescence assay for HCoV-NL63 nsp3 and nsp4. LLC-MK2 cells were infected with HCoV-NL63, fixed, permeabilized at 24 hpi, and stained with anti-R3 or anti-R4 sera, anti-rabbit Ig Alexa-Fluor-488 secondary antibody, and Sytox orange to stain nuclei as described in Materials and Methods. (C) Detection of nsp3 by immunoprecipitation. HCoV-NL63 (NL)- or mock (M)-infected cells were radiolabeled with [<sup>35</sup>
S]methionine for 24 h from 72 to 96 hpi. Cells were harvested, and the cell lysates were subjected to immunoprecipitation with anti-R3 sera or preimmune sera. Products were analyzed by 10% SDS-PAGE and subjected to autoradiography. (D) Detection of nsp3 and nsp4 by a Western blot assay with anti-R3 and anti-R4 antibodies. LLC-MK2 cells infected with HCoV-NL63 were lysed at 96 hpi, and whole-cell lysates were separated by SDS-PAGE. Immunoblotting was performed using anti-R3 and anti-R4 antibodies. Molecular mass markers (in kDa) are shown on the left of each gel.</p>
</caption>
<graphic xlink:href="zjv0110791680001"></graphic>
</fig>
<fig position="float" id="f2"><label>FIG. 2.</label>
<caption><p>Identification of proteases that process the amino-terminal region of the HCoV-NL63 replicase polyprotein. (A) Schematic representation of predicted processing at CS1 and CS2 by PLP1 and PLP2 (top panel) and constructs tested in the <italic>cis</italic>
-cleavage assay. nsp3 conserved domains: Ac, acidic domain; ADRP, ADP-ribose-1′′-phosphatase (<xref ref-type="bibr" rid="r49">49</xref>
); TM, transmembrane domain; Y, Y domain (<xref ref-type="bibr" rid="r70">70</xref>
). The proposed catalytic residues (C1602 for PLP1 and C1678 for PLP2) are indicated. (B) <italic>cis</italic>
-Cleavage assay for CS1 and CS2. Constructs were transfected into HeLa-MHVR cells infected with vTF7.3. Newly synthesized proteins were labeled with [<sup>35</sup>
S]methionine from 4.5 to 9.5 hpi. Lysates were prepared and subjected to immunoprecipitation (IP) with anti-V5 (top) or anti-Flag M2 (bottom) antibody. Immunoprecipitated proteins were separated by 10% SDS-PAGE or 8 to 16% Criterion gel and subjected to autoradiography. The precursors and cleaved products are indicated. Molecular mass markers (in kDa) are shown on the left of the gel.</p>
</caption>
<graphic xlink:href="zjv0110791680002"></graphic>
</fig>
<fig position="float" id="f3"><label>FIG. 3.</label>
<caption><p>Processing of HCoV-NL63 replicase CS3 by PLP2. (A) Schematic diagram of the predicted processing at CS3 by PLP2 and the constructs used in the <italic>cis</italic>
-cleavage assay. The PLP2 catalytic residues (C1678 and H1836) are indicated. (B) Detection of processing at CS3 by PLP2 and identification of PLP2 catalytic sites. <italic>cis</italic>
-Cleavage assays were performed as described for Fig. <xref ref-type="fig" rid="f2">2</xref>
. The processed product (nsp4) and the precursors are indicated. The precursors and processed products were detected with anti-V5 antibody from cells transfected with wild-type PLP2 (WT) (lane 1), the C1678A mutant (lane 2), the C1684A mutant (lane 3), or the H1836A mutant (lane 4). (C) Identification of HCoV-NL63 PLP2 core domain. The processing activities, as detected by immunoprecipitation (IP) of nsp4, from pNL-4 (lane 1), pNL-5 (lane 2), and pNL-6 (lane 3), are shown. Molecular mass markers (in kDa) are shown on the left of each gel.</p>
</caption>
<graphic xlink:href="zjv0110791680003"></graphic>
</fig>
<fig position="float" id="f4"><label>FIG. 4.</label>
<caption><p>Identification of CS2 and CS3 cleavage sites. (A) Purified wild-type HCoV-NL63 six-His-PLP2 (lane 2) and mutant PLP2-C1678A (lane 3) are shown resolved on a Coomassie-stained SDS-PAGE gel. Twelve micrograms of purified protein was loaded in each lane. The molecular masses of the marker proteins (lane 1) are shown to the left of the gel in kDa. The expected molecular mass of six-His-PLP2 is 38.6 kDa. HPLC separation of cleavage products from 12-mer peptides representing CS2 (FTKLAGGKISFS) (B) and CS3 (VAKQGAGFKRTY) (C) incubated in the presence (dashed lines) and absence (solid lines) of purified HCoV-NL63 PLP2. The identity of each cleavage product was confirmed by MS (data not shown), as indicated above the peaks.</p>
</caption>
<graphic xlink:href="zjv0110791680004"></graphic>
</fig>
<fig position="float" id="f5"><label>FIG. 5.</label>
<caption><p>Effect of mutagenesis of critical determinants in CS2 and CS3 on PLP2 recognition and processing. (A) Cleavage site sequences of CS2 and CS3. The arrow indicates the cleavage site identified in the peptide-based cleavage assay. The P1 and P2 residues are highlighted. (B) P1 and P2 residues are critical for processing at CS2. pNL-3 constructs encoding the wild-type (WT) sequence or amino acid substitutions were tested for processing activity as described for Fig. <xref ref-type="fig" rid="f2">2</xref>
. The site of substitution is indicated above each lane. (C) Critical determinants for processing at CS3. pNL-6 constructs (lanes 1 to 4) and pNL-4 constructs (lanes 5 and 6) were analyzed for processing at CS3. Specific substitutions are indicated above each lane. IP, immunoprecipitation.</p>
</caption>
<graphic xlink:href="zjv0110791680005"></graphic>
</fig>
<fig position="float" id="f6"><label>FIG. 6.</label>
<caption><p>HCoV-NL63 PLP2 has DUB activity. (A) Processing of K48-Ub<sub>6</sub>
by PLP2. Lane 1, K48-Ub<sub>6</sub>
incubated in the absence of PLP2. Lane 2, a molecular-mass (MW) ladder with 250-, 150-, 100-, 75-, 50-, 37-, 25-, 20-, 15-, and 10-kDa markers. Lanes 3 to 5, K48-Ub<sub>6</sub>
incubated in the presence of wild-type (WT) PLP2 for the indicated time points. Lane 6, K48-Ub<sub>6</sub>
incubated in the presence of PLP2-C1678A for 60 min. The positions of bovine serum albumin and the various Ub multimers are indicated to the right of the gel. (B) Schematic diagram of the adduct formed between DUBs and HA-Ub-VS. (C) Western blot detection of HA-Ub-PLP2. Purified protein and substrate were incubated for 10 min at 37°C, and the products were separated by SDS-PAGE and analyzed by Western blot analysis using anti-HA antibody.</p>
</caption>
<graphic xlink:href="zjv0110791680006"></graphic>
</fig>
<fig position="float" id="f7"><label>FIG. 7.</label>
<caption><p>Model for processing the HCoV-NL63 replicase polyprotein by PLP1 and PLP2. PLP1 processing at CS1 and PLP2 processing at CS2 and CS3 are indicated by arrows. Critical determinants at the P1 and P2 positions of the cleavage sites are indicated in bold.</p>
</caption>
<graphic xlink:href="zjv0110791680007"></graphic>
</fig>
<table-wrap position="float" id="t1"><label>TABLE 1.</label>
<caption><p>Primers used for amplification or mutagenesis of HCoV-NL63 sequences</p>
</caption>
<table frame="hsides" rules="groups"><thead><tr><th colspan="1" rowspan="1" align="center" valign="bottom">Purpose</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Primer</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Oligonucleotide sequence<xref ref-type="table-fn" rid="t1fn1"><italic>a</italic>
</xref>
</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Nucleotide no.<xref ref-type="table-fn" rid="t1fn2"><italic>b</italic>
</xref>
</th>
<th colspan="1" rowspan="1" align="center" valign="bottom">Polarity or mutation</th>
</tr>
</thead>
<tbody><tr><td colspan="1" rowspan="1" align="left" valign="top">Generation of GST-R3 fusion protein</td>
<td colspan="1" rowspan="1" align="left" valign="top">R3-1</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CCG</underline>
</underline>
<underline><underline>GAA TTC</underline>
</underline>
GGT AAA ATA TCT TTT TCT GAT GAT G 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">2981-3005</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">R3-2</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CCG</underline>
</underline>
<underline><underline>CTC GAG</underline>
</underline>
ACC ATT ACT ATC ATT ACT AAT AGG 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">3257-3280</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Generation of GST-R4 fusion protein</td>
<td colspan="1" rowspan="1" align="left" valign="top">R4-1</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CCG</underline>
</underline>
<underline><underline>GAA TTC</underline>
</underline>
GAG CTT TTA CCT AAT GTT TTT AAG 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">8774-8797</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">R4-2</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline>CCG CTC GAG</underline>
TTG TAA GGT GGA ATT GTA GCT AAT AG 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">9078-9103</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Expression of six-His-tagged PLP2 in <italic>E. coli</italic>
</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC1</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline>CGC GGA TCC</underline>
<underline>G</underline>
GT TGT AGA GAG TAA TGT TAT GG 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">4979-5000</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC2</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CTA TTG CTC AGC</underline>
</underline>
ACC AGT ATC AAG TTT ATC CAT AAC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5950-5968</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Cloning of pNL-1</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC3</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CCG GAA TTC ACC ATG</underline>
</underline>
GCT GTT GCA AGT GAT TCG 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">311-328</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC4</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline>AAG CTC GAG</underline>
<underline>CG</underline>
A CAG TCG TTA ACA TCC ATA ACA TTA C 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">4989-5014</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Amplification of PLP2 for cloning into pNL-1 to make pNL-2</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC7</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline>C CGC TCG AGT</underline>
TTT AAG AAT GAT AAT GTA GTT TTG 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">5015-5038</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC8</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CG CGG GCC CTC</underline>
</underline>
ACT TTT AGA ACA AGC TAC ACA GTC 3′ (ApaI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">6689-6712</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Amplification of nsp123 to make pNL3 by addition of PLP2 to C terminus</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC5</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CCG GAA TTC ACC ATG</underline>
</underline>
GCT GTT GCA AGT GAT TCG 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">311-328</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC6</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>AA CT CGA GCG</underline>
</underline>
TTG CAA CTG TAC AAG TGT GG 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">3483-3502</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Cloning of 5′ Flag</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC9</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CGG GGT ACC ATG</underline>
</underline>
GAC TAC AAA GAC CAT GAC GGT G 3′ (KpnI)</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC10</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline>CCG GAA TTC CAT ATT CAT ATT CAT CAT CAT</underline>
GGC CGC AAG CTT GTC ATC GTC ATC 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Cloning of pNL-4</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC11</td>
<td colspan="1" rowspan="1" align="left" valign="top"><underline><underline>5′ CCG GAA TTC</underline>
</underline>
ACC ATG GAT ATT TCA CAC CTT GTA AAC TGT G 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">4100-4124</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC12</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>AAG CTC GAG</underline>
</underline>
CGT TGT AAG GTG GAA TTG TAG CTA ATA G 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">9078-9103</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Cloning of PLP2</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC13</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>C CGG AAT TC ACC ATG</underline>
</underline>
GTT GTA GAG AGT AAT GTT ATG 3′ (EcoRI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">4979-4999</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC14</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CC GCT CGA GCG</underline>
</underline>
ACC AGT ATC AAG TTT ATC C 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">5950-5968</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Cloning of pNL-5</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC15</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>C CGC TCG AGT</underline>
</underline>
GCT AGA CTT AAA CGT GTA CCA CTT C 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">6713-6737</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC16</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CG CGG GCC CTC</underline>
</underline>
TTG TAA GGT GGA ATT GTA GCT AAT AG 3′ (ApaI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">9078-9103</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Cloning of pNL-6</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC17</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline>C CGC TCG AGT</underline>
ACA ACT AAA GCA AAG GGT TTG AC 3′ (XhoI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">7574-7596</td>
<td colspan="1" rowspan="1" align="left" valign="top">Forward</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC18</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ <underline><underline>CG CGG GCC CTC</underline>
</underline>
TTG TAA GGT GGA ATT GTA GCT AAT AG 3′ (ApaI)</td>
<td colspan="1" rowspan="1" align="left" valign="top">9078-9103</td>
<td colspan="1" rowspan="1" align="left" valign="top">Reverse</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top">Site-directed mutagenesis</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> PLP1 C1062A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC19</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ ATA ATG CTT GGA TTA GTA CCA CAC TTG TAC AGT TG 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">3465-3499</td>
<td colspan="1" rowspan="1" align="left" valign="top">C1062A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC20</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ TCC AAG CAT TAT TAT CAG ATT GAT CTA AAA CAC G 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">3443-3476</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> PLP2 C1678A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC21</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ CCA CCG ACA ATA AT<underline><underline>G</underline>
</underline>
<underline><underline>CT</underline>
</underline>
T GGG TTA ATG CAA C 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5304-5334</td>
<td colspan="1" rowspan="1" align="left" valign="top">C1678A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC22</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GTT GCA TTA ACC CA<underline>A</underline>
<underline>GC</underline>
A TTA TTG TCG GTG G 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5304-5334</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> PLP2 C1684A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC23</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GTT GGG TTA ATG CAA CT<underline>G</underline>
<underline>CT</underline>
A TAA TTT TAC AGT ATC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5319-5354</td>
<td colspan="1" rowspan="1" align="left" valign="top">C1684A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC24</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GAT ACT GTA AAA TTA T<underline>AG</underline>
<underline>C</underline>
AG TTG CAT TAA CCC AAC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5319-5354</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> PLP2 H1836A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC25</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GGT TCT TTT GAT AAC GGT <underline>GCC</underline>
TAT GTA GTT TAT GAT GC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5774-5811</td>
<td colspan="1" rowspan="1" align="left" valign="top">H1836A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC26</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GCA TCA TAA ACT ACA TA<underline><underline>G</underline>
</underline>
<underline><underline>GC</underline>
</underline>
A CCG TTA TCA AAA GAA CC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">5774-5811</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS3 G2461A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC27</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GTA TAG TTG CAA AAC AG<underline><underline>G</underline>
</underline>
<underline><underline>CT</underline>
</underline>
G CTG GTT TTA AAC GTA C 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">7650-7686</td>
<td colspan="1" rowspan="1" align="left" valign="top">G2461A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC28</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GTA CGT TTA AAA CCA GC<underline><underline>A</underline>
</underline>
<underline><underline>GC</underline>
</underline>
C TGT TTT GCA ACT ATA C 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">7650-7686</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS3 A2462N</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC29</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GTT GCA AAA CAG GGT <underline><underline>AAC</underline>
</underline>
GGT TTT AAA CGT ACT 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">7655-7687</td>
<td colspan="1" rowspan="1" align="left" valign="top">A2462N</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC30</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ AGT ACG TTT AAA ACC <underline>GTT</underline>
ACC CTG TTT TGC AAC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">7655-7687</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS3 LXGG</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC31</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ TT GCA <underline>CT</underline>
A CAG GGT G<underline>G</underline>
T GGT TTT AAA CGT ACT TAT AA 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">7656-7692</td>
<td colspan="1" rowspan="1" align="left" valign="top">LXGG</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC32</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ A ACC A<underline>C</underline>
C ACC CTG T<underline>AG</underline>
TGC AAC TAT ACT AGT AG 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">7644-7676</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS2 L896A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC33</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ ACA AAA <underline>GCA</underline>
GCT GGT GGT AAA ATA TCT TTT TCT GAT G 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2966-3002</td>
<td colspan="1" rowspan="1" align="left" valign="top">L896A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC34</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ ACC AGC <underline><underline>TGC</underline>
</underline>
TTT TGT AAA AGC AAC AGG CAA TAC ACC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2945-2980</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS2 A897N</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC35</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ AAA TTA <underline><underline>AAT</underline>
</underline>
GGT GGT AAA ATA TCT TTT TCT GAT GAT G 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2969-3005</td>
<td colspan="1" rowspan="1" align="left" valign="top">A897N</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC36</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ ACC ACC <underline><underline>ATT</underline>
</underline>
TAA TTT TGT AAA AGC AAC AGG CAA TAC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2948-2983</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS2 G898A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC37</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ TTA GCT <underline><underline>GCT</underline>
</underline>
GGT AAA ATA TCT TTT TCT GAT GAT GTT 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2972-3007</td>
<td colspan="1" rowspan="1" align="left" valign="top">G898A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC38</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ TTT ACC <underline>AGC</underline>
AGC TAA TTT TGT AAA AGC AAC AGG CAA 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2951-2986</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS2 G899A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC39</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GCT GGT <underline>GCT</underline>
AAA ATA TCT TTT TCT GAT GAT GTT ATA G 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2975-3011</td>
<td colspan="1" rowspan="1" align="left" valign="top">G899A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC40</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ TAT TTT <underline>AGC</underline>
ACC AGC TAA TTT TGT AAA AGC AAC AGG 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">2954-2989</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS1 A109N</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC41</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ CAT GGT <underline>AAC</underline>
GGA AGT GTG GTT TTT GTG GAT AAG TAC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">605-640</td>
<td colspan="1" rowspan="1" align="left" valign="top">A109N</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC42</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ ACT TCC <underline><underline>GTT</underline>
</underline>
ACC ATG GCC AAA AAC AAC ATC AAA GTC 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">584-619</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"> CS1 G110A</td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC43</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ GGT GCA <underline><underline>GCA</underline>
</underline>
AGT GTG GTT TTT GTG GAT AAG TAC ATG 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">608-643</td>
<td colspan="1" rowspan="1" align="left" valign="top">G110A</td>
</tr>
<tr><td colspan="1" rowspan="1" align="left" valign="top"></td>
<td colspan="1" rowspan="1" align="left" valign="top">ZC44</td>
<td colspan="1" rowspan="1" align="left" valign="top">5′ CAC ACT <underline><underline>TGC</underline>
</underline>
TGC ACC ATG GCC AAA AAC AAC ATC AAA G 3′</td>
<td colspan="1" rowspan="1" align="left" valign="top">586-622</td>
<td colspan="1" rowspan="1" align="left" valign="top"></td>
</tr>
</tbody>
</table>
<table-wrap-foot><fn id="t1fn1"><label>a</label>
<p>Underlined nucleotides were added for cloning purposes or mutated sequences. Restriction enzymes are indicated in parentheses.</p>
</fn>
<fn id="t1fn2"><label>b</label>
<p>All nucleotide numbers are based on the NL63 genome sequence (GenBank accession no. NC_005831).</p>
</fn>
</table-wrap-foot>
</table-wrap>
</floats-wrap>
</pmc>
</record>
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