Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase
Identifieur interne : 000963 ( Pmc/Corpus ); précédent : 000962; suivant : 000964Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase
Auteurs : Tadanobu Takahashi ; Chairul A. Nidom ; Mai Thi Quynh Le ; Takashi Suzuki ; Yoshihiro KawaokaSource :
- FEBS Open Bio [ 2211-5463 ] ; 2012.
Abstract
Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans.
Url:
DOI: 10.1016/j.fob.2012.08.007
PubMed: 23650608
PubMed Central: 3642167
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PMC:3642167Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase</title><author><name sortKey="Takahashi, Tadanobu" sort="Takahashi, Tadanobu" uniqKey="Takahashi T" first="Tadanobu" last="Takahashi">Tadanobu Takahashi</name><affiliation><nlm:aff id="aff0001">Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff0002">Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and Global COE Program for Innovation in Human Health Sciences, Japan</nlm:aff></affiliation></author><author><name sortKey="Nidom, Chairul A" sort="Nidom, Chairul A" uniqKey="Nidom C" first="Chairul A." last="Nidom">Chairul A. Nidom</name><affiliation><nlm:aff id="aff0003">Faculty of Veterinary Medicine, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia</nlm:aff></affiliation></author><author><name sortKey="Quynh Le, Mai Thi" sort="Quynh Le, Mai Thi" uniqKey="Quynh Le M" first="Mai Thi" last="Quynh Le">Mai Thi Quynh Le</name><affiliation><nlm:aff id="aff0004">National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam</nlm:aff></affiliation></author><author><name sortKey="Suzuki, Takashi" sort="Suzuki, Takashi" uniqKey="Suzuki T" first="Takashi" last="Suzuki">Takashi Suzuki</name><affiliation><nlm:aff id="aff0002">Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and Global COE Program for Innovation in Human Health Sciences, Japan</nlm:aff></affiliation></author><author><name sortKey="Kawaoka, Yoshihiro" sort="Kawaoka, Yoshihiro" uniqKey="Kawaoka Y" first="Yoshihiro" last="Kawaoka">Yoshihiro Kawaoka</name><affiliation><nlm:aff id="aff0001">Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff0005">Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan</nlm:aff></affiliation><affiliation><nlm:aff id="aff0006">ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">23650608</idno><idno type="pmc">3642167</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3642167</idno><idno type="RBID">PMC:3642167</idno><idno type="doi">10.1016/j.fob.2012.08.007</idno><date when="2012">2012</date><idno type="wicri:Area/Pmc/Corpus">000963</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000963</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase</title><author><name sortKey="Takahashi, Tadanobu" sort="Takahashi, Tadanobu" uniqKey="Takahashi T" first="Tadanobu" last="Takahashi">Tadanobu Takahashi</name><affiliation><nlm:aff id="aff0001">Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff0002">Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and Global COE Program for Innovation in Human Health Sciences, Japan</nlm:aff></affiliation></author><author><name sortKey="Nidom, Chairul A" sort="Nidom, Chairul A" uniqKey="Nidom C" first="Chairul A." last="Nidom">Chairul A. Nidom</name><affiliation><nlm:aff id="aff0003">Faculty of Veterinary Medicine, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia</nlm:aff></affiliation></author><author><name sortKey="Quynh Le, Mai Thi" sort="Quynh Le, Mai Thi" uniqKey="Quynh Le M" first="Mai Thi" last="Quynh Le">Mai Thi Quynh Le</name><affiliation><nlm:aff id="aff0004">National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam</nlm:aff></affiliation></author><author><name sortKey="Suzuki, Takashi" sort="Suzuki, Takashi" uniqKey="Suzuki T" first="Takashi" last="Suzuki">Takashi Suzuki</name><affiliation><nlm:aff id="aff0002">Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and Global COE Program for Innovation in Human Health Sciences, Japan</nlm:aff></affiliation></author><author><name sortKey="Kawaoka, Yoshihiro" sort="Kawaoka, Yoshihiro" uniqKey="Kawaoka Y" first="Yoshihiro" last="Kawaoka">Yoshihiro Kawaoka</name><affiliation><nlm:aff id="aff0001">Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA</nlm:aff></affiliation><affiliation><nlm:aff id="aff0005">Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan</nlm:aff></affiliation><affiliation><nlm:aff id="aff0006">ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan</nlm:aff></affiliation></author></analytic><series><title level="j">FEBS Open Bio</title><idno type="eISSN">2211-5463</idno><imprint><date when="2012">2012</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Basler, C F" uniqKey="Basler C">C.F. Basler</name></author><author><name sortKey="Aguilar, P V" uniqKey="Aguilar P">P.V. Aguilar</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Uchida, Y" uniqKey="Uchida Y">Y. Uchida</name></author><author><name sortKey="Chaichoune, K" uniqKey="Chaichoune K">K. Chaichoune</name></author><author><name sortKey="Wiriyarat, W" uniqKey="Wiriyarat W">W. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">FEBS Open Bio</journal-id><journal-id journal-id-type="iso-abbrev">FEBS Open Bio</journal-id><journal-title-group><journal-title>FEBS Open Bio</journal-title></journal-title-group><issn pub-type="epub">2211-5463</issn><publisher><publisher-name>Elsevier</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">23650608</article-id><article-id pub-id-type="pmc">3642167</article-id><article-id pub-id-type="publisher-id">FOB46</article-id><article-id pub-id-type="doi">10.1016/j.fob.2012.08.007</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Amino acid determinants conferring stable sialidase activity at low pH for H5N1 influenza A virus neuraminidase</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Takahashi</surname><given-names>Tadanobu</given-names></name><xref rid="aff0001" ref-type="aff">a</xref><xref rid="aff0002" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Nidom</surname><given-names>Chairul A.</given-names></name><xref rid="aff0003" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Quynh Le</surname><given-names>Mai thi</given-names></name><xref rid="aff0004" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Suzuki</surname><given-names>Takashi</given-names></name><email>suzukit@u-shizuoka-ken.ac.jp</email><xref rid="aff0002" ref-type="aff">b</xref><xref rid="cor0001" ref-type="corresp">*</xref></contrib><contrib contrib-type="author"><name><surname>Kawaoka</surname><given-names>Yoshihiro</given-names></name><email>kawaokay@svm.vetmed.wisc.edu</email><xref rid="aff0001" ref-type="aff">a</xref><xref rid="aff0005" ref-type="aff">e</xref><xref rid="aff0006" ref-type="aff">f</xref><xref rid="cor0001" ref-type="corresp">*</xref></contrib></contrib-group><aff id="aff0001"><label>a</label>Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA</aff><aff id="aff0002"><label>b</label>Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, and Global COE Program for Innovation in Human Health Sciences, Japan</aff><aff id="aff0003"><label>c</label>Faculty of Veterinary Medicine, Tropical Disease Centre, Airlangga University, Surabaya, Indonesia</aff><aff id="aff0004"><label>d</label>National Institute of Hygiene and Epidemiology, Hanoi, Viet Nam</aff><aff id="aff0005"><label>e</label>Division of Virology, Department of Microbiology and Immunology and International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan</aff><aff id="aff0006"><label>f</label>ERATO Infection-Induced Host Responses Project, Japan Science and Technology Agency, Saitama, Japan</aff><author-notes><corresp id="cor0001"><label>*</label>Takashi Suzuki; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 422-8526, Japan (T. Suzuki),Yoshihiro Kawaoka; Influenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53711, USA (Y. Kawaoka). Tel.: +81 54 264 5725; fax: +81 54 264 5723 (T. Suzuki), Tel.: +1 608 265 4925; fax: +1 608 262 9641 (Y. Kawaoka). <email>suzukit@u-shizuoka-ken.ac.jp</email><email>kawaokay@svm.vetmed.wisc.edu</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>5</day><month>9</month><year>2012</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="epub"><day>5</day><month>9</month><year>2012</year></pub-date><pub-date pub-type="collection"><year>2012</year></pub-date><volume>2</volume><fpage>261</fpage><lpage>266</lpage><history><date date-type="received"><day>25</day><month>6</month><year>2012</year></date><date date-type="rev-recd"><day>29</day><month>8</month><year>2012</year></date><date date-type="accepted"><day>29</day><month>8</month><year>2012</year></date></history><permissions><copyright-statement>© 2012 Published by Elsevier B.V. on behalf of Federation of European Biochemical Societies.</copyright-statement><copyright-year>2012</copyright-year><copyright-holder>Federation of European Biochemical Societies</copyright-holder><license><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non- commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.</license-p></license></permissions><abstract><p>Avian influenza A viruses (IAVs) and human 1918, 1957, and 1968 pandemic IAVs all have neuraminidases (NAs) that are stable at low pH sialidase activity, yet most human epidemic IAVs do not. We examined the pH stability of H5N1 highly pathogenic avian IAV (HPAI) NAs and identified amino acids responsible for conferring stability at low pH. We found that, unlike other avian viruses, most H5N1 IAVs isolated since 2003 had NAs that were unstable at low pH, similar to human epidemic IAVs. These H5N1 viruses are thus already human virus-like and, therefore, have the frequent infections of humans.</p></abstract><abstract abstract-type="graphical"><title>Highlights</title><p>▸ All neuraminidases (NAs) of avian influenza A viruses (IAVs) are stable at low pH. ▸ Human 1918, 1957, and 1968 pandemic IAVs also have NAs that are stable at low pH. ▸ Most human epidemic IAVs have NAs that are unstable at low pH. ▸ The NAs of most H5N1 IAVs isolated since 2003 are unstable at low pH. ▸ Instability of H5N1 IAV NAs at low pH might explain frequent human infections.</p></abstract><kwd-group><title>Keywords</title><kwd>Avian influenza A virus</kwd><kwd>H5N1</kwd><kwd>Highly pathogenic</kwd><kwd>Low-pH stability</kwd><kwd>Neuraminidase</kwd><kwd>Sialidase</kwd></kwd-group><kwd-group><title>Abbreviations</title><kwd>FBS, fetal bovine serum</kwd><kwd>HA, hemagglutinin</kwd><kwd>HPAI, highly pathogenic avian influenza A virus</kwd><kwd>IAV, influenza A virus</kwd><kwd>NA, neuraminidase</kwd><kwd>PBS, phosphate-buffered saline</kwd><kwd>TGF-β, transforming growth factor-beta</kwd></kwd-group></article-meta></front><body><sec id="sec0001"><label>1</label><title>Introduction</title><p>Since the first isolation of H5N1 highly pathogenic avian influenza A virus (HPAI) from a 3-year-old boy in Hong Kong in 1997, more than 600 confirmed cases of human H5N1 HPAI infection have been reported, mainly from Indonesia, Viet Nam, and Egypt (as of June, 2012). Since 2004, frequent human infections have raised worldwide concerns regarding the pandemic potential of H5N1 HPAI and its high fatality rate (approximately 60% as of 2012 June) [<xref rid="bib1" ref-type="bibr">1</xref>]. The viral envelope glycoprotein hemagglutinin (HA) has been investigated extensively and is essential to the high virulence and transmission of HPAI to humans, because it possesses receptor binding ability (recognizing both avian- and human-type sialoglycoconjugates). It also has a multi-basic cleavage site that facilitates systemic infection in chicken and mice <xref rid="bib2" ref-type="bibr">[2</xref>,<xref rid="bib11" ref-type="bibr">3</xref>]. Another viral envelope glycoprotein, neuraminidase (NA), has sialidase activity that enhances virus release from the cell surface by removing sialic acid from cellular glycoconjugates and viral glycoproteins. Deletions in the NA stalk and a reduction in the level of NA-induced transforming growth factor-beta (TGF-β) influence the high virulence of HPAI viruses [<xref rid="bib3" ref-type="bibr">4</xref>,<xref rid="bib4" ref-type="bibr">5</xref>]. However, the sialidase activity of H5N1 HPAI NA <italic>per se</italic> has not been compared with that of human influenza A virus (IAV) NA or other avian IAV NA <xref rid="bib10" ref-type="bibr">[</xref><xref rid="bib6" ref-type="bibr">6</xref>].</p><p>We previously showed that influenza viruses differ in their stability at low pH. All avian IAV NAs tested to date are highly stable at low pH; their sialidase activities are retained at pH 5.0 or less [<xref rid="bib5" ref-type="bibr">7</xref>]. This property may be associated with avian virus replication in intestines, because for a virus to reach the intestine, it must pass through the acidic environment of the gizzard. The NAs of pandemic human IAVs, such as 1918 H1N1, 1957 H2N2, and 1968 H3N2 IAV, are also stable at low pH. Viruses possessing a low-pH-stable NA from a pandemic IAV in the background of A/WSN/33 (WSN; H1N1) replicated more efficiently in cell culture and mouse lungs compared with a WSN virus possessing a NA unstable at low pH [<xref rid="bib6" ref-type="bibr">8</xref>]. This enhanced replicative ability was associated with retention of the sialidase activity under the acidic conditions of the endocytic pathway during virus entry [<xref rid="bib6" ref-type="bibr">8</xref>]. On the other hand, the NAs of most seasonal human IAVs are unstable at low pH [<xref rid="bib5" ref-type="bibr">7</xref>,<xref rid="bib7" ref-type="bibr">9</xref>,<xref rid="bib8" ref-type="bibr">10</xref>].</p><p>Here, we examined the sialidase activity of 42 H5N1 HPAI viruses isolated from humans, chickens, ducks, and other waterfowl and found that most of the NAs, with the exception of three H5N1 viruses, were not stable at low pH. We also identified amino acid determinants that could confer low-pH stability to H5N1 HPAI NAs.</p></sec><sec sec-type="materials|methods" id="sec0002"><label>2</label><title>Materials and methods</title><sec id="sec0003"><label>2.1</label><title>Cells</title><p>Human embryonic kidney 293T cells were maintained in high glucose Dulbecco's modified medium supplemented with 10% fetal bovine serum (FBS).</p></sec><sec id="sec0004"><label>2.2</label><title>NA genes and plasmids</title><p>All NA genes inserted into the pCAGGS expression vector and virus abbreviations are listed in <xref rid="sec0011" ref-type="sec">Supplementary Table 1</xref>. Chimeric NAs between A/duck/Guangdong/1/01 (H5N1) (DKG/1) and A/Hong Kong/213/03 (H5N1) (HK/213) were generated by ligating the <italic>Mfe</italic> I and <italic>Bam</italic>H I sites (at positions 595 and 1145 in the NA gene, respectively) or by ligating two PCR fragments derived from primers with the <italic>Bsa</italic> I site (at position 842 in the NA gene). The Y155H, I289T, and Q313R mutations of DKG/1 NA and the H155Y, T289I, and R313Q mutations of HK/213 NA were introduced by means of PCR.</p></sec><sec id="sec0005"><label>2.3</label><title>Sialidase activity of cell-expressed NA</title><p>293T cells (1.5 × 10<sup>5</sup> cells/well) in a 24-well tissue culture plate were cultured overnight. The 70% confluent cells were transfected with a plasmid (1 μg/well) for NA expression by using TransIT-293 (Mirus, Madison, WI). After a 24-h incubation at 37 °C, the transfected cells were suspended in phosphate-buffered saline (PBS; 1.2 ml/well), and 50 μl of each cell suspension was transferred into microtubes and centrifuged at 100 ×<italic>g</italic> for 10 min. The cell pellets were incubated with 57 μl of 10 mM acetate buffer (pH 4.0, 5.0, or 6.0) at 37 °C for 10 min. Fifty microliters of each suspension was then transferred to a 96-well black plate on ice and reacted with 2.5 μl of 2 mM 2′-(4-methylumbelliferyl)-<italic>N</italic>-acetylneuraminic acid (4MU-Neu5Ac; Sigma-Aldrich Corp., St. Louis, MI) at 37 °C for 30 min. The reaction was stopped by the addition of 200 μl of 100 mM sodium carbonate buffer (pH 10.7). The fluorescent intensity (Ex, 355 nm; Em, 460 nm) was measured with an Infinite M1000 microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The sialidase activities of the cell-expressed NAs were expressed as a percentage of the activity at pH 6.0.</p></sec></sec><sec sec-type="results" id="sec0006"><label>3</label><title>Results</title><sec id="sec0007"><label>3</label><title>1. Low-pH stabilities of the sialidase activities of H5N1 HPAI NAs</title><p>We tested the low-pH stabilities of the sialidase activity of cells expressing each of the NA genes of 42 H5N1 HPAI viruses isolated from human and avian hosts, as well as 10 other non-H5N1 avian IAVs and eight human H1N1 IAVs. The NAs of all avian IAVs tested were highly stable at low-pH, as described in our previous reports [<xref rid="bib5" ref-type="bibr">7</xref>,<xref rid="bib7" ref-type="bibr">9</xref>,<xref rid="bib8" ref-type="bibr">10</xref>]. Three H5N1 HPAI NAs of A/Hong Kong/483/97 (HK/483), DKG/1, and CKK/3, were also stable at low pH; at pH 4.0, their NAs had more than 30% of the sialidase activity at pH 6.0. Thirteen H5N1 HPAI NAs were also moderately stable at low pH, retaining more than 30% of their sialidase activity at pH 5.0 compared with that at pH 6.0. However, the NAs of the remaining 26 H5N1 HPAIVs, which were isolated since 1997, were not low-pH-stable, like seasonal human IAVs. No distinct host species-specific differences in their low-pH stabilities were apparent among the H5N1 viruses isolated from different hosts such as humans (<xref rid="fig0001" ref-type="fig">Fig. 1</xref>A), aquatic birds (<xref rid="fig0001" ref-type="fig">Fig. 1</xref>B), or chickens (<xref rid="fig0001" ref-type="fig">Fig. 1</xref>C). There were also no differences in the low-pH stabilities of H5N1 HPAI NAs between aquatic and terrestrial birds (e.g., chickens). We also measured the sialidase activities of avian IAV NAs (<xref rid="fig0001" ref-type="fig">Fig. 1</xref>D) and human H1N1 IAV NAs (<xref rid="fig0001" ref-type="fig">Fig. 1</xref>E) as controls of avian-like and human-like low-pH stability. All avian IAV NAs tested were highly stable at low pH, whereas most human H1N1 IAV NAs, except Spanish IAV A/Brevig Mission/1/18 (H1N1) NA (BM/1), were not low-pH-stable, consistent with our previous work [<xref rid="bib5" ref-type="bibr">7</xref>,<xref rid="bib7" ref-type="bibr">9</xref>,<xref rid="bib8" ref-type="bibr">10</xref>].</p><p>When we measured the sialidase activity after pre-incubating the NAs under acidic conditions, BM/1 NA lost its activity in a time-dependent manner [<xref rid="bib8" ref-type="bibr">10</xref>], whereas avian IAV NAs retained their sialidase activity even after 60 min of pre-incubation at pH 4.0 [<xref rid="bib8" ref-type="bibr">10</xref>] (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>G and H). The low-pH-stable NAs of H5N1 HPAI DKG/1 and A/chicken/Kyoto/3/04 (CKK/3) lost their sialidase activities, similarly to BM/1, as a function of increasing pre-incubation time under acidic conditions (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>A and C). The low-pH-unstable H5N1 HK/213, A/whooper swan/Mongolia/6/05 (WSM/6), and A/Viet Nam/1203/04 (VN/1203) NAs also lost their sialidase activity, as did human H1N1 IAV NAs, in this assay (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>B, D, I, and J). These results indicate that most H5N1 HPAIs have low-pH-unstable NAs, like seasonal human IAVs, and that H5N1 HPAIs with low-pH-stable NAs are the exception, like human pandemic Spanish IAVs, and their low-pH stability profiles differ from the highly stable NAs at low pH in more common avian IAVs (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>).</p></sec><sec id="sec0008"><label>3</label><title>2. Identification of amino acid residues responsible for the low-pH stability of an H5N1 HPAI NA</title><p>The amino acid sequence of the low-pH-stable DKG/1 NA was most closely related to that of the low-pH-unstable HK/213 NA among the viruses tested here (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 2</xref>). Therefore, to determine the amino acid residues responsible for the low-pH stability of H5N1 HPAI NAs, we used the DKG/1 NA and the HK/213 NA to generate chimeric NAs. There are eight amino acid differences between the DKG/1 NA and the HK/213 NA in their ectodomains (amino acids 83–468), at positions 155, 224, 257, 267, 289, 313, 341, and 415 (DKG/1 NA numbering) (<xref rid="tbl0001" ref-type="table">Table 1</xref>). First, we generated eight chimeric NAs (Chimeras 1–8) by using the <italic>Mfe</italic> I and <italic>BamH</italic> sites and PCR primers with the <italic>Bsa</italic> I site (<xref rid="fig0002" ref-type="fig">Fig. 2</xref>A) and tested their low-pH stability. Based on the properties of chimeras 3 and 4, residues at positions 155, 224, 257, 267, 289, 313 and 341 were identified as candidate determinants of the low-pH stability of DKG/1 NA, whereas the results with chimeras 7 and 8 suggested a role for the amino acids at positions at 155, 289, 313, 341, and 415 in the low-pH stability of DKG/1 NA. Taken together, the residues at positions 155, 289, 313, and 341 appeared to be determinants for the DKG/1 NA's low-pH stability (<xref rid="fig0002" ref-type="fig">Fig. 2</xref>A, <xref rid="tbl0001" ref-type="table">Table 1</xref>). We therefore generated an additional eight chimeric NAs in which Tyr or His was substituted at position 155 in the different genetic backbones (chimeras 9–16) (<xref rid="fig0002" ref-type="fig">Fig. 2</xref>B). A striking difference in low-pH stability between chimeras 13 and 14 suggested that the residues at positions 289, 313, and 341 were likely determinants of low-pH stability. Finally, we generated three NAs with substitutions at positions 289, 313, and 341, respectively, in addition to the substitution at position 155 in the DKG/1 NA (<xref rid="fig0002" ref-type="fig">Fig. 2</xref>C) or the HK/213 NA (<xref rid="fig0002" ref-type="fig">Fig. 2</xref>D). The dual substitutions at positions 155 and 341 substantially changed the enzymatic stabilities at pH 4.0 and 5.0 (<xref rid="tbl0001" ref-type="table">Table 1</xref>). We also examined the low-pH stability of the HK/213 NA mutant with substitutions at positions 155 and 341 after it was incubated under acidic conditions in a time-dependent manner. The HK/213 NA with both the H155Y and K341N mutations lost its sialidase activity in a time-dependent manner, like the DKG/1 NA (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>A and F), but it was not as stable as the highly stable NAs at low pH in the more common avian IAVs (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>G and H). The DKG/1 NA with both the Y155H and N341K mutations lost its sialidase activity within five minutes of exposure to pH 4.0 and 5.0, like the HK/213 NA (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 1</xref>B and E). Therefore, these results show that histidine at position 155 and lysine at position 341 are responsible for the low-pH instability of H5N1 HPAI NAs.</p></sec></sec><sec sec-type="discussion" id="sec0009"><label>4</label><title>Discussion</title><p>Here, we showed that most H5N1 HPAI NAs are unstable at low pH, like seasonal human IAV NAs. Thus, the low-pH stability of H5N1 HPAI NAs more closely resembles that of human IAV NAs than that of avian IAV NAs, regardless of the host (i.e., humans and birds) from which the virus is isolated. Although human infections with avian H5N1, H7N3, H7N7, and H9N2 IAVs have occurred, by far the large number of human infections has been reported for H5N1 HPAI [<xref rid="bib12" ref-type="bibr">11</xref>]. Our previous research suggested that NA that are unstable at low pH may be more suitable for the adaptation of IAV to humans [<xref rid="bib7" ref-type="bibr">9</xref>]. The low-pH instability of H5N1 HPAI NAs among avian IAV NAs might contribute to the frequent transmission of H5N1 HPAI to humans.</p><p>For the DKG/1 NA and the HK/213 NA, both tyrosine at position 155 and asparagine at position 341 were responsible for the stability at low pH; histidine at position 155 together with lysine at position 341 destabilized NA activity at low pH. In the three-dimensional structure of VN/1203 NA (<xref rid="fig0003" ref-type="fig">Fig. 3</xref>) [<xref rid="bib13" ref-type="bibr">12</xref>], the residue at position 155 was near the monomer interface, suggesting that it may be involved in stabilization of the homotetrameric structure. The residue at position 341 was located near the calcium ion-binding site, which is thought to be involved in the conformation of the enzymatic active site [<xref rid="bib14" ref-type="bibr">13</xref>,<xref rid="bib15" ref-type="bibr">14</xref>]. We previously identified residues at positions 430, 435, and 454 (BM/1 N1 numbering) as important for the low-pH stability of N1 NAs (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 3</xref>A) [<xref rid="bib8" ref-type="bibr">10</xref>] and residues at positions 344 and 466 (N2 numbering) as important for the low-pH stability of N2 NAs (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 3</xref>B) [<xref rid="bib9" ref-type="bibr">15</xref>]; these residues are also located near the enzymatic active site, the calcium ion-binding site, and the subunit interfaces.</p><p>Of the 42 H5N1 NAs tested, we found that the tyrosine at position 155 that is responsible for the low-pH stability of DKG/1 NA was present in only three NAs [those of viruses HK/483, A/Hong Kong/486/97 (HK/486), and DKG/1], whereas the remaining 39 H5N1 HPAI NAs, including the low-pH-stable CKK/3 NA, had histidine at this position, indicating that the low-pH stability of CKK/3 NA does not require tyrosine at this position. The lysine at position 341 that was responsible for the low-pH instability of HK/213 NA was present only in this NA; the remaining 41 NAs tested here had asparagine at this position. Therefore, these substitutions are not necessarily responsible for the low-pH stabilities of all H5N1 HPAI NAs.</p><p>Several moderately stable NAs at low pH were found among the 42 H5N1 HPAI NAs (<xref rid="sec0011" ref-type="sec">Supplementary Fig. 2</xref>). The amino acid sequences of the low-pH-unstable A/Viet Nam/UT3028II/03 (VN/UT3028II) NA and the moderately stable A/Viet Nam/UT3028/03 (VN/UT3028) NA at low pH differ by only a single substitution at position 344 (DKG/1 NA numbering; cysteine for VN/UT3028II and glycine for VN/UT3028). Also, only two amino acid differences were found between the unstable A/duck/Viet Nam/5001/04 (DKVN/5001) NA and the moderately stable A/Viet Nam/UT30259/04 (VN/UT30259) NA at low pH; the former having lysine and serine and the latter having asparagine and glycine at positions 150 and 249 (DKG/1 NA numbering), respectively. These results indicate that amino acids at positions other than 155 and 341 affect the low-pH stability of H5N1 HPAI NAs.</p><p>Among the residues responsible for the low-pH stability of the NAs of H5N1 HPAI viruses, the residues at positions 249 and 344 are located at or near the calcium ion-binding site, whereas the residues at positions 150 and 249 are located near the enzymatic active site. These results are consistent with our previous findings with BM/1 N1 and human N2 [<xref rid="bib8" ref-type="bibr">10</xref>,<xref rid="bib9" ref-type="bibr">15</xref>].</p><p>Unlike avian IAV NAs, most human IAV NAs are not stable at low pH. Therefore, most H5N1 HPAI NAs isolated since 2003 are more like the NAs of human IAVs in this regard, which might explain their frequent infection of humans. Further studies of the low-pH stability of IAV NAs may enable us to predict low-pH stability on the basis of the amino acid residues located near the active site, the calcium ion-binding site, and the subunit interfaces, which may, in turn, help us to assess the adaptability of avian IAVs to humans.</p></sec></body><back><ref-list><title>References</title><ref id="bib1"><label>1</label><mixed-citation publication-type="other" id="ceotherref1">WHO (2012) Cumulative Number of Confirmed Human Cases of Avian Influenza A/ (H5N1) Reported to WHO as of 7 June 2012. 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Sialidase activities of NA-expressing cells transfected with each NA gene were measured after pre-incubation for 5, 15, 30, and 60 min at pH 4.0 (filled column), 5.0 (hatched column) and 6.0 (open column). Sialidase activities were expressed as described in the legend to <xref rid="fig0001" ref-type="fig">Fig. 1</xref>. The low-pH stabilities of DKG/1 NA (A), HK/213 NA (B), CKK/3 NA (C), WSM/6 NA (D), DKG/1 NA with Y155H and N341K mutations (E), and HK/213 NA with H155Y and K341N mutations (F) were measured. Avian IAV NAs of DKA/80 (G) and CKV/85 (H) served as representative low-pH-stable NAs. H5N1 VN/1203 NA (I) and human H1N1 K/173 NA (J) served as representative low-pH-unstable NAs.</p></caption><media xlink:href="mmc1.doc" mimetype="application" mime-subtype="msword"></media></supplementary-material><supplementary-material content-type="local-data" id="ec2"><caption><title>Supplementary Fig. 2</title><p>Phylogenic tree of amino acid sequences of H5N1 HPAI NAs. The phylogenic tree was generated from the amino acid sequences of 42 H5N1 HPAI NAs by using DNASTAR Lasergene software (DNASTAR, Inc. Madison, WI). The bar at the bottom indicates amino acid differences. Red and orange colors indicate NAs that retained more than 30% of their sialidase activity at pH 4.0 and pH 5.0, respectively, relative to that at pH 6.0, respectively.</p></caption><media xlink:href="mmc1.doc" mimetype="application" mime-subtype="msword"></media></supplementary-material><supplementary-material content-type="local-data" id="ec3"><caption><title>Supplementary Fig. 3</title><p>Location of previously reported amino acid residues responsible for the low-pH stabilities of NAs. We previously showed that amino acid substitutions at positions 430, 435, and 454 in the N1 NA of BM/1 virus and A/USSR/92/77 (H1N1) virus (BM/1 N1 numbering) [<xref rid="bib8" ref-type="bibr">10</xref>] or mutations at positions 344 and 466 of the N2 NA of A/Hong Kong/1/68 (H3N2) virus and A/Texas/68 (H2N2) virus (A/Tokyo/3/67 N2 numbering) [<xref rid="bib9" ref-type="bibr">15</xref>] confer marked changes in stability at low pH. Red, purple, and cyan in the figures indicate the active site, the calcium-ion binding site, and the subunit interfaces, respectively. A, Dimer structure of BM/1 N1NA (3BEQ. pdb). One subunit of NA is colored in gray. Residues at positions 430, 435, and 454, are colored in blue, green, and yellow, respectively. Residues at positions 430 and 435 in the N1 NA are near to both the active site and the primary calcium ion-binding site, and the residue at position 454 is part of the subunit interfaces. B, Monomeric structure of A/Tokyo/3/67 N2 NA (2BAT. pdb). Residues at positions 344 and 466 are colored in green and blue, respectively. The residue at position 344 is near the secondary calcium ion-binding site, and the residue at position 466 is near to both the primary calcium ion-binding site and the subunit interfaces. Pictures were generated by using the Pymol Molecular Graphics System Ver. 1.1r1 (DeLano Scientific LLC).</p></caption><media xlink:href="mmc1.doc" mimetype="application" mime-subtype="msword"></media></supplementary-material><supplementary-material content-type="local-data" id="ec4"><caption><title>Supplementary Table 1</title><p>Abbreviations of influenza A virus strains.</p></caption><media xlink:href="mmc2.doc" mimetype="application" mime-subtype="msword"></media></supplementary-material></p></sec><ack><title>Acknowledgments</title><p>We thank Susan Watson for scientific editing. This work was supported, in part, by <funding-source id="GS1">MEXT/JSPS KAKENHI</funding-source> Grant Number (C; 23590549), by a Sasakawa Scientific Research Grant from The Japan Science Society, by a SRI (Shizuoka Research Institute) academic research grant, by the Global COE Program from the Japan Society for the Promotion of Science, by ERATO (Japan Science and Technology Agency), by a grant-in-aid for Specially Promoted Research from the Ministries of Education, Culture, Sports, Science, and Technology, by grants-in-aid from Health, Labor, and Welfare of Japan, by the Japan Initiative for Global Research Network on Infectious Diseases, and by National Institute of Allergy and Infectious Disease Public Health Service research grants.</p></ack></back><floats-group><fig id="fig0001"><label>Fig. 1</label><caption><p>The low-pH stabilities of the sialidase activities of H5N1 HPAI NAs, human H1N1 IAV NAs, and non-H5N1 avian IAV NAs. Sialidase activities of NA-expressing cells transfected with each NA gene were measured at pH 4.0 (filled columns), 5.0 (gray columns) and 6.0 (open columns). Sialidase activities are expressed as a percentage of each activity at pH 6.0. The NA genes used were derived from H5N1 HPAIs isolated from humans (A), aquatic birds (B), chickens (C), other avian IAVs (D) and from human H1N1 IAVs (E).</p></caption><graphic xlink:href="gr1"></graphic></fig><fig id="fig0002"><label>Fig. 2</label><caption><p>Identification of amino acid residues responsible for the low-pH stability of DKG/1 NA and HK/213 NA. Sialidase activities were measured as described in the legend to Fig. 1. A, Low-pH stabilities of chimeric NAs. B, Low-pH stabilities of chimeric NAs with the Y155H or H155Y mutation at position 155. C, Low-pH stabilities of mutated DKG/1 NAs. D, Low-pH stabilities of mutated HK/213 NAs.</p></caption><graphic xlink:href="gr2"></graphic></fig><fig id="fig0003"><label>Fig. 3</label><caption><p>Locations of the amino acid residues responsible for the low-pH stabilities of H5N1 HPAI NAs. One subunit of the NA homotetramer structure (2HTY. pdb, VN/1203) is shown in gray. In the surface model of NA, red and purple indicate the active site and the calcium ion-binding site, respectively. The residues at positions 155 and 341 are colored in green and blue, respectively (DKG/1 NA numbering). Pictures were generated by using the Pymol Molecular Graphics System Ver. 1.1r1 (DeLano Scientific LLC).</p></caption><graphic xlink:href="gr3"></graphic></fig><table-wrap id="tbl0001" position="float"><label>Table 1</label><caption><p>Amino acid comparison of the NA globular domains between DKG/1 and HK/213.</p></caption><table frame="hsides" rules="groups"><tbody><tr><td align="center"><inline-graphic xlink:href="fx1.gif"></inline-graphic></td></tr></tbody></table><table-wrap-foot><fn><p><sup>a</sup>Amino acid position is based on DKG/1 NA numbering.</p><p><sup>b</sup>Positions in the NA amino acid sequence involved in creating the chimeric NAs.</p><p><sup>c</sup>Boxed amino acids are responsible for the low-pH stability of DKG/1 NA and HK/213 NA.</p></fn></table-wrap-foot></table-wrap></floats-group></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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