Corpus
Pmc
Racine
Corpus
Checkpoint
Pmc
Racine
Pmc
Exploration
Accueil
Racine
Racine

Serveur d'exploration SRAS

Attention, ce site est en cours de développement !
Attention, site généré par des moyens informatiques à partir de corpus bruts.
Les informations ne sont donc pas validées.

Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7

Identifieur interne : 001376 ( Pmc/Corpus ); précédent : 001375; suivant : 001377

Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7

Auteurs : Yaoqing Chen ; Luyang Cao ; Maohua Zhong ; Yan Zhang ; Chen Han ; Qiaoli Li ; Jingyi Yang ; Dihan Zhou ; Wei Shi ; Benxia He ; Fang Liu ; Jie Yu ; Ying Sun ; Yuan Cao ; Yaoming Li ; Wenxin Li ; Deying Guo ; Zhijian Cao ; Huimin Yan

Source :

RBID : PMC:3334916

Abstract

For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC50 value of 2.76 µg/ml (1.65 µM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1.


Url:
DOI: 10.1371/journal.pone.0034947
PubMed: 22536342
PubMed Central: 3334916

Links to Exploration step

PMC:3334916

Le document en format XML

<record>
<TEI>
<teiHeader>
<fileDesc>
<titleStmt>
<title xml:lang="en">Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7</title>
<author>
<name sortKey="Chen, Yaoqing" sort="Chen, Yaoqing" uniqKey="Chen Y" first="Yaoqing" last="Chen">Yaoqing Chen</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Luyang" sort="Cao, Luyang" uniqKey="Cao L" first="Luyang" last="Cao">Luyang Cao</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhong, Maohua" sort="Zhong, Maohua" uniqKey="Zhong M" first="Maohua" last="Zhong">Maohua Zhong</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhang, Yan" sort="Zhang, Yan" uniqKey="Zhang Y" first="Yan" last="Zhang">Yan Zhang</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Han, Chen" sort="Han, Chen" uniqKey="Han C" first="Chen" last="Han">Chen Han</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Li, Qiaoli" sort="Li, Qiaoli" uniqKey="Li Q" first="Qiaoli" last="Li">Qiaoli Li</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yang, Jingyi" sort="Yang, Jingyi" uniqKey="Yang J" first="Jingyi" last="Yang">Jingyi Yang</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhou, Dihan" sort="Zhou, Dihan" uniqKey="Zhou D" first="Dihan" last="Zhou">Dihan Zhou</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Shi, Wei" sort="Shi, Wei" uniqKey="Shi W" first="Wei" last="Shi">Wei Shi</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="He, Benxia" sort="He, Benxia" uniqKey="He B" first="Benxia" last="He">Benxia He</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Liu, Fang" sort="Liu, Fang" uniqKey="Liu F" first="Fang" last="Liu">Fang Liu</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yu, Jie" sort="Yu, Jie" uniqKey="Yu J" first="Jie" last="Yu">Jie Yu</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Sun, Ying" sort="Sun, Ying" uniqKey="Sun Y" first="Ying" last="Sun">Ying Sun</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Yuan" sort="Cao, Yuan" uniqKey="Cao Y" first="Yuan" last="Cao">Yuan Cao</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Li, Yaoming" sort="Li, Yaoming" uniqKey="Li Y" first="Yaoming" last="Li">Yaoming Li</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Li, Wenxin" sort="Li, Wenxin" uniqKey="Li W" first="Wenxin" last="Li">Wenxin Li</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Guo, Deying" sort="Guo, Deying" uniqKey="Guo D" first="Deying" last="Guo">Deying Guo</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Zhijian" sort="Cao, Zhijian" uniqKey="Cao Z" first="Zhijian" last="Cao">Zhijian Cao</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yan, Huimin" sort="Yan, Huimin" uniqKey="Yan H" first="Huimin" last="Yan">Huimin Yan</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
</titleStmt>
<publicationStmt>
<idno type="wicri:source">PMC</idno>
<idno type="pmid">22536342</idno>
<idno type="pmc">3334916</idno>
<idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3334916</idno>
<idno type="RBID">PMC:3334916</idno>
<idno type="doi">10.1371/journal.pone.0034947</idno>
<date when="2012">2012</date>
<idno type="wicri:Area/Pmc/Corpus">001376</idno>
<idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001376</idno>
</publicationStmt>
<sourceDesc>
<biblStruct>
<analytic>
<title xml:lang="en" level="a" type="main">Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7</title>
<author>
<name sortKey="Chen, Yaoqing" sort="Chen, Yaoqing" uniqKey="Chen Y" first="Yaoqing" last="Chen">Yaoqing Chen</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Luyang" sort="Cao, Luyang" uniqKey="Cao L" first="Luyang" last="Cao">Luyang Cao</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhong, Maohua" sort="Zhong, Maohua" uniqKey="Zhong M" first="Maohua" last="Zhong">Maohua Zhong</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhang, Yan" sort="Zhang, Yan" uniqKey="Zhang Y" first="Yan" last="Zhang">Yan Zhang</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Han, Chen" sort="Han, Chen" uniqKey="Han C" first="Chen" last="Han">Chen Han</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Li, Qiaoli" sort="Li, Qiaoli" uniqKey="Li Q" first="Qiaoli" last="Li">Qiaoli Li</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yang, Jingyi" sort="Yang, Jingyi" uniqKey="Yang J" first="Jingyi" last="Yang">Jingyi Yang</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Zhou, Dihan" sort="Zhou, Dihan" uniqKey="Zhou D" first="Dihan" last="Zhou">Dihan Zhou</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Shi, Wei" sort="Shi, Wei" uniqKey="Shi W" first="Wei" last="Shi">Wei Shi</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="He, Benxia" sort="He, Benxia" uniqKey="He B" first="Benxia" last="He">Benxia He</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Liu, Fang" sort="Liu, Fang" uniqKey="Liu F" first="Fang" last="Liu">Fang Liu</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yu, Jie" sort="Yu, Jie" uniqKey="Yu J" first="Jie" last="Yu">Jie Yu</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Sun, Ying" sort="Sun, Ying" uniqKey="Sun Y" first="Ying" last="Sun">Ying Sun</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Yuan" sort="Cao, Yuan" uniqKey="Cao Y" first="Yuan" last="Cao">Yuan Cao</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Li, Yaoming" sort="Li, Yaoming" uniqKey="Li Y" first="Yaoming" last="Li">Yaoming Li</name>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Li, Wenxin" sort="Li, Wenxin" uniqKey="Li W" first="Wenxin" last="Li">Wenxin Li</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Guo, Deying" sort="Guo, Deying" uniqKey="Guo D" first="Deying" last="Guo">Deying Guo</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Cao, Zhijian" sort="Cao, Zhijian" uniqKey="Cao Z" first="Zhijian" last="Cao">Zhijian Cao</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
<author>
<name sortKey="Yan, Huimin" sort="Yan, Huimin" uniqKey="Yan H" first="Huimin" last="Yan">Huimin Yan</name>
<affiliation>
<nlm:aff id="aff1">
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
<affiliation>
<nlm:aff id="aff2">
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</nlm:aff>
</affiliation>
</author>
</analytic>
<series>
<title level="j">PLoS ONE</title>
<idno type="eISSN">1932-6203</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>For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC
<sub>50</sub>
value of 2.76 µg/ml (1.65 µM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1.</p>
</div>
</front>
<back>
<div1 type="bibliography">
<listBibl>
<biblStruct>
<analytic>
<author>
<name sortKey="Unaids" uniqKey="Unaids">UNAIDS</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Titti, F" uniqKey="Titti F">F Titti</name>
</author>
<author>
<name sortKey="Cafaro, A" uniqKey="Cafaro A">A Cafaro</name>
</author>
<author>
<name sortKey="Ferrantelli, F" uniqKey="Ferrantelli F">F Ferrantelli</name>
</author>
<author>
<name sortKey="Tripiciano, A" uniqKey="Tripiciano A">A Tripiciano</name>
</author>
<author>
<name sortKey="Moretti, S" uniqKey="Moretti S">S Moretti</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Buchbinder, Sp" uniqKey="Buchbinder S">SP Buchbinder</name>
</author>
<author>
<name sortKey="Mehrotra, Dv" uniqKey="Mehrotra D">DV Mehrotra</name>
</author>
<author>
<name sortKey="Duerr, A" uniqKey="Duerr A">A Duerr</name>
</author>
<author>
<name sortKey="Fitzgerald, Dw" uniqKey="Fitzgerald D">DW Fitzgerald</name>
</author>
<author>
<name sortKey="Mogg, R" uniqKey="Mogg R">R Mogg</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Rerks Ngarm, S" uniqKey="Rerks Ngarm S">S Rerks-Ngarm</name>
</author>
<author>
<name sortKey="Pitisuttithum, P" uniqKey="Pitisuttithum P">P Pitisuttithum</name>
</author>
<author>
<name sortKey="Nitayaphan, S" uniqKey="Nitayaphan S">S Nitayaphan</name>
</author>
<author>
<name sortKey="Kaewkungwal, J" uniqKey="Kaewkungwal J">J Kaewkungwal</name>
</author>
<author>
<name sortKey="Chiu, J" uniqKey="Chiu J">J Chiu</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Dalgleish, Ag" uniqKey="Dalgleish A">AG Dalgleish</name>
</author>
<author>
<name sortKey="Beverley, Pc" uniqKey="Beverley P">PC Beverley</name>
</author>
<author>
<name sortKey="Clapham, Pr" uniqKey="Clapham P">PR Clapham</name>
</author>
<author>
<name sortKey="Crawford, Dh" uniqKey="Crawford D">DH Crawford</name>
</author>
<author>
<name sortKey="Greaves, Mf" uniqKey="Greaves M">MF Greaves</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Deng, H" uniqKey="Deng H">H Deng</name>
</author>
<author>
<name sortKey="Liu, R" uniqKey="Liu R">R Liu</name>
</author>
<author>
<name sortKey="Ellmeier, W" uniqKey="Ellmeier W">W Ellmeier</name>
</author>
<author>
<name sortKey="Choe, S" uniqKey="Choe S">S Choe</name>
</author>
<author>
<name sortKey="Unutmaz, D" uniqKey="Unutmaz D">D Unutmaz</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Doranz, Bj" uniqKey="Doranz B">BJ Doranz</name>
</author>
<author>
<name sortKey="Rucker, J" uniqKey="Rucker J">J Rucker</name>
</author>
<author>
<name sortKey="Yi, Y" uniqKey="Yi Y">Y Yi</name>
</author>
<author>
<name sortKey="Smyth, Rj" uniqKey="Smyth R">RJ Smyth</name>
</author>
<author>
<name sortKey="Samson, M" uniqKey="Samson M">M Samson</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Feng, Y" uniqKey="Feng Y">Y Feng</name>
</author>
<author>
<name sortKey="Broder, Cc" uniqKey="Broder C">CC Broder</name>
</author>
<author>
<name sortKey="Kennedy, Pe" uniqKey="Kennedy P">PE Kennedy</name>
</author>
<author>
<name sortKey="Berger, Ea" uniqKey="Berger E">EA Berger</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Neurath, Ar" uniqKey="Neurath A">AR Neurath</name>
</author>
<author>
<name sortKey="Strick, N" uniqKey="Strick N">N Strick</name>
</author>
<author>
<name sortKey="Li, Yy" uniqKey="Li Y">YY Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Telwatte, S" uniqKey="Telwatte S">S Telwatte</name>
</author>
<author>
<name sortKey="Moore, K" uniqKey="Moore K">K Moore</name>
</author>
<author>
<name sortKey="Johnson, A" uniqKey="Johnson A">A Johnson</name>
</author>
<author>
<name sortKey="Tyssen, D" uniqKey="Tyssen D">D Tyssen</name>
</author>
<author>
<name sortKey="Sterjovski, J" uniqKey="Sterjovski J">J Sterjovski</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Torre, Vs" uniqKey="Torre V">VS Torre</name>
</author>
<author>
<name sortKey="Marozsan, Aj" uniqKey="Marozsan A">AJ Marozsan</name>
</author>
<author>
<name sortKey="Albright, Jl" uniqKey="Albright J">JL Albright</name>
</author>
<author>
<name sortKey="Collins, Kr" uniqKey="Collins K">KR Collins</name>
</author>
<author>
<name sortKey="Hartley, O" uniqKey="Hartley O">O Hartley</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Mccormack, S" uniqKey="Mccormack S">S McCormack</name>
</author>
<author>
<name sortKey="Ramjee, G" uniqKey="Ramjee G">G Ramjee</name>
</author>
<author>
<name sortKey="Kamali, A" uniqKey="Kamali A">A Kamali</name>
</author>
<author>
<name sortKey="Rees, H" uniqKey="Rees H">H Rees</name>
</author>
<author>
<name sortKey="Crook, Am" uniqKey="Crook A">AM Crook</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Pearce Pratt, R" uniqKey="Pearce Pratt R">R Pearce-Pratt</name>
</author>
<author>
<name sortKey="Phillips, Dm" uniqKey="Phillips D">DM Phillips</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Tao, J" uniqKey="Tao J">J Tao</name>
</author>
<author>
<name sortKey="Hu, Q" uniqKey="Hu Q">Q Hu</name>
</author>
<author>
<name sortKey="Yang, J" uniqKey="Yang J">J Yang</name>
</author>
<author>
<name sortKey="Li, R" uniqKey="Li R">R Li</name>
</author>
<author>
<name sortKey="Li, X" uniqKey="Li X">X Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Vzorov, An" uniqKey="Vzorov A">AN Vzorov</name>
</author>
<author>
<name sortKey="Bozja, J" uniqKey="Bozja J">J Bozja</name>
</author>
<author>
<name sortKey="Dixon, Dw" uniqKey="Dixon D">DW Dixon</name>
</author>
<author>
<name sortKey="Marzilli, Lg" uniqKey="Marzilli L">LG Marzilli</name>
</author>
<author>
<name sortKey="Compans, Rw" uniqKey="Compans R">RW Compans</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Tao, W" uniqKey="Tao W">W Tao</name>
</author>
<author>
<name sortKey="Richards, C" uniqKey="Richards C">C Richards</name>
</author>
<author>
<name sortKey="Hamer, D" uniqKey="Hamer D">D Hamer</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Boman, Hg" uniqKey="Boman H">HG Boman</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hancock, Re" uniqKey="Hancock R">RE Hancock</name>
</author>
<author>
<name sortKey="Lehrer, R" uniqKey="Lehrer R">R Lehrer</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Fjell, Cd" uniqKey="Fjell C">CD Fjell</name>
</author>
<author>
<name sortKey="Hancock, Re" uniqKey="Hancock R">RE Hancock</name>
</author>
<author>
<name sortKey="Cherkasov, A" uniqKey="Cherkasov A">A Cherkasov</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Jenssen, H" uniqKey="Jenssen H">H Jenssen</name>
</author>
<author>
<name sortKey="Hamill, P" uniqKey="Hamill P">P Hamill</name>
</author>
<author>
<name sortKey="Hancock, Re" uniqKey="Hancock R">RE Hancock</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Martin, E" uniqKey="Martin E">E Martin</name>
</author>
<author>
<name sortKey="Ganz, T" uniqKey="Ganz T">T Ganz</name>
</author>
<author>
<name sortKey="Lehrer, Ri" uniqKey="Lehrer R">RI Lehrer</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Wang, Z" uniqKey="Wang Z">Z Wang</name>
</author>
<author>
<name sortKey="Wang, G" uniqKey="Wang G">G Wang</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zasloff, M" uniqKey="Zasloff M">M Zasloff</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Ma, Y" uniqKey="Ma Y">Y Ma</name>
</author>
<author>
<name sortKey="Zhao, R" uniqKey="Zhao R">R Zhao</name>
</author>
<author>
<name sortKey="He, Y" uniqKey="He Y">Y He</name>
</author>
<author>
<name sortKey="Li, S" uniqKey="Li S">S Li</name>
</author>
<author>
<name sortKey="Liu, J" uniqKey="Liu J">J Liu</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Possani, Ld" uniqKey="Possani L">LD Possani</name>
</author>
<author>
<name sortKey="Merino, E" uniqKey="Merino E">E Merino</name>
</author>
<author>
<name sortKey="Corona, M" uniqKey="Corona M">M Corona</name>
</author>
<author>
<name sortKey="Bolivar, F" uniqKey="Bolivar F">F Bolivar</name>
</author>
<author>
<name sortKey="Becerril, B" uniqKey="Becerril B">B Becerril</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zhijian, C" uniqKey="Zhijian C">C Zhijian</name>
</author>
<author>
<name sortKey="Feng, L" uniqKey="Feng L">L Feng</name>
</author>
<author>
<name sortKey="Yingliang, W" uniqKey="Yingliang W">W Yingliang</name>
</author>
<author>
<name sortKey="Xin, M" uniqKey="Xin M">M Xin</name>
</author>
<author>
<name sortKey="Wenxin, L" uniqKey="Wenxin L">L Wenxin</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Torres Larios, A" uniqKey="Torres Larios A">A Torres-Larios</name>
</author>
<author>
<name sortKey="Gurrola, Gb" uniqKey="Gurrola G">GB Gurrola</name>
</author>
<author>
<name sortKey="Zamudio, Fz" uniqKey="Zamudio F">FZ Zamudio</name>
</author>
<author>
<name sortKey="Possani, Ld" uniqKey="Possani L">LD Possani</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Conde, R" uniqKey="Conde R">R Conde</name>
</author>
<author>
<name sortKey="Zamudio, Fz" uniqKey="Zamudio F">FZ Zamudio</name>
</author>
<author>
<name sortKey="Rodriguez, Mh" uniqKey="Rodriguez M">MH Rodriguez</name>
</author>
<author>
<name sortKey="Possani, Ld" uniqKey="Possani L">LD Possani</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Moerman, L" uniqKey="Moerman L">L Moerman</name>
</author>
<author>
<name sortKey="Bosteels, S" uniqKey="Bosteels S">S Bosteels</name>
</author>
<author>
<name sortKey="Noppe, W" uniqKey="Noppe W">W Noppe</name>
</author>
<author>
<name sortKey="Willems, J" uniqKey="Willems J">J Willems</name>
</author>
<author>
<name sortKey="Clynen, E" uniqKey="Clynen E">E Clynen</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Dai, L" uniqKey="Dai L">L Dai</name>
</author>
<author>
<name sortKey="Yasuda, A" uniqKey="Yasuda A">A Yasuda</name>
</author>
<author>
<name sortKey="Naoki, H" uniqKey="Naoki H">H Naoki</name>
</author>
<author>
<name sortKey="Corzo, G" uniqKey="Corzo G">G Corzo</name>
</author>
<author>
<name sortKey="Andriantsiferana, M" uniqKey="Andriantsiferana M">M Andriantsiferana</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Dai, C" uniqKey="Dai C">C Dai</name>
</author>
<author>
<name sortKey="Ma, Y" uniqKey="Ma Y">Y Ma</name>
</author>
<author>
<name sortKey="Zhao, Z" uniqKey="Zhao Z">Z Zhao</name>
</author>
<author>
<name sortKey="Zhao, R" uniqKey="Zhao R">R Zhao</name>
</author>
<author>
<name sortKey="Wang, Q" uniqKey="Wang Q">Q Wang</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zhao, Z" uniqKey="Zhao Z">Z Zhao</name>
</author>
<author>
<name sortKey="Ma, Y" uniqKey="Ma Y">Y Ma</name>
</author>
<author>
<name sortKey="Dai, C" uniqKey="Dai C">C Dai</name>
</author>
<author>
<name sortKey="Zhao, R" uniqKey="Zhao R">R Zhao</name>
</author>
<author>
<name sortKey="Li, S" uniqKey="Li S">S Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Albiol Matanic, Vc" uniqKey="Albiol Matanic V">VC Albiol Matanic</name>
</author>
<author>
<name sortKey="Castilla, V" uniqKey="Castilla V">V Castilla</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Sun, L" uniqKey="Sun L">L Sun</name>
</author>
<author>
<name sortKey="Finnegan, Cm" uniqKey="Finnegan C">CM Finnegan</name>
</author>
<author>
<name sortKey="Kish Catalone, T" uniqKey="Kish Catalone T">T Kish-Catalone</name>
</author>
<author>
<name sortKey="Blumenthal, R" uniqKey="Blumenthal R">R Blumenthal</name>
</author>
<author>
<name sortKey="Garzino Demo, P" uniqKey="Garzino Demo P">P Garzino-Demo</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Carriel Gomes, Mc" uniqKey="Carriel Gomes M">MC Carriel-Gomes</name>
</author>
<author>
<name sortKey="Kratz, Jm" uniqKey="Kratz J">JM Kratz</name>
</author>
<author>
<name sortKey="Barracco, Ma" uniqKey="Barracco M">MA Barracco</name>
</author>
<author>
<name sortKey="Bachere, E" uniqKey="Bachere E">E Bachere</name>
</author>
<author>
<name sortKey="Barardi, Cr" uniqKey="Barardi C">CR Barardi</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Yan, R" uniqKey="Yan R">R Yan</name>
</author>
<author>
<name sortKey="Zhao, Z" uniqKey="Zhao Z">Z Zhao</name>
</author>
<author>
<name sortKey="He, Y" uniqKey="He Y">Y He</name>
</author>
<author>
<name sortKey="Wu, L" uniqKey="Wu L">L Wu</name>
</author>
<author>
<name sortKey="Cai, D" uniqKey="Cai D">D Cai</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Mohan, Kv" uniqKey="Mohan K">KV Mohan</name>
</author>
<author>
<name sortKey="Rao, Ss" uniqKey="Rao S">SS Rao</name>
</author>
<author>
<name sortKey="Atreya, Cd" uniqKey="Atreya C">CD Atreya</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Li, Q" uniqKey="Li Q">Q Li</name>
</author>
<author>
<name sortKey="Zhao, Z" uniqKey="Zhao Z">Z Zhao</name>
</author>
<author>
<name sortKey="Zhou, D" uniqKey="Zhou D">D Zhou</name>
</author>
<author>
<name sortKey="Chen, Y" uniqKey="Chen Y">Y Chen</name>
</author>
<author>
<name sortKey="Hong, W" uniqKey="Hong W">W Hong</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zeng, Xc" uniqKey="Zeng X">XC Zeng</name>
</author>
<author>
<name sortKey="Wang, Sx" uniqKey="Wang S">SX Wang</name>
</author>
<author>
<name sortKey="Zhu, Y" uniqKey="Zhu Y">Y Zhu</name>
</author>
<author>
<name sortKey="Zhu, Sy" uniqKey="Zhu S">SY Zhu</name>
</author>
<author>
<name sortKey="Li, Wx" uniqKey="Li W">WX Li</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Rost, B" uniqKey="Rost B">B Rost</name>
</author>
<author>
<name sortKey="Sander, C" uniqKey="Sander C">C Sander</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Cory, A" uniqKey="Cory A">A Cory</name>
</author>
<author>
<name sortKey="Owen, T" uniqKey="Owen T">T Owen</name>
</author>
<author>
<name sortKey="Barltrop, J" uniqKey="Barltrop J">J Barltrop</name>
</author>
<author>
<name sortKey="Cory, J" uniqKey="Cory J">J Cory</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Choudhry, V" uniqKey="Choudhry V">V Choudhry</name>
</author>
<author>
<name sortKey="Zhang, My" uniqKey="Zhang M">MY Zhang</name>
</author>
<author>
<name sortKey="Sidorov, Ia" uniqKey="Sidorov I">IA Sidorov</name>
</author>
<author>
<name sortKey="Louis, Jm" uniqKey="Louis J">JM Louis</name>
</author>
<author>
<name sortKey="Harris, I" uniqKey="Harris I">I Harris</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Liu, R" uniqKey="Liu R">R Liu</name>
</author>
<author>
<name sortKey="Paxton, Wa" uniqKey="Paxton W">WA Paxton</name>
</author>
<author>
<name sortKey="Choe, S" uniqKey="Choe S">S Choe</name>
</author>
<author>
<name sortKey="Ceradini, D" uniqKey="Ceradini D">D Ceradini</name>
</author>
<author>
<name sortKey="Martin, Sr" uniqKey="Martin S">SR Martin</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Arien, Kk" uniqKey="Arien K">KK Arien</name>
</author>
<author>
<name sortKey="Jespers, V" uniqKey="Jespers V">V Jespers</name>
</author>
<author>
<name sortKey="Vanham, G" uniqKey="Vanham G">G Vanham</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Li, M" uniqKey="Li M">M Li</name>
</author>
<author>
<name sortKey="Gao, F" uniqKey="Gao F">F Gao</name>
</author>
<author>
<name sortKey="Mascola, Jr" uniqKey="Mascola J">JR Mascola</name>
</author>
<author>
<name sortKey="Stamatatos, L" uniqKey="Stamatatos L">L Stamatatos</name>
</author>
<author>
<name sortKey="Polonis, Vr" uniqKey="Polonis V">VR Polonis</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Cole, Al" uniqKey="Cole A">AL Cole</name>
</author>
<author>
<name sortKey="Yang, Oo" uniqKey="Yang O">OO Yang</name>
</author>
<author>
<name sortKey="Warren, Ad" uniqKey="Warren A">AD Warren</name>
</author>
<author>
<name sortKey="Waring, Aj" uniqKey="Waring A">AJ Waring</name>
</author>
<author>
<name sortKey="Lehrer, Ri" uniqKey="Lehrer R">RI Lehrer</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Brogden, Ka" uniqKey="Brogden K">KA Brogden</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Oren, Z" uniqKey="Oren Z">Z Oren</name>
</author>
<author>
<name sortKey="Shai, Y" uniqKey="Shai Y">Y Shai</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Ehrenstein, G" uniqKey="Ehrenstein G">G Ehrenstein</name>
</author>
<author>
<name sortKey="Lecar, H" uniqKey="Lecar H">H Lecar</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Wei, X" uniqKey="Wei X">X Wei</name>
</author>
<author>
<name sortKey="Decker, Jm" uniqKey="Decker J">JM Decker</name>
</author>
<author>
<name sortKey="Liu, H" uniqKey="Liu H">H Liu</name>
</author>
<author>
<name sortKey="Zhang, Z" uniqKey="Zhang Z">Z Zhang</name>
</author>
<author>
<name sortKey="Arani, Rb" uniqKey="Arani R">RB Arani</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Platt, Ej" uniqKey="Platt E">EJ Platt</name>
</author>
<author>
<name sortKey="Wehrly, K" uniqKey="Wehrly K">K Wehrly</name>
</author>
<author>
<name sortKey="Kuhmann, Se" uniqKey="Kuhmann S">SE Kuhmann</name>
</author>
<author>
<name sortKey="Chesebro, B" uniqKey="Chesebro B">B Chesebro</name>
</author>
<author>
<name sortKey="Kabat, D" uniqKey="Kabat D">D Kabat</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Wei, X" uniqKey="Wei X">X Wei</name>
</author>
<author>
<name sortKey="Decker, Jm" uniqKey="Decker J">JM Decker</name>
</author>
<author>
<name sortKey="Wang, S" uniqKey="Wang S">S Wang</name>
</author>
<author>
<name sortKey="Hui, H" uniqKey="Hui H">H Hui</name>
</author>
<author>
<name sortKey="Kappes, Jc" uniqKey="Kappes J">JC Kappes</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Adachi, A" uniqKey="Adachi A">A Adachi</name>
</author>
<author>
<name sortKey="Gendelman, He" uniqKey="Gendelman H">HE Gendelman</name>
</author>
<author>
<name sortKey="Koenig, S" uniqKey="Koenig S">S Koenig</name>
</author>
<author>
<name sortKey="Folks, T" uniqKey="Folks T">T Folks</name>
</author>
<author>
<name sortKey="Willey, R" uniqKey="Willey R">R Willey</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Hamilton, Ma" uniqKey="Hamilton M">MA Hamilton</name>
</author>
<author>
<name sortKey="Mckenzie Pollock, M" uniqKey="Mckenzie Pollock M">M McKenzie-Pollock</name>
</author>
<author>
<name sortKey="Heath, Me" uniqKey="Heath M">ME Heath</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Sakai, K" uniqKey="Sakai K">K Sakai</name>
</author>
<author>
<name sortKey="Ma, Xy" uniqKey="Ma X">XY Ma</name>
</author>
<author>
<name sortKey="Gordienko, I" uniqKey="Gordienko I">I Gordienko</name>
</author>
<author>
<name sortKey="Volsky, Dj" uniqKey="Volsky D">DJ Volsky</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Montefiori, Dc" uniqKey="Montefiori D">DC Montefiori</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Zhang, Y" uniqKey="Zhang Y">Y Zhang</name>
</author>
<author>
<name sortKey="Yang, J" uniqKey="Yang J">J Yang</name>
</author>
<author>
<name sortKey="Bao, R" uniqKey="Bao R">R Bao</name>
</author>
<author>
<name sortKey="Chen, Y" uniqKey="Chen Y">Y Chen</name>
</author>
<author>
<name sortKey="Zhou, D" uniqKey="Zhou D">D Zhou</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Abdiche, Y" uniqKey="Abdiche Y">Y Abdiche</name>
</author>
<author>
<name sortKey="Malashock, D" uniqKey="Malashock D">D Malashock</name>
</author>
<author>
<name sortKey="Pinkerton, A" uniqKey="Pinkerton A">A Pinkerton</name>
</author>
<author>
<name sortKey="Pons, J" uniqKey="Pons J">J Pons</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Do, T" uniqKey="Do T">T Do</name>
</author>
<author>
<name sortKey="Ho, F" uniqKey="Ho F">F Ho</name>
</author>
<author>
<name sortKey="Heidecker, B" uniqKey="Heidecker B">B Heidecker</name>
</author>
<author>
<name sortKey="Witte, K" uniqKey="Witte K">K Witte</name>
</author>
<author>
<name sortKey="Chang, L" uniqKey="Chang L">L Chang</name>
</author>
</analytic>
</biblStruct>
<biblStruct>
<analytic>
<author>
<name sortKey="Reed, Lj" uniqKey="Reed L">LJ Reed</name>
</author>
<author>
<name sortKey="Muench, H" uniqKey="Muench H">H Muench</name>
</author>
</analytic>
</biblStruct>
</listBibl>
</div1>
</back>
</TEI>
<pmc article-type="research-article">
<pmc-dir>properties open_access</pmc-dir>
<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">22536342</article-id>
<article-id pub-id-type="pmc">3334916</article-id>
<article-id pub-id-type="publisher-id">PONE-D-11-25439</article-id>
<article-id pub-id-type="doi">10.1371/journal.pone.0034947</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Research Article</subject>
</subj-group>
<subj-group subj-group-type="Discipline-v2">
<subject>Biology</subject>
<subj-group>
<subject>Biochemistry</subject>
<subj-group>
<subject>Neurochemistry</subject>
<subj-group>
<subject>Neurochemicals</subject>
<subj-group>
<subject>Neuropeptides</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
<subj-group>
<subject>Microbiology</subject>
<subj-group>
<subject>Virology</subject>
<subj-group>
<subject>Viral classification</subject>
<subj-group>
<subject>Retroviruses</subject>
<subject>RNA viruses</subject>
</subj-group>
</subj-group>
<subj-group>
<subject>Antivirals</subject>
</subj-group>
</subj-group>
</subj-group>
<subj-group>
<subject>Proteomics</subject>
<subj-group>
<subject>Synthetic Peptide</subject>
</subj-group>
</subj-group>
<subj-group>
<subject>Toxicology</subject>
<subj-group>
<subject>Toxin Binding</subject>
</subj-group>
</subj-group>
</subj-group>
<subj-group subj-group-type="Discipline-v2">
<subject>Medicine</subject>
<subj-group>
<subject>Drugs and Devices</subject>
<subj-group>
<subject>Drug Research and Development</subject>
</subj-group>
</subj-group>
<subj-group>
<subject>Infectious Diseases</subject>
<subj-group>
<subject>Viral Diseases</subject>
<subj-group>
<subject>HIV</subject>
</subj-group>
</subj-group>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7</article-title>
<alt-title alt-title-type="running-head">Anti-HIV-1 Activity of a Venom Peptide Kn2-7</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Chen</surname>
<given-names>Yaoqing</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cao</surname>
<given-names>Luyang</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhong</surname>
<given-names>Maohua</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhang</surname>
<given-names>Yan</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Han</surname>
<given-names>Chen</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Li</surname>
<given-names>Qiaoli</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yang</surname>
<given-names>Jingyi</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhou</surname>
<given-names>Dihan</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Shi</surname>
<given-names>Wei</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>He</surname>
<given-names>Benxia</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Liu</surname>
<given-names>Fang</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yu</surname>
<given-names>Jie</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Sun</surname>
<given-names>Ying</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cao</surname>
<given-names>Yuan</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Li</surname>
<given-names>Yaoming</given-names>
</name>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Li</surname>
<given-names>Wenxin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Guo</surname>
<given-names>Deying</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cao</surname>
<given-names>Zhijian</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="corresp" rid="cor1">
<sup>*</sup>
</xref>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yan</surname>
<given-names>Huimin</given-names>
</name>
<xref ref-type="aff" rid="aff1">
<sup>1</sup>
</xref>
<xref ref-type="aff" rid="aff2">
<sup>2</sup>
</xref>
<xref ref-type="corresp" rid="cor1">
<sup>*</sup>
</xref>
</contrib>
</contrib-group>
<aff id="aff1">
<label>1</label>
<addr-line>State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, People's Republic of China</addr-line>
</aff>
<aff id="aff2">
<label>2</label>
<addr-line>State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, People's Republic of China</addr-line>
</aff>
<contrib-group>
<contrib contrib-type="editor">
<name>
<surname>Cole</surname>
<given-names>Alexander M.</given-names>
</name>
<role>Editor</role>
<xref ref-type="aff" rid="edit1"></xref>
</contrib>
</contrib-group>
<aff id="edit1">University of Central Florida College of Medicine, United States of America</aff>
<author-notes>
<corresp id="cor1">* E-mail:
<email>zjcao@whu.edu.cn</email>
(ZC);
<email>hmyan@wh.iov.cn</email>
(HY)</corresp>
<fn fn-type="con">
<p>Conceived and designed the experiments: Y. Chen HY ZC. Performed the experiments: Y. Chen LC MZ YZ CH QL J. Yang DZ WS BH FL J. Yu YS Y. Cao YL. Analyzed the data: Y. Chen HY ZC MZ. Contributed reagents/materials/analysis tools: Y. Chen HY ZC WL DL. Wrote the paper: Y. Chen HY.</p>
</fn>
</author-notes>
<pub-date pub-type="collection">
<year>2012</year>
</pub-date>
<pub-date pub-type="epub">
<day>19</day>
<month>4</month>
<year>2012</year>
</pub-date>
<volume>7</volume>
<issue>4</issue>
<elocation-id>e34947</elocation-id>
<history>
<date date-type="received">
<day>17</day>
<month>12</month>
<year>2011</year>
</date>
<date date-type="accepted">
<day>7</day>
<month>3</month>
<year>2012</year>
</date>
</history>
<permissions>
<copyright-statement>Chen et al.</copyright-statement>
<copyright-year>2012</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>For over 30 years, HIV/AIDS has wreaked havoc in the world. In the absence of an effective vaccine for HIV, development of new anti-HIV agents is urgently needed. We previously identified the antiviral activities of the scorpion-venom-peptide-derived mucroporin-M1 for three RNA viruses (measles viruses, SARS-CoV, and H5N1). In this investigation, a panel of scorpion venom peptides and their derivatives were designed and chosen for assessment of their anti-HIV activities. A new scorpion venom peptide derivative Kn2-7 was identified as the most potent anti-HIV-1 peptide by screening assays with an EC
<sub>50</sub>
value of 2.76 µg/ml (1.65 µM) and showed low cytotoxicity to host cells with a selective index (SI) of 13.93. Kn2-7 could inhibit all members of a standard reference panel of HIV-1 subtype B pseudotyped virus (PV) with CCR5-tropic and CXCR4-tropic NL4-3 PV strain. Furthermore, it also inhibited a CXCR4-tropic replication-competent strain of HIV-1 subtype B virus. Binding assay of Kn2-7 to HIV-1 PV by Octet Red system suggested the anti-HIV-1 activity was correlated with a direct interaction between Kn2-7 and HIV-1 envelope. These results demonstrated that peptide Kn2-7 could inhibit HIV-1 by direct interaction with viral particle and may become a promising candidate compound for further development of microbicide against HIV-1.</p>
</abstract>
<counts>
<page-count count="9"></page-count>
</counts>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Introduction</title>
<p>Nearly 34 million people were living with human immunodeficiency virus (HIV) at the end of 2010 in the globe
<xref rid="pone.0034947-UNAIDS1" ref-type="bibr">[1]</xref>
and half of them were women. Unfortunately, there are still no effective vaccine or other countermeasure to eliminate HIV transmission
<xref rid="pone.0034947-Titti1" ref-type="bibr">[2]</xref>
. The Merck STEP
<xref rid="pone.0034947-Buchbinder1" ref-type="bibr">[3]</xref>
and the Thai RV144 HIV vaccine
<xref rid="pone.0034947-RerksNgarm1" ref-type="bibr">[4]</xref>
trials confirmed that we still have a long way to go before developing a prophylactic HIV vaccine. Meanwhile, HIV virus spreads fast and the HIV/AIDS pandemic still stands as a serious public health problem worldwide
<xref rid="pone.0034947-UNAIDS1" ref-type="bibr">[1]</xref>
. Current situation clearly indicates the necessity of developing new anti-HIV agents which can be used for prevention of HIV/AIDS dissemination.</p>
<p>HIV-1 initially infects T cells through CD4 receptor
<xref rid="pone.0034947-Dalgleish1" ref-type="bibr">[5]</xref>
and either of the two chemokine co-receptors CXCR4 (X4) or CCR5 (R5) (or both)
<xref rid="pone.0034947-Deng1" ref-type="bibr">[6]</xref>
,
<xref rid="pone.0034947-Doranz1" ref-type="bibr">[7]</xref>
,
<xref rid="pone.0034947-Feng1" ref-type="bibr">[8]</xref>
. It has been suggested that R5 is the major co-receptor involved in sexual transmission of HIV-1
<xref rid="pone.0034947-Deng1" ref-type="bibr">[6]</xref>
. Some microbicides tested can indeed inhibit infection by X4-tropic HIV-1 but insufficiently inhibit R5-tropic HIV-1 to the same extent
<xref rid="pone.0034947-Neurath1" ref-type="bibr">[9]</xref>
,
<xref rid="pone.0034947-Telwatte1" ref-type="bibr">[10]</xref>
. SPL7013 is a dendrimer which had HIV-1 virucidal activity against X4 and R5X4 HIV-1 strains but not R5 virus strains
<xref rid="pone.0034947-Telwatte1" ref-type="bibr">[10]</xref>
. Effectiveness, safety and broad spectrum are very important to an anti-HIV microbicide. The chemokine analogue PSC-RANTES had strong inhibition activity at R5-tropic HIV strains but might induce local inflammation
<xref rid="pone.0034947-Torre1" ref-type="bibr">[11]</xref>
. The sulfonated polymer PRO2000 is safe but cannot provide efficacious protection against sexual HIV transmission
<xref rid="pone.0034947-McCormack1" ref-type="bibr">[12]</xref>
. Polyanioun had been suggested as potential microbicides
<xref rid="pone.0034947-PearcePratt1" ref-type="bibr">[13]</xref>
,
<xref rid="pone.0034947-Tao1" ref-type="bibr">[14]</xref>
,
<xref rid="pone.0034947-Vzorov1" ref-type="bibr">[15]</xref>
. Unfortunately, a recent phase III trial of cellulose sulfate was terminated because of its increasing rate of HIV infection than women using a placebo
<xref rid="pone.0034947-Tao2" ref-type="bibr">[16]</xref>
. Therefore, more sources of antiviral reagent to prevent HIV-1 transmission are needed for efficient protection of our body from HIV infection.</p>
<p>Natural antimicrobial peptides (AMPs) are widely expressed and rapidly induced on epithelial surfaces to repel invasion from diverse infectious agents including bacteria, viruses, fungi and parasites
<xref rid="pone.0034947-Boman1" ref-type="bibr">[17]</xref>
,
<xref rid="pone.0034947-Hancock1" ref-type="bibr">[18]</xref>
. So far, more than 1700 AMPs of different origins have been identified or predicted
<xref rid="pone.0034947-Fjell1" ref-type="bibr">[19]</xref>
. Most AMPs maintain certain common features such as being small (10–50 amino acids), containing positive charge of 2 to 9 and an amphipathic structure
<xref rid="pone.0034947-Jenssen1" ref-type="bibr">[20]</xref>
,
<xref rid="pone.0034947-Martin1" ref-type="bibr">[21]</xref>
,
<xref rid="pone.0034947-Wang1" ref-type="bibr">[22]</xref>
,
<xref rid="pone.0034947-Zasloff1" ref-type="bibr">[23]</xref>
. Scorpion venom is a cocktail of peptides and proteins with diverse bioactivities, which represent a tremendous potential for use in drug design and development
<xref rid="pone.0034947-Ma1" ref-type="bibr">[24]</xref>
,
<xref rid="pone.0034947-Possani1" ref-type="bibr">[25]</xref>
,
<xref rid="pone.0034947-Zhijian1" ref-type="bibr">[26]</xref>
. AMPs from scorpion venom such as hadrurin
<xref rid="pone.0034947-TorresLarios1" ref-type="bibr">[27]</xref>
, scorpine
<xref rid="pone.0034947-Conde1" ref-type="bibr">[28]</xref>
, opistoporins, parabutoporin
<xref rid="pone.0034947-Moerman1" ref-type="bibr">[29]</xref>
, ISCTs
<xref rid="pone.0034947-Dai1" ref-type="bibr">[30]</xref>
and mucroporin
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
are paid more and more attention due to their biological activity
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
,
<xref rid="pone.0034947-Zhao1" ref-type="bibr">[32]</xref>
. Some of these molecules have activities against viral pathogens such as junin virus, herpes simplex virus, adenovirus, rotavirus, vaccinia virus, HCV and measles virus
<xref rid="pone.0034947-AlbiolMatanic1" ref-type="bibr">[33]</xref>
,
<xref rid="pone.0034947-Sun1" ref-type="bibr">[34]</xref>
,
<xref rid="pone.0034947-CarrielGomes1" ref-type="bibr">[35]</xref>
,
<xref rid="pone.0034947-Yan1" ref-type="bibr">[36]</xref>
,
<xref rid="pone.0034947-Mohan1" ref-type="bibr">[37]</xref>
.</p>
<p>We had reported previously that mucroporin cloned from the venom of the scorpion
<italic>Lychas mucronatus</italic>
and its optimized derivative mucroporin-M1 showed antimicrobial activity on bacteria and measles virus
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
,
<xref rid="pone.0034947-Li1" ref-type="bibr">[38]</xref>
. Another scorpion peptide BmKn2 which was cloned from the venom of
<italic>Mesobuthus martensii Karsch</italic>
had also showed a strong antimicrobial activity against bacteria
<xref rid="pone.0034947-Zeng1" ref-type="bibr">[39]</xref>
. As a further development of BmKn2 peptide, a new peptide named Kn2-7 was designed by substituting Glycine Alanine and Serine with Lysine or Arginine (G3K, A4R, and S10R) to enhance the net positive charge and α-helix structure. In this study, five scorpion venom peptides and their derivatives were screened for their anti-HIV-1 activities, and the results showed that three of them (mocroporin-M1, BmKn2 and Kn2-7) exhibited potent anti-HIV-1 activity, in which Kn2-7 showed the highest level of anti-HIV-1 activity. So, we speculated that Kn2-7 might be a potential lead peptide and was tested for antiviral effects by using a standard reference panel of subtype B HIV-1 pseudotyped virus (PV) and a replication-competent strain of HIV-1 virus. The binding assay of Kn2-7 to HIV-1 PV was performed for exploring the related antiviral mechanism by Octet Red system.</p>
</sec>
<sec id="s2">
<title>Results</title>
<sec id="s2a">
<title>Molecular design, synthesis, purification and characterization of the peptides</title>
<p>Kn2-7 (FIKRIARLLRKIF) was designed based on the peptide sequence of BmKn2 (FIGAIARLLSKIF) to enhance the net positive charge of the hydrophilic side. Kn2-7 peptide contains five positive charges while BmKn2 carries only two positive charges (
<xref ref-type="table" rid="pone-0034947-t001">Table 1</xref>
). Glycine, Alanine and Serine residues of BmKn2 were replaced with Lysine or Arginine (G3K, A4R, and S10R). The secondary structures were predicted by PHD method
<xref rid="pone.0034947-Rost1" ref-type="bibr">[40]</xref>
on the website (
<ext-link ext-link-type="uri" xlink:href="http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html">http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html</ext-link>
), suggesting that Kn2-7 has higher α-helix percentage than BmKn2, which are 84.62% and 76.92%, respectively. Information of mucroporin, mucroporin-M1 and mucroporin-S1 were describe by our previous study
<xref rid="pone.0034947-Li1" ref-type="bibr">[38]</xref>
.</p>
<table-wrap id="pone-0034947-t001" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.t001</object-id>
<label>Table 1</label>
<caption>
<title>The charge, first and predict secondary structure of scorpion venom peptides.</title>
</caption>
<alternatives>
<graphic id="pone-0034947-t001-1" xlink:href="pone.0034947.t001"></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>
<col align="center" span="1"></col>
<col align="center" span="1"></col>
</colgroup>
<thead>
<tr>
<td align="left" rowspan="1" colspan="1">Peptide</td>
<td align="left" rowspan="1" colspan="1">Amino Acid Sequence</td>
<td align="left" rowspan="1" colspan="1">Charge</td>
<td align="left" rowspan="1" colspan="1">Cationic(%)</td>
<td align="left" rowspan="1" colspan="1">Structure Prediction</td>
</tr>
</thead>
<tbody>
<tr>
<td align="left" rowspan="1" colspan="1">BmKn2</td>
<td align="left" rowspan="1" colspan="1">FIGAIARLLSKIF</td>
<td align="left" rowspan="1" colspan="1">2</td>
<td align="left" rowspan="1" colspan="1">15.38</td>
<td align="left" rowspan="1" colspan="1">chhhhhhhhhhcc</td>
</tr>
<tr>
<td align="left" rowspan="1" colspan="1">Kn2-7</td>
<td align="left" rowspan="1" colspan="1">FIKRIARLLRKIF</td>
<td align="left" rowspan="1" colspan="1">5</td>
<td align="left" rowspan="1" colspan="1">38.46</td>
<td align="left" rowspan="1" colspan="1">chhhhhhhhhhhc</td>
</tr>
<tr>
<td align="left" rowspan="1" colspan="1">mucroporin</td>
<td align="left" rowspan="1" colspan="1">LFGLIPSLIGGLVSAFK</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">5.88</td>
<td align="left" rowspan="1" colspan="1">cccchhhhhhhhhhhhc</td>
</tr>
<tr>
<td align="left" rowspan="1" colspan="1">mucroporin-M1</td>
<td align="left" rowspan="1" colspan="1">LFRLIKSLIKRLVSAFK</td>
<td align="left" rowspan="1" colspan="1">5</td>
<td align="left" rowspan="1" colspan="1">29.41</td>
<td align="left" rowspan="1" colspan="1">chhhhhhhhhhhhhhhc</td>
</tr>
<tr>
<td align="left" rowspan="1" colspan="1">mucropotin-S1</td>
<td align="left" rowspan="1" colspan="1">SLIGGLVSAFK</td>
<td align="left" rowspan="1" colspan="1">1</td>
<td align="left" rowspan="1" colspan="1">9.09</td>
<td align="left" rowspan="1" colspan="1">ccccccceccc</td>
</tr>
</tbody>
</table>
</alternatives>
<table-wrap-foot>
<fn id="nt101">
<label></label>
<p>h: alpha helix, c: random coil, e: extended strand.</p>
</fn>
</table-wrap-foot>
</table-wrap>
<p>The five peptides were successfully synthesized on an Abimed AMS 422 synthesizer by Fmoc solid-phase peptide synthesis. The purities of all five peptides showed reliable quality of more than 95%. The molecule weights measured by mass spectrometry (MS) were completely matched with the calculated molecule weights of these peptides.</p>
</sec>
<sec id="s2b">
<title>Kn2-7 was identified as the most effective scorpion venom peptide against HIV-1 PV by screening assay</title>
<p>To assess anti-HIV activities of scorpion venom peptides, pseudotyped virus-based assays with TZM-bl system were adopted for the screening of anti-HIV-1 effect of different peptides. The results showed that mucroporin-M1, BmKn2 and Kn2-7 could significantly decrease infectivity of an HIV-1 clade B b12-resistant pseudotyped virus CAAN5342 compare to the mucroporin, mucroporin-S1, BSA and no-peptide virus mock control, in which peptide Kn2-7 showed the highest level of anti-HIV-1 activity and almost completely inhibited viral infection (
<xref ref-type="fig" rid="pone-0034947-g001">Fig. 1</xref>
). This gave us a hint that peptide Kn2-7 is the most potential for developing anti-HIV agent. Therefore, Kn2-7 peptide was chosen for further study to clarify its anti-HIV effect. Mucroporin-S1 was chosen as a negative control for the further study because it shows no anti-bacterial and antiviral activity in our previous research
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
but has similar number of amino acid with Kn2-7 peptide.</p>
<fig id="pone-0034947-g001" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.g001</object-id>
<label>Figure 1</label>
<caption>
<title>Kn2-7 was the most effective peptide against HIV-1 PV by screening assay.</title>
<p>The antiviral effect of the peptides against HIV-1 PV was performed by TZM-bl detection systems as described in
<xref ref-type="sec" rid="s4">Materials and Methods</xref>
. Briefly, 10 µg/ml of peptides were incubated with 200 TCID
<sub>50</sub>
of HIV-1 PV for 1 h at 37°C and then added to TZM-bl cells. The luciferase activity was measured in a 96-well black solid plate by Turner Biosystems Modulus II microplate reader 48 h later. Significant reduction in the RLU value of groups treated by different peptides compared to virus group in which cells were exposed to BSA are denoted by a * (p<0.05), ** (p<0.01), and *** (p<0.001). Data are expressed as mean ± SD of three replicate samples.</p>
</caption>
<graphic xlink:href="pone.0034947.g001"></graphic>
</fig>
</sec>
<sec id="s2c">
<title>Kn2-7 acted sensitively and rapidly against HIV-1 PV</title>
<p>To confirm whether anti-HIV-1 activity of Kn2-7 was dependent on concentration, several concentrations of Kn2-7 anti-HIV-1 activity were measured by pseudotyped virus-based assays with TZM-bl detection system. As shown in
<xref ref-type="fig" rid="pone-0034947-g002">Fig. 2A</xref>
, the antiviral-activity of Kn2-7 against HIV-1 PV was in a dose-dependent manner and an inhibition of 40.1% was achieved at a concentration of 2 µg/ml and its EC
<sub>50</sub>
value was calculated as 2.76 µg/ml. Up to 98.7% inhibition could be achieved when concentration rose to 16 µg/ml.</p>
<fig id="pone-0034947-g002" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.g002</object-id>
<label>Figure 2</label>
<caption>
<title>Dose- and time-dependent effects of Kn2-7 peptide against HIV-1 PV.</title>
<p>(A) For dose-dependent effect experiments, serial dilution from 0.25 µg/ml to 30 µg/ml of Kn2-7 and mucroporin-S1 were incubated with 200 TCID
<sub>50</sub>
HIV-1 PV for 1 h at 37°C and then added to TZM-bl cells, respectively. (B) For time-dependent effect experiments, 3 µg/ml of Kn2-7 and mucroporin-S1 were incubated with 200 TCID
<sub>50</sub>
of HIV-1 PV for 3 min, 10 min, 30 min, 60 min and 90 min, respectively. The viruses treated without any peptide were used as the control. Data are presented as mean ± SD of three replicate samples for one representative experiment.</p>
</caption>
<graphic xlink:href="pone.0034947.g002"></graphic>
</fig>
<p>To determine the effective time needed for Kn2-7 to act on HIV-1 PV, 3 µg/ml peptide was incubated with 200 TCID
<sub>50</sub>
of HIV-1 PV for 3 min, 10 min, 30 min, 60 min and 90 min, respectively. As shown in
<xref ref-type="fig" rid="pone-0034947-g002">Fig. 2B</xref>
, from 3 min to 60 min, the longer time treatment, the higher inhibition was achieved but no significant difference was observed between 60 min treatment group and 90 min treatment group. The inhibition could be up to 42.3% when Kn2-7 was incubated with HIV-1 PV for only 3 min. Therefore, the time needed for Kn2-7 to act on HIV-1 PV was very short and the activity peaked at the time point of 60 min.</p>
<p>According to this result, the concentration of 3 µg/ml (nearby the EC
<sub>50</sub>
value) and 60 min treatment were chosen for further assay of Kn2-7 peptide on anti-HIV-1 PV activity in the following experiments.</p>
</sec>
<sec id="s2d">
<title>The inhibitory effect of Kn2-7 was selective to HIV-1 PV</title>
<p>To distinguish selective antiviral activity from nonselective cytotoxicity, we tested the cytotoxicity of the peptides on TZM-bl cells. TZM-bl cells were treated with several concentrations of Kn2-7 peptide for 24 hours and the viability of TZM-bl cells was measured using the MTS assay
<xref rid="pone.0034947-Cory1" ref-type="bibr">[41]</xref>
. The absorbance was measured at 490 nm using an ELISA plate reader and the CC
<sub>50</sub>
value were calculated by SPSS. As shown in
<xref ref-type="table" rid="pone-0034947-t002">Table 2</xref>
, CC
<sub>50</sub>
of Kn2-7 on TZM-bl cells was 38.46 µg/ml, which is 13.93 times over the EC
<sub>50</sub>
value. Therefore, the antiviral activity of the peptide Kn2-7 against HIV-1 does not result from the cytotoxicity of the peptide on TZM-bl cells.</p>
<table-wrap id="pone-0034947-t002" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.t002</object-id>
<label>Table 2</label>
<caption>
<title>Anti-HIV-1 PV activities and cytotoxicity of Kn2-7 peptide on TZM-bl cells.</title>
</caption>
<alternatives>
<graphic id="pone-0034947-t002-2" xlink:href="pone.0034947.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>
<col align="center" span="1"></col>
</colgroup>
<thead>
<tr>
<td align="left" rowspan="1" colspan="1"></td>
<td align="left" rowspan="1" colspan="1">EC
<sub>50</sub>
<xref ref-type="table-fn" rid="nt103">a</xref>
(µg/ml)</td>
<td align="left" rowspan="1" colspan="1">CC
<sub>50</sub>
<xref ref-type="table-fn" rid="nt103">a</xref>
(µg/ml)</td>
<td align="left" rowspan="1" colspan="1">SI</td>
</tr>
</thead>
<tbody>
<tr>
<td align="left" rowspan="1" colspan="1">Kn2-7</td>
<td align="left" rowspan="1" colspan="1">2.76</td>
<td align="left" rowspan="1" colspan="1">38.46</td>
<td align="left" rowspan="1" colspan="1">13.93</td>
</tr>
<tr>
<td align="left" rowspan="1" colspan="1">mucroporin-S1</td>
<td align="left" rowspan="1" colspan="1">I</td>
<td align="left" rowspan="1" colspan="1">>100</td>
<td align="left" rowspan="1" colspan="1"></td>
</tr>
</tbody>
</table>
</alternatives>
<table-wrap-foot>
<fn id="nt102">
<label></label>
<p>EC
<sub>50</sub>
: peptide concentration required to reduce virus infection by 50%; CC
<sub>50</sub>
: peptide concentration required to reduce cell viability by 50%; SI (selectivity index) = CC
<sub>50</sub>
/EC
<sub>50</sub>
; I: inactive.</p>
</fn>
<fn id="nt103">
<label>a</label>
<p>All the data represent mean values for at least three independent experiments.</p>
</fn>
</table-wrap-foot>
</table-wrap>
</sec>
<sec id="s2e">
<title>Kn2-7 directly targeted HIV-1 PV</title>
<p>In order to gain some insight into the mechanism of anti-HIV-1 activity of Kn2-7, the interaction time and order among Kn2-7, HIV-1 PV and TZM-b1 cells were varied by four different ways described in
<xref ref-type="sec" rid="s4">Materials and Methods</xref>
. Mucroporin-S1 were prepared in the same manner and set as mock group. As shown in
<xref ref-type="fig" rid="pone-0034947-g003">Fig. 3</xref>
, Kn2-7 incubating with HIV-1 PV for 1 h before inoculating to TZM-b1 cells (peptide/virus pre-incubation group) showed prominent inhibitory effect, agreed with the results above. Although in a low degree, Kn2-7 and HIV-1 PV adding to cells simultaneously but without pre-incubation (peptide/virus mixture group) also showed a significant inhibition. However, neither the peptide pretreated group, in which TZM-bl cells were pre-treated with Kn2-7 for 1 h, nor the infection first group, in which TZM-bl cells were infected with HIV-1 PV first and then treated with Kn2-7 showed any antiviral effect (
<xref ref-type="fig" rid="pone-0034947-g003">Fig. 3</xref>
).</p>
<fig id="pone-0034947-g003" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.g003</object-id>
<label>Figure 3</label>
<caption>
<title>Directly inactivated mechanism of Kn2-7 peptide to HIV-1 PV.</title>
<p>Four action modes of treatment between peptides with HIV-1 PV were carried out. (1) Peptide pretreated group, TZM-bl cells were pretreated with 10 µg/ml peptide at 37°C for 1 h and then treated with HIV-1 PV. (2) Infection first group, cells were infected with HIV-1 PV first by inoculating at 37°C for 1 h and then treated with 10 µg/ml peptide. (3) Peptide/virus mixture group, cells were treated with mixture of HIV-1 PV and 10 µg/ml peptides immediately after mix but without inoculation. (4) Peptide/virus pre-incubation group, cells were infected with mixture of HIV-1 PV and 10 µg/ml peptides pre-incubating at 37°C for 1 h. The inhibitory effects of different groups were determined in TZM-bl detection system. Significant reduction in the RLU value of Kn2-7 treatment group compared with that of mucoporin-S1 treatment group are denoted by a * (p<0.05), ** (p<0.01), and *** (p<0.001).</p>
</caption>
<graphic xlink:href="pone.0034947.g003"></graphic>
</fig>
<p>Based on the results of four treatment modes of Kn2-7, we assumed that the anti-HIV activity of Kn2-7 attributed to its interaction with HIV-1 viral particle directly. To confirm the hypothesis, a binding assay based on Biolayer Interferometry was performed to study the interaction between Kn2-7 and HIV-1 PV. HIV-1 PV was loaded to APS biosensor and the sensor tip was transferred to Kn2-7 or mucroporin-S1 with concentration of 10 µg/ml or 40 µg/ml. Expectedly, we observed rapid direct binding between HIV-1 PV and Kn2-7 and the binding capacity of 40 µg/ml Kn2-7 was stronger than that of 10 µg/ml. Mucroporin-S1 showed no significant binding with HIV-1 PV (
<xref ref-type="fig" rid="pone-0034947-g004">Fig. 4</xref>
). Therefore, Kn2-7 peptide executes anti-HIV-1 activity by direct targeting HIV-1 particle.</p>
<fig id="pone-0034947-g004" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.g004</object-id>
<label>Figure 4</label>
<caption>
<title>Direct binding of peptide Kn2-7 to HIV-1 PV.</title>
<p>HIV-1 PV was bound and analyzed, in parallel, by five Octet APS sensors. APS sensors were pre-wet for 120 s, and then coupled with 200 µl HIV-1 PV for 300 s. After clearance of unabsorbed PV, APS sensors were associated with 10 µg/ml and 40 µg/ml of Kn2-7 or mucroporin-S1 for 900 s (Measured in nanometers).</p>
</caption>
<graphic xlink:href="pone.0034947.g004"></graphic>
</fig>
</sec>
<sec id="s2f">
<title>Kn2-7 had broad-spectrum of antiviral activity against HIV-1 PV</title>
<p>To define whether the anti-viral effect of Kn2-7 peptide against HIV-1 PV is broad-spectrum or not, the Kn2-7 (3 µg/ml and 10 µg/ml) against a standard reference panel of HIV-1 subtype B isolates were measured using TZM-bl detection system and mucroporin-S1 was used as the negative control. Inhibitions were calculated compared to the HIV-1 PV control group. 3 µg/ml and 10 µg/ml of Kn2-7 peptide showed comparable antiviral potencies against 13 strains of HIV-1 subtype B PV including 12 R5-tropic (6535, QH0692, SC422661, PVO, TRO, AC10, PHPA4259, THRO4156, REJO4551, TRJO4551, WITO4160, CAAN5342) and 1 X4-tropic (NL4-3). The negative control of mucroporin-S1 show no inhibitory effect at corresponding concentrations (3 µg/ml of mucroporin-S1 data not shown) (
<xref ref-type="fig" rid="pone-0034947-g005">Fig. 5</xref>
). Consequently, Kn2-7 peptide had a broad-spectrum antiviral activity against HIV-1 PV regardless of tropism.</p>
<fig id="pone-0034947-g005" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.g005</object-id>
<label>Figure 5</label>
<caption>
<title>Broad-spectrum antiviral activity of Kn2-7 against 13 strains of HIV-1 subtype B PV.</title>
<p>The inhibitory effect of the peptide was determined in TZM-bl luciferase detection system. Briefly, 3 µg/ml and 10 µg/ml of Kn2-7 was incubated with different strains of HIV-1 PV (200 TCID
<sub>50</sub>
) for 1 h at 37°C and then added to TZM-bl cells. The group treated with mucroporin-S1 and without any peptide in the same manner served as the negative and background control, respectively.</p>
</caption>
<graphic xlink:href="pone.0034947.g005"></graphic>
</fig>
</sec>
<sec id="s2g">
<title>Kn2-7 had anti-viral effect against replication-competent HIV-1 virus</title>
<p>To confirm the potent inhibition of Kn2-7 to HIV-1, a replication-competent HIV-1 virus was also adopted for the anti-HIV activity assay. Serial dilutions of Kn2-7 or mucroporin-S1 were incubated with 20 µl X4-tropic proviral DNA (pKS242) derived HIV-1 virus (15 ng p24) for 1 h at 37°C and then infected CEM×174 cells for 17 days. Supernatant and cell samples were collected at day 7, 12, 13, 14, 15, 16 and 17. P24 antigen expression was analyzed by ELISA and infectivity was further detected by TZM-bl detection system. 10 µg/ml and 15 µg/ml of Kn2-7 suppressed p24 expression completely from 7
<sup>th</sup>
day to 17
<sup>th</sup>
day in infected cell (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6A</xref>
) as well as in supernatant (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6B</xref>
). 5 µg/ml of Kn2-7 suppressed p24 expression in cell sample in early stage (7
<sup>th</sup>
day to 14
<sup>th</sup>
day), but not in the later time points, with the value of p24 rose at 15
<sup>th</sup>
day, till to 17
<sup>th</sup>
day (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6A</xref>
). The p24 antigen level in supernatants showed the same trend as that of cell samples by 5 µg/ml of Kn2-7, but with a delayed reaction (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6B</xref>
). The negative control peptide, mucroporin-S1, did not show any inhibitory effect. To further determine antiviral activity of Kn2-7, the above samples was added to TZM-bl cells for detection of their infectivity. Luciferase activities of infected TZM-bl cells were measured after 48 h incubation. Similar trends of inhibitory effect were observed (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6C</xref>
). However, 15 µg/ml of Kn2-7 inhibited virus replication completely but 10 µg/ml of Kn2-7 did not (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6C</xref>
) while both 10 and 15 µg/ml of Kn2-7 showed completed suppression by p24 assay (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6A, 6B</xref>
). The results further demonstrated that the Kn2-7 can inhibit HIV infectivity and its consequent replication. To rule out potential inhibitions attributable to cytotoxicity to host cells, the cytotoxicity of the peptides on CEM×174 cells were measured by using the MTS assay from 5 µg/ml to 100 µg/ml and the CC
<sub>50</sub>
value of Kn2-7 on CEM×174 cells was 43.18 µg/ml, which shown that the antiviral activity of Kn2-7 against HIV-1 does not result from the cytotoxicity of the peptide on CEM×174 cells.</p>
<fig id="pone-0034947-g006" position="float">
<object-id pub-id-type="doi">10.1371/journal.pone.0034947.g006</object-id>
<label>Figure 6</label>
<caption>
<title>Antiviral activity of Kn2-7 against a replication-competent HIV-1 virus.</title>
<p>Three dilutions of Kn2-7 or mucroporin-S1 were incubated with HIV-1 virus for 1 h at 37°C, and then were added to CEM×174 cells in 12-well plates in duplicates for 17 days. Samples were collected at day 7, 12, 13, 14, 15, 16, and 17 after infection and p24 protein concentration of the cells and supernatants were detected by ELISA. The infectivity was measured by TZM-bl detection luciferase assay. (A) CEM×174 cells samples were measured for p24 protein by ELSIA. (B) CEM×174 supernatant samples were measured for p24 protein by ELSIA. (C) Samples were detected for infectivity by TZM-bl detection system.</p>
</caption>
<graphic xlink:href="pone.0034947.g006"></graphic>
</fig>
<p>In brief, the scorpion venom peptide derivative Kn2-7 not only significantly inhibits HIV-1 PV, but also has antiviral effect against replication-competent HIV-1 virus with broad-spectrum by direct interaction with viral particle and may become a promising candidate compound for further development of anti-HIV-1 agent.</p>
</sec>
</sec>
<sec id="s3">
<title>Discussion</title>
<p>For three decades, HIV/AIDS has wreaked havoc on the population of the world. Although the number of new infections has been falling, levels of new infections overall are still high. The vast majority of people newly infected with HIV are infected during unprotected sexual intercourse
<xref rid="pone.0034947-Choudhry1" ref-type="bibr">[42]</xref>
. The quest for effective microbicide and drug to prevent initial HIV-1 infection and treat AIDS patients is essential for countering the spread of the diseases worldwide when there is no effective prophylactic HIV vaccine available at present. Tremendous efforts have been directed towards development of effective anti-HIV-1 microbicides, which are defined as products that can be applied topically for the prevention of HIV-1 and other sexually transmitted infections by creating chemical, biological and-/or physical barriers. An effective microbicide against HIV should be efficient with anti-HIV-1 activity, safe with low cytotoxicity to host cells and broad-spectrum against both R5-tropic and X4-tropic HIV-1
<xref rid="pone.0034947-Deng1" ref-type="bibr">[6]</xref>
,
<xref rid="pone.0034947-Liu1" ref-type="bibr">[43]</xref>
,
<xref rid="pone.0034947-Arien1" ref-type="bibr">[44]</xref>
. For satisfying all the criteria above, more resources of possible anti-HIV-1 reagents should be explored.</p>
<p>In previous studies, we isolated several kinds of cationic antimicrobial peptides from the cDNA libraries of the venomous gland of scorpions and identified their anti-bacterial activity against pathogens
<xref rid="pone.0034947-Ma1" ref-type="bibr">[24]</xref>
,
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
. Recently we reported that mucroporin-M1, one kind of modified scorpion venom peptide, can inhibit some RNA viruses such as measles virus, SARS-CoV and influenza H5N1 viruses
<xref rid="pone.0034947-Li1" ref-type="bibr">[38]</xref>
. In this study, we screened the anti-HIV-1 efficacy of five scorpion venom peptides and their derivatives. The results showed that Kn2-7, BmKn2 and mucorporin-M1 exhibited varying degrees of antiviral effects and among them Kn2-7 peptide was the most effective with an EC
<sub>50</sub>
value of 2.76 µg/ml (
<xref ref-type="fig" rid="pone-0034947-g001">Fig. 1</xref>
). According to the most potent anti-HIV-1 activity and low toxicity on TZM-bl cells and the mouse skin model (unpublished data), Kn2-7 was chosen for further investigation to clarify its anti-HIV-1 effect. Kn2-7 was modified from BmKn2 by enhancing the net positive charge of the hydrophilic side and the amphiphilic helix through amino acid replacing (G3K, A4R, and S10R). We found Kn2-7 had higher anti-HIV-1 activity than BmKn2, but with similar CC
<sub>50</sub>
to cells and even decreased toxicity response by the mouse skin model test (unpublished data). So, the modification improved the specific anti-viral activity in a certain extend. However, the mechanism needs to study further.</p>
<p>Since CCR5 is the major co-receptor involved in sexual transmission which becomes the leading infection route of HIV-1, antiviral activity against R5-tropic HIV-1 is more important
<xref rid="pone.0034947-Deng1" ref-type="bibr">[6]</xref>
,
<xref rid="pone.0034947-Liu1" ref-type="bibr">[43]</xref>
. Therefore, CAAN5342, a R5-tropic HIV-1 PV, the most tolerated HIV-1 PV to monoclonal antibody B12 and 2G12 in the standard reference panel of HIV-1 subtype B pseudotyped virus was chosen to test the antiviral activity of Kn2-7
<xref rid="pone.0034947-Li2" ref-type="bibr">[45]</xref>
. Kn2-7 showed potent antiviral activity against CAAN5342 in a dose- and time-dependent manner (
<xref ref-type="fig" rid="pone-0034947-g002">Fig. 2</xref>
). Further studies demonstrated that Kn2-7 was effective against HIV-1 subtype B PV regardless of their tropism with a selective index (SI) of 13.93. It was also highly effective to suppress replication-competent HIV-1 strain (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6</xref>
). The results demonstrated that Kn2-7 was broad-spectrum against HIV-1 which was in accordance with our previous findings that scorpion venom peptides are broad-spectrum biocides
<xref rid="pone.0034947-Ma1" ref-type="bibr">[24]</xref>
,
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
,
<xref rid="pone.0034947-Li1" ref-type="bibr">[38]</xref>
.</p>
<p>On the other hand, Kn2-7 was proven to be active to both cell-free and cell-associated replication-competent HIV-1 exhibited by its suppressing capability on infected TZM-bl cells, in which 15 µg/ml of Kn2-7 almost completely inhibited viral replication (
<xref ref-type="fig" rid="pone-0034947-g006">Fig. 6C</xref>
). As shown by Cole et al., HIV-1 evolves little resistance during continued passaging in the presence of defensins, another kind of cationic antimicrobial peptide
<xref rid="pone.0034947-Cole1" ref-type="bibr">[46]</xref>
. Kn2-7 may also possess this particular property which can reduce the likelihood of the emergence of resistance and the subsequent spread of infection.</p>
<p>As reported earlier, scorpion venom peptides display anti-pathogen activity by breaking bacteria directly
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
,
<xref rid="pone.0034947-Li1" ref-type="bibr">[38]</xref>
. We assumed that Kn2-7 may implement the inhibition of HIV-1 with the similar mechanism. The experiment of four different treatment groups of Kn2-7 to HIV-1 PV, in which the viral infectivity was inhibited only when Kn2-7 had chance to interact directly to HIV-1 PV, gave us a hint that Kn2-7 may exert its antiviral activity by damaging HIV virus particle itself. To clarify the presumption, we tried the binding assay for possible interaction between Kn2-7 and HIV-1 PV with Octet Red system by using APS sensors. The result confirmed that Kn2-7 could directly bind to HIV-1. However, whether viral particle could be broken by this binding is not clear. In experiments for effective action time of Kn2-7 to HIV-1 PV, it was observed that 3 min interaction allowed Kn2-7 showing almost maximum inhibition to HIV-1 PV (
<xref ref-type="fig" rid="pone-0034947-g002">Fig. 2B</xref>
). This told us that the anti-HIV-1 activity of Kn2-7 was rapid and direct which is similar to the action manner of mucroporin to bacteria
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
. Barrel-stave model might explain what happened between Kn2-7 and HIV-1 virus to some extent
<xref rid="pone.0034947-Brogden1" ref-type="bibr">[47]</xref>
,
<xref rid="pone.0034947-Oren1" ref-type="bibr">[48]</xref>
,
<xref rid="pone.0034947-Ehrenstein1" ref-type="bibr">[49]</xref>
. We may deduce the action principles as following: once Kn2-7 encounters with HIV-1, Kn2-7 binds to the envelope of HIV-1 directly and immediately. The attached Kn2-7 aggregates and inserts into viral envelope so that the hydrophobic peptide regions align with the lipid core region and the hydrophilic peptide regions form the interior region of the pore, with the help of positive charge of peptide somehow. However, more investigations need to be done for further clarification.</p>
<p>The epidemic of HIV-1 combined with the lack of an effective vaccine urge us to find more new resources of antiviral agents to help control HIV-1 transmission. In this study, a rationally designed peptide, Kn2-7, shows potent antiviral activities against 13 strains of HIV-1 subtype B strains by direct interaction with virus regardless of their tropism with low cytotoxicity. The data presented here suggest that Kn2-7 is a potential microbicide candidate against HIV-1 virus and even become a powerful anti-HIV-1 drug component when it is conjugated with HIV-1 specific antibodies. All in all, scorpion venom peptides can be at least served as molecular templates for designing antiviral peptides for HIV-1. Further pharmacokinetic, pharmacodynamic, and toxicological studies in animal models are worth being conducted to define safety parameters and application potential for the practical use of Kn2-7 as preventive agents for HIV-1 transmission.</p>
</sec>
<sec sec-type="materials|methods" id="s4">
<title>Materials and Methods</title>
<sec id="s4a">
<title>Molecular design, synthesis, purification and characterization of scorpion venom peptides</title>
<p>BmKn2 is a 13 amino acid antimicrobial peptide which cloned from the venom of
<italic>Mesobuthus martensii Karsch</italic>
<xref rid="pone.0034947-Zeng1" ref-type="bibr">[39]</xref>
. Kn2-7 is an optimizing design of BmKn2 by substituting Glycine Alanine and Serine with Lysine or Arginine (G3K, A4R, and S10R). Mucroporin is a 17 amino acid peptide from the venom of the scorpion
<italic>Lychas mucronatus</italic>
. Mucroporin-M1 is a derivative of mucroporin by substituting Glycine and Proline residues with Lysine or Arginine (G3R, P6K, G10K, and G11R)
<xref rid="pone.0034947-Dai2" ref-type="bibr">[31]</xref>
. Mucroporin-S1 is an 11-amino peptide by removing 6 N-terminal amino acids from mucroporin as a control in this study.</p>
<p>Kn2-7 (FIKRIARLLRKIF), BmKn2 (FIGAIARLLSKIF), mucroporin (LFGLIPSLIGGLVSAFK), mucroporin-M1 (LFRLIKSLIKRLVSAFK), and mucroporin-S1 (SLIGGLVSAFK), were synthesized on an Abimed AMS 422 synthesizer by Fmoc solid–phase peptide synthesis. The purification and characterization were carried out as described before
<xref rid="pone.0034947-Li1" ref-type="bibr">[38]</xref>
. Briefly, all peptides were purified on a C18 column (Elite HPLC, China) using a linear gradient and peaks were detected by the absorbance at 215 nm and collected manually. Thereafter, peptides were lyophilized and the purity was tested by high-performance liquid chromatography (HPLC) and mass spectrometry.</p>
</sec>
<sec id="s4b">
<title>Cells</title>
<p>TZM-bl cells (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health) are a genetically engineered HeLa cell clone that express CD4, CCR5 and CXCR4, and was further engineered with an HIV-1-based vector to contain Tat-responsive reporter genes for firefly luciferase (Luc) as contributed by John Kappes and Xiaoyun Wu
<xref rid="pone.0034947-Wei1" ref-type="bibr">[50]</xref>
,
<xref rid="pone.0034947-Platt1" ref-type="bibr">[51]</xref>
. 293 T, Vero and CEM×174 cell lines were obtained from American Type Culture Collection (Rockville, MD, U.S.). TZM-bl, 293 T and Vero cells were cultured in Dulbecco's modified Eagle's medium (Gibco DMEM) containing 10% fetal bovine serum (FBS, 56°C heat-inactivated, Gibco), 1% L-glutamine and 1% penicillin/streptomycin (Gibco). The cells were incubated at 37°C in a humidified atmosphere containing 5% CO
<sub>2</sub>
. The CEM×174 cells were cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS, 1% L-glutamine, and 1% penicillin/streptomycin.</p>
</sec>
<sec id="s4c">
<title>Preparation and titration of single-cycle HIV-1 PV and replication-competent HIV-1 virus from proviral DNA</title>
<p>All clones for pseudotyped virus-based assays were provided by NIH AIDS Research and Reference Reagent Program. Stocks of Env-pseudotyped virus were produced in 293 T cells briefly as following. 293 T cells (5×10
<sup>6</sup>
cells/ml) were co-transfected with 6 µg of an Env-deficient HIV-1 backbone vector (pSG3ΔEnv) and 4 µg of a plasmid encoding envelope protein of one strain of HIV-1 subtype B (6535, QH0692, SC422661, PVO, TRO, AC10, PHPA4259, THRO4156, REJO4551, TRJO4551, WITO4160, CAAN5342
<xref rid="pone.0034947-Li2" ref-type="bibr">[45]</xref>
,
<xref rid="pone.0034947-Wei1" ref-type="bibr">[50]</xref>
,
<xref rid="pone.0034947-Wei2" ref-type="bibr">[52]</xref>
, NL4-3
<xref rid="pone.0034947-Adachi1" ref-type="bibr">[53]</xref>
) using Lipo2000 agent (Invitrogen) in 6-well plates. PV-containing culture supernatants were harvested 48 h after transfection, centrifuged, purified by 0.45 µm filter and stored at −80°C in 200 µl aliquots. The 50% tissue culture infectious dose (TCID
<sub>50</sub>
) measurements were done in triplicates using 8 serial four-fold dilutions of each HIV-1 PV. For each dilution, every well of a 96-well culture plate was added with 100 µl diluted PV firstly and then with trypsinized TZM-bl cells (10
<sup>4</sup>
cells in 100 µl of growth medium containing 15 µg/ml DEAE-dextran (Sigma)). Plates were incubated at 37°C in a humidified, 5% CO
<sub>2</sub>
atmosphere for 48 h. To measure luciferase activity, culture medium was removed, monolayers were washed once with 200 µl PBS, and 100 µl cell culture lysis buffer (Promega) was added to cells and incubated for 20 min at room temperature to allow cell lysis. After that, 50 µl of cell lysate was transferred to 96-well black solid plate (Promega) and relative luminescence units (RLU) activity was measured by a Luciferase Assay System according to the manufacturer's instructions (Promega) using Tuner Biosystems Modulus II. Samples producing relative light units (RLU)>2.5×background wells (containing cells only) were scored as positive infection. An endpoint virus titer of TCID
<sub>50</sub>
was calculated by the Spearman-Karber method
<xref rid="pone.0034947-Hamilton1" ref-type="bibr">[54]</xref>
.</p>
<p>Except in broad-spectrum antiviral effect assay, a panel of HIV-1 PV (R5-tropic (6535, QH0692, SC422661, PVO, TRO, AC10, PHPA4259, THRO4156, REJO4551, TRJO4551, WITO4160, CAAN5342) and X4-tropic (NL4-3) HIV-1 PV) was used, a R5-tropic HIV isolate CAAN5342 was used in all other pseudotyped virus-related experiments.</p>
<p>An HIV-1 subtype B, X4-tropic proviral DNA (pKS242 molecular clone)
<xref rid="pone.0034947-Sakai1" ref-type="bibr">[55]</xref>
was used to produce replication-competent virus by transfecting Vero cells with Lipo2000 agent, and virus was propagated in CEM×174 cells. Cultures were maintained and HIV-containing CEM×174 cells and supernatant were collected. HIV-1 replication-competent viruses were quantified by p24 concentration.</p>
</sec>
<sec id="s4d">
<title>Cell viability assay</title>
<p>
<italic>In vitro</italic>
cytotoxicity test of Kn2-7 and mucroporin-S1, were performed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) procedure
<xref rid="pone.0034947-Cory1" ref-type="bibr">[41]</xref>
. Briefly, TZM-bl or CEM×174 cells were seeded into 96-well plates at a density of 1×10
<sup>4</sup>
cells/well in triplicates and incubated at 37°C for 24 h. Thereafter, culture medium was replaced by 100 µl serial dilutions of the peptides (from 5 µg/ml to 100 µg/ml) in DMEM culture, and the cells were incubated for another 24 h. Each dilution was tested in triplicates and all the experiments were repeated three times. Then 20 µl of CellTiter 96® AQueous One Solution Reagent (Promega) was added to each well of the 96-well assay plate containing 100 µl sample in culture medium. The plate was further incubated for 2 h at 37°C in a humidified, 5% CO
<sub>2</sub>
atmosphere. The absorbance was measured at 490 nm using an ELISA plate reader (Multiskan MK3, Thermo Labsystems). The relative cell viability (%) related to control wells containing cell culture medium without peptide was calculated by [A] test/[A] control×100%. The cytotoxicity of each peptide is expressed as the 50% of cytotoxic concentration (CC
<sub>50</sub>
), which is the concentration that inhibits the growth of 50% of cells relative to non-treated control cells.</p>
</sec>
<sec id="s4e">
<title>Antiviral effect assay of the peptides against HIV-1 PV</title>
<p>To test the antiviral effect of the peptides against HIV-1, pseudotyped virus-based assays with TZM-bl system were performed as described previously
<xref rid="pone.0034947-Montefiori1" ref-type="bibr">[56]</xref>
. Briefly, 200 TCID
<sub>50</sub>
of HIV-1 PV were incubated with indicated concentration of test scorpion venom peptides in a total volume of 100 µl for 60 min or indicated time intervals when considered with time-course effects at 37°C in triplicates in 96-well plates. After incubation, freshly trypsinized TZM-bl cells (10
<sup>4</sup>
cells in 100 µl of growth medium containing 15 µg/ml DEAE-dextran) were added to each well and incubated for 48 h. The luciferase activities of the TZM-bl cells were measured for antiviral activities of tested peptides, in which virus control and blank control were set up as wells of cells plus virus and cells only respectively.</p>
<p>In screening assay of anti-virus effect, 10 µg/ml peptides were used. For the following anti-HIV-1 PV effect assay, 3 µg/ml of the lead peptide Kn2-7 and the truncated control peptide mucroporin-S1 were used except for the dose-dependent effect experiment, in which serial dilutions from 0.25 µg/ml to 30 µg/ml were used.</p>
<p>In the time-dependent effect experiment, 3 µg/ml of peptides were incubated with 200 TCID
<sub>50</sub>
of HIV-1 PV for 3 min, 10 min, 30 min, 60 min, and 90 min, respectively.</p>
</sec>
<sec id="s4f">
<title>Measurement of antiviral activity of the peptides against replication-competent HIV-1 virus</title>
<p>Three dilutions of peptide (5 µg/ml, 10 µg/ml, and 15 µg/ml) were incubated with HIV-1 replication-competent virus (15 ng p24) for 1 h at 37°C. Thereafter, the compounds were added to CEM×174 cells (10
<sup>5</sup>
cells/ml) in duplicates in 12-well plate. Samples were collected at day 7, 12, 13, 14, 15, 16, and 17 after infection. Supernatant and cell samples were separated by centrifuge 1000 rpm for 5 min (Thermo). Samples were detected for p24 by ELISA and measured for infectivity by TZM-bl detection systems as described above.</p>
</sec>
<sec id="s4g">
<title>Action mode of the peptides to HIV-1 PV</title>
<p>In order to help determine the possible antiviral mechanism of Kn2-7 peptide, four different treatment groups were designed for comparison of the antiviral effect as following: (1) peptide pretreated group, TZM-bl cells were pretreated with 10 µg/ml peptide at 37°C for 1 h and then treated with HIV-1 PV; (2) infection first group, TZM-bl cells were infected with HIV-1 PV first by inoculating at 37°C for 1 h and then treated with 10 µg/ml peptide; (3) peptide/virus mixture group, TZM-bl cells were treated with mixture of HIV-1 PV and 10 µg/ml peptides immediately after mix but without inoculation; and (4) peptide/virus pre-incubation group, TZM-bl cells were infected with mixture of HIV-1 PV and 10 µg/ml peptides pre-incubating at 37°C for 1 h. To compare the effects of peptide to viral infection, TZM-bl cells were measured by luciferase activity assay after 48 h incubation as described above. 200 TCID
<sub>50</sub>
of HIV-1 PV (CAAN5342) was used in different treatment groups. In parallel, mucroporin-S1 control groups were set up in the same four manners.</p>
</sec>
<sec id="s4h">
<title>p24 protein detection by ELISA</title>
<p>For p24 protein detection, a double antibody sandwich ELISA was used
<xref rid="pone.0034947-Zhang1" ref-type="bibr">[57]</xref>
. Briefly, ELISA plates (Greiner Bio-one Microplate) were coated with rabbit anti-p24 polyclone-antibody (3 µg/ml in carbonate buffer 0.1 M NaHCO
<sub>3</sub>
, 0.1 M Na
<sub>2</sub>
CO
<sub>3</sub>
, pH 9.6) at 37°C for 2.5 h. Thereafter, plates were blocked using 10% FBS in PBS at 4°C overnight. Samples were serially diluted with PBS containing 10% FBS and 1% (v/v) TritonX-100 and treated for 3 h at 37°C to release p24 from the HIV-1 PV particles. P24 standard samples were treated in the same way to create a standard curve. Then treated sample dilutions were placed in wells for 2 h at 37°C. The plates were washed 6 times with PBS containing 0.05% Tween 20 (PBS-T). Mouse anti-p24-HRP antibody (1∶1200) was added and incubated for 1 h at 37°C and then washed 6 times with PBS-T. 100 µl of 3, 3′, 5, 5′-tetramethylbenzidine (TMB, Sigma Aldrich) was added to each well and incubated for 10–20 minutes at room temperature depending on the degree of colorimetric conversion of standard samples. The reaction was stopped with 1 M H
<sub>2</sub>
SO
<sub>4</sub>
, and the plate was read at 450 nm and 570 nm. OD value = (OD value at 450 nm) - (OD value at 570 nm). The p24 standard curve was drawn and a linear regression equation was made. The linear range was from 5 pg/ml to 100 pg/ml and the p24 concentration of the sample was computed using the equation.</p>
</sec>
<sec id="s4i">
<title>Biolayer Interferomtery</title>
<p>Binding assays were performed in 96-well microplates at 25°C by Octet Red system (Ferbio)
<xref rid="pone.0034947-Abdiche1" ref-type="bibr">[58]</xref>
,
<xref rid="pone.0034947-Do1" ref-type="bibr">[59]</xref>
. All the measurement processes are under computer control. The run was carried out by placing the APS sensors in the wells and measuring changes in layer thickness (in nanometers, nm) with time. Serial dilutions of peptides (Kn2-7 and mucroporin-S1) were run in 200 µl volumes under the same assay. Firstly, APS sensors were rinsed in DMEM buffer for 120 s which served as the background buffer. Secondly, APS sensors were coupled with 200 µl culture containing HIV-1 PV for 300 s. Thirdly, APS sensors were moved into DMEM buffer and incubated for another 120 s to clear unabsorbed HIV-1 PV. Lastly, APS sensors were exposed to Kn2-7 or mucoporin-S1 at concentrations of 10 µg/ml or 40 µg/ml. Association was monitored for 900 s followed by dissociation in DMEM buffer alone for another 400 s. The standard curve was measured at the beginning and the end of the assay to confirm that it was reproducible and valid over the time taken to run all rows of samples. Data were processed automatically using the Octet User Software version 3.1.</p>
</sec>
<sec id="s4j">
<title>Data analysis</title>
<p>The virus titer was calculated within each individual experiment using the method of Reed and Muench
<xref rid="pone.0034947-Reed1" ref-type="bibr">[60]</xref>
. All statistical analyses were conducted using GraphPad Prism, version 5. Statistical comparisons were conducted using the 2-tailed unpaired
<italic>t</italic>
test. P values <0.05 were considered significant. Data were presented as one representative of at least three repeated experiments and expressed as mean ± standard deviation (SD) of triplicates. The value of the TCID
<sub>50</sub>
, CC
<sub>50</sub>
and EC
<sub>50</sub>
were analog computation by SPSS statistical software version 13 (SPSS, Chicago, IL).</p>
</sec>
</sec>
</body>
<back>
<ack>
<p>The following reagent was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: 6535, QH0692, SC422661, PVO, TRO, AC10, PHPA4259, THRO4156, REJO4551, TRJO4551, WITO4160, CAAN5342.</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 was financially supported by the National Natural Science Foundation of China (NSFC) (No. 30670097 to HY), the National Basic Research Program of China (973 Program) (Nos. 2005CB522903, 2010CB530100, and 2012CB518904), and the National S&T Major Project on Major Infectious Diseases (Nos. 2008ZX10001-010, 2012ZX10001-008) from the Ministry of Science and Technology of the People's Republic of China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.</p>
</fn>
</fn-group>
<ref-list>
<title>References</title>
<ref id="pone.0034947-UNAIDS1">
<label>1</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>UNAIDS</surname>
</name>
</person-group>
<year>2010</year>
<volume>23</volume>
<comment>UNAIDS Report on the Global AIDS Epidemic 2010.Available:
<ext-link ext-link-type="uri" xlink:href="http://www.unaids.org/globalreport/documents/20101123_GlobalReport_full_en.pdf">http://www.unaids.org/globalreport/documents/20101123_GlobalReport_full_en.pdf</ext-link>
. Accessed 2010 Nov</comment>
</element-citation>
</ref>
<ref id="pone.0034947-Titti1">
<label>2</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Titti</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Cafaro</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Ferrantelli</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Tripiciano</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Moretti</surname>
<given-names>S</given-names>
</name>
<etal></etal>
</person-group>
<year>2007</year>
<article-title>Problems and emerging approaches in HIV/AIDS vaccine development.</article-title>
<source>Expert Opin Emerg Drugs</source>
<volume>12</volume>
<fpage>23</fpage>
<lpage>48</lpage>
<pub-id pub-id-type="pmid">17355212</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Buchbinder1">
<label>3</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Buchbinder</surname>
<given-names>SP</given-names>
</name>
<name>
<surname>Mehrotra</surname>
<given-names>DV</given-names>
</name>
<name>
<surname>Duerr</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Fitzgerald</surname>
<given-names>DW</given-names>
</name>
<name>
<surname>Mogg</surname>
<given-names>R</given-names>
</name>
<etal></etal>
</person-group>
<year>2008</year>
<article-title>Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial.</article-title>
<source>Lancet</source>
<volume>372</volume>
<fpage>1881</fpage>
<lpage>1893</lpage>
<pub-id pub-id-type="pmid">19012954</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-RerksNgarm1">
<label>4</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Rerks-Ngarm</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Pitisuttithum</surname>
<given-names>P</given-names>
</name>
<name>
<surname>Nitayaphan</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Kaewkungwal</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Chiu</surname>
<given-names>J</given-names>
</name>
<etal></etal>
</person-group>
<year>2009</year>
<article-title>Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand.</article-title>
<source>N Engl J Med</source>
<volume>361</volume>
<fpage>2209</fpage>
<lpage>2220</lpage>
<pub-id pub-id-type="pmid">19843557</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Dalgleish1">
<label>5</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dalgleish</surname>
<given-names>AG</given-names>
</name>
<name>
<surname>Beverley</surname>
<given-names>PC</given-names>
</name>
<name>
<surname>Clapham</surname>
<given-names>PR</given-names>
</name>
<name>
<surname>Crawford</surname>
<given-names>DH</given-names>
</name>
<name>
<surname>Greaves</surname>
<given-names>MF</given-names>
</name>
<etal></etal>
</person-group>
<year>1984</year>
<article-title>The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus.</article-title>
<source>Nature</source>
<volume>312</volume>
<fpage>763</fpage>
<lpage>767</lpage>
<pub-id pub-id-type="pmid">6096719</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Deng1">
<label>6</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Deng</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Ellmeier</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Choe</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Unutmaz</surname>
<given-names>D</given-names>
</name>
<etal></etal>
</person-group>
<year>1996</year>
<article-title>Identification of a major co-receptor for primary isolates of HIV-1.</article-title>
<source>Nature</source>
<volume>381</volume>
<fpage>661</fpage>
<lpage>666</lpage>
<pub-id pub-id-type="pmid">8649511</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Doranz1">
<label>7</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Doranz</surname>
<given-names>BJ</given-names>
</name>
<name>
<surname>Rucker</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Yi</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Smyth</surname>
<given-names>RJ</given-names>
</name>
<name>
<surname>Samson</surname>
<given-names>M</given-names>
</name>
<etal></etal>
</person-group>
<year>1996</year>
<article-title>A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors.</article-title>
<source>Cell</source>
<volume>85</volume>
<fpage>1149</fpage>
<lpage>1158</lpage>
<pub-id pub-id-type="pmid">8674120</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Feng1">
<label>8</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Feng</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Broder</surname>
<given-names>CC</given-names>
</name>
<name>
<surname>Kennedy</surname>
<given-names>PE</given-names>
</name>
<name>
<surname>Berger</surname>
<given-names>EA</given-names>
</name>
</person-group>
<year>1996</year>
<article-title>HIV-1 entry cofactor: functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor.</article-title>
<source>Science</source>
<volume>272</volume>
<fpage>872</fpage>
<lpage>877</lpage>
<pub-id pub-id-type="pmid">8629022</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Neurath1">
<label>9</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Neurath</surname>
<given-names>AR</given-names>
</name>
<name>
<surname>Strick</surname>
<given-names>N</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>YY</given-names>
</name>
</person-group>
<year>2002</year>
<article-title>Anti-HIV-1 activity of anionic polymers: a comparative study of candidate microbicides.</article-title>
<source>BMC Infect Dis</source>
<volume>2</volume>
<fpage>27</fpage>
<pub-id pub-id-type="pmid">12445331</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Telwatte1">
<label>10</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Telwatte</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Moore</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Johnson</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Tyssen</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Sterjovski</surname>
<given-names>J</given-names>
</name>
<etal></etal>
</person-group>
<year>2011</year>
<article-title>Virucidal activity of the dendrimer microbicide SPL7013 against HIV-1.</article-title>
<source>Antiviral Research</source>
<volume>90</volume>
<fpage>195</fpage>
<lpage>199</lpage>
<pub-id pub-id-type="pmid">21459115</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Torre1">
<label>11</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Torre</surname>
<given-names>VS</given-names>
</name>
<name>
<surname>Marozsan</surname>
<given-names>AJ</given-names>
</name>
<name>
<surname>Albright</surname>
<given-names>JL</given-names>
</name>
<name>
<surname>Collins</surname>
<given-names>KR</given-names>
</name>
<name>
<surname>Hartley</surname>
<given-names>O</given-names>
</name>
<etal></etal>
</person-group>
<year>2000</year>
<article-title>Variable sensitivity of CCR5-tropic human immunodeficiency virus type 1 isolates to inhibition by RANTES analogs.</article-title>
<source>J Virol</source>
<volume>74</volume>
<fpage>4868</fpage>
<lpage>4876</lpage>
<pub-id pub-id-type="pmid">10775626</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-McCormack1">
<label>12</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>McCormack</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Ramjee</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Kamali</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Rees</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Crook</surname>
<given-names>AM</given-names>
</name>
<etal></etal>
</person-group>
<year>2010</year>
<article-title>PRO2000 vaginal gel for prevention of HIV-1 infection (Microbicides Development Programme 301): a phase 3, randomised, double-blind, parallel-group trial.</article-title>
<source>Lancet</source>
<volume>376</volume>
<fpage>1329</fpage>
<lpage>1337</lpage>
<pub-id pub-id-type="pmid">20851460</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-PearcePratt1">
<label>13</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Pearce-Pratt</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Phillips</surname>
<given-names>DM</given-names>
</name>
</person-group>
<year>1996</year>
<article-title>Sulfated polysaccharides inhibit lymphocyte-to-epithelial transmission of human immunodeficiency virus-1.</article-title>
<source>Biol Reprod</source>
<volume>54</volume>
<fpage>173</fpage>
<lpage>182</lpage>
<pub-id pub-id-type="pmid">8838015</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Tao1">
<label>14</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tao</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Hu</surname>
<given-names>Q</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>X</given-names>
</name>
<etal></etal>
</person-group>
<year>2007</year>
<article-title>In vitro anti-HIV and -HSV activity and safety of sodium rutin sulfate as a microbicide candidate.</article-title>
<source>Antiviral Res</source>
<volume>75</volume>
<fpage>227</fpage>
<lpage>233</lpage>
<pub-id pub-id-type="pmid">17459492</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Vzorov1">
<label>15</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Vzorov</surname>
<given-names>AN</given-names>
</name>
<name>
<surname>Bozja</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Dixon</surname>
<given-names>DW</given-names>
</name>
<name>
<surname>Marzilli</surname>
<given-names>LG</given-names>
</name>
<name>
<surname>Compans</surname>
<given-names>RW</given-names>
</name>
</person-group>
<year>2007</year>
<article-title>Parameters of inhibition of HIV-1 infection by small anionic microbicides.</article-title>
<source>Antiviral Res</source>
<volume>73</volume>
<fpage>60</fpage>
<lpage>68</lpage>
<pub-id pub-id-type="pmid">16949681</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Tao2">
<label>16</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Tao</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Richards</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Hamer</surname>
<given-names>D</given-names>
</name>
</person-group>
<year>2008</year>
<article-title>Enhancement of HIV infection by cellulose sulfate.</article-title>
<source>AIDS Res Hum Retroviruses</source>
<volume>24</volume>
<fpage>925</fpage>
<lpage>929</lpage>
<pub-id pub-id-type="pmid">18627218</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Boman1">
<label>17</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Boman</surname>
<given-names>HG</given-names>
</name>
</person-group>
<year>2000</year>
<article-title>Innate immunity and the normal microflora.</article-title>
<source>Immunol Rev</source>
<volume>173</volume>
<fpage>5</fpage>
<lpage>16</lpage>
<pub-id pub-id-type="pmid">10719663</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Hancock1">
<label>18</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hancock</surname>
<given-names>RE</given-names>
</name>
<name>
<surname>Lehrer</surname>
<given-names>R</given-names>
</name>
</person-group>
<year>1998</year>
<article-title>Cationic peptides: a new source of antibiotics.</article-title>
<source>Trends Biotechnol</source>
<volume>16</volume>
<fpage>82</fpage>
<lpage>88</lpage>
<pub-id pub-id-type="pmid">9487736</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Fjell1">
<label>19</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Fjell</surname>
<given-names>CD</given-names>
</name>
<name>
<surname>Hancock</surname>
<given-names>RE</given-names>
</name>
<name>
<surname>Cherkasov</surname>
<given-names>A</given-names>
</name>
</person-group>
<year>2007</year>
<article-title>AMPer: a database and an automated discovery tool for antimicrobial peptides.</article-title>
<source>Bioinformatics</source>
<volume>23</volume>
<fpage>1148</fpage>
<lpage>1155</lpage>
<pub-id pub-id-type="pmid">17341497</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Jenssen1">
<label>20</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Jenssen</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Hamill</surname>
<given-names>P</given-names>
</name>
<name>
<surname>Hancock</surname>
<given-names>RE</given-names>
</name>
</person-group>
<year>2006</year>
<article-title>Peptide antimicrobial agents.</article-title>
<source>Clin Microbiol Rev</source>
<volume>19</volume>
<fpage>491</fpage>
<lpage>511</lpage>
<pub-id pub-id-type="pmid">16847082</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Martin1">
<label>21</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Martin</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Ganz</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Lehrer</surname>
<given-names>RI</given-names>
</name>
</person-group>
<year>1995</year>
<article-title>Defensins and other endogenous peptide antibiotics of vertebrates.</article-title>
<source>J Leukoc Biol</source>
<volume>58</volume>
<fpage>128</fpage>
<lpage>136</lpage>
<pub-id pub-id-type="pmid">7643008</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Wang1">
<label>22</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wang</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>G</given-names>
</name>
</person-group>
<year>2004</year>
<article-title>APD: the Antimicrobial Peptide Database.</article-title>
<source>Nucleic Acids Res</source>
<volume>32</volume>
<fpage>D590</fpage>
<lpage>592</lpage>
<pub-id pub-id-type="pmid">14681488</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Zasloff1">
<label>23</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zasloff</surname>
<given-names>M</given-names>
</name>
</person-group>
<year>2002</year>
<article-title>Antimicrobial peptides of multicellular organisms.</article-title>
<source>Nature</source>
<volume>415</volume>
<fpage>389</fpage>
<lpage>395</lpage>
<pub-id pub-id-type="pmid">11807545</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Ma1">
<label>24</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ma</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>R</given-names>
</name>
<name>
<surname>He</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>J</given-names>
</name>
<etal></etal>
</person-group>
<year>2009</year>
<article-title>Transcriptome analysis of the venom gland of the scorpion Scorpiops jendeki: implication for the evolution of the scorpion venom arsenal.</article-title>
<source>BMC Genomics</source>
<volume>10</volume>
<fpage>290</fpage>
<pub-id pub-id-type="pmid">19570192</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Possani1">
<label>25</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Possani</surname>
<given-names>LD</given-names>
</name>
<name>
<surname>Merino</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Corona</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Bolivar</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Becerril</surname>
<given-names>B</given-names>
</name>
</person-group>
<year>2000</year>
<article-title>Peptides and genes coding for scorpion toxins that affect ion-channels.</article-title>
<source>Biochimie</source>
<volume>82</volume>
<fpage>861</fpage>
<lpage>868</lpage>
<pub-id pub-id-type="pmid">11086216</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Zhijian1">
<label>26</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zhijian</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Feng</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Yingliang</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Xin</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Wenxin</surname>
<given-names>L</given-names>
</name>
</person-group>
<year>2006</year>
<article-title>Genetic mechanisms of scorpion venom peptide diversification.</article-title>
<source>Toxicon</source>
<volume>47</volume>
<fpage>348</fpage>
<lpage>355</lpage>
<pub-id pub-id-type="pmid">16387337</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-TorresLarios1">
<label>27</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Torres-Larios</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Gurrola</surname>
<given-names>GB</given-names>
</name>
<name>
<surname>Zamudio</surname>
<given-names>FZ</given-names>
</name>
<name>
<surname>Possani</surname>
<given-names>LD</given-names>
</name>
</person-group>
<year>2000</year>
<article-title>Hadrurin, a new antimicrobial peptide from the venom of the scorpion Hadrurus aztecus.</article-title>
<source>Eur J Biochem</source>
<volume>267</volume>
<fpage>5023</fpage>
<lpage>5031</lpage>
<pub-id pub-id-type="pmid">10931184</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Conde1">
<label>28</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Conde</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Zamudio</surname>
<given-names>FZ</given-names>
</name>
<name>
<surname>Rodriguez</surname>
<given-names>MH</given-names>
</name>
<name>
<surname>Possani</surname>
<given-names>LD</given-names>
</name>
</person-group>
<year>2000</year>
<article-title>Scorpine, an anti-malaria and anti-bacterial agent purified from scorpion venom.</article-title>
<source>FEBS Lett</source>
<volume>471</volume>
<fpage>165</fpage>
<lpage>168</lpage>
<pub-id pub-id-type="pmid">10767415</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Moerman1">
<label>29</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Moerman</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Bosteels</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Noppe</surname>
<given-names>W</given-names>
</name>
<name>
<surname>Willems</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Clynen</surname>
<given-names>E</given-names>
</name>
<etal></etal>
</person-group>
<year>2002</year>
<article-title>Antibacterial and antifungal properties of alpha-helical, cationic peptides in the venom of scorpions from southern Africa.</article-title>
<source>Eur J Biochem</source>
<volume>269</volume>
<fpage>4799</fpage>
<lpage>4810</lpage>
<pub-id pub-id-type="pmid">12354111</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Dai1">
<label>30</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dai</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Yasuda</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Naoki</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Corzo</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Andriantsiferana</surname>
<given-names>M</given-names>
</name>
<etal></etal>
</person-group>
<year>2001</year>
<article-title>IsCT, a novel cytotoxic linear peptide from scorpion Opisthacanthus madagascariensis.</article-title>
<source>Biochem Biophys Res Commun</source>
<volume>286</volume>
<fpage>820</fpage>
<lpage>825</lpage>
<pub-id pub-id-type="pmid">11520071</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Dai2">
<label>31</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Dai</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Ma</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>Q</given-names>
</name>
<etal></etal>
</person-group>
<year>2008</year>
<article-title>Mucroporin, the first cationic host defense peptide from the venom of Lychas mucronatus.</article-title>
<source>Antimicrob Agents Chemother</source>
<volume>52</volume>
<fpage>3967</fpage>
<lpage>3972</lpage>
<pub-id pub-id-type="pmid">18779362</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Zhao1">
<label>32</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zhao</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Ma</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Dai</surname>
<given-names>C</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>S</given-names>
</name>
<etal></etal>
</person-group>
<year>2009</year>
<article-title>Imcroporin, a new cationic antimicrobial peptide from the venom of the scorpion Isometrus maculates.</article-title>
<source>Antimicrob Agents Chemother</source>
<volume>53</volume>
<fpage>3472</fpage>
<lpage>3477</lpage>
<pub-id pub-id-type="pmid">19451300</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-AlbiolMatanic1">
<label>33</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Albiol Matanic</surname>
<given-names>VC</given-names>
</name>
<name>
<surname>Castilla</surname>
<given-names>V</given-names>
</name>
</person-group>
<year>2004</year>
<article-title>Antiviral activity of antimicrobial cationic peptides against Junin virus and herpes simplex virus.</article-title>
<source>Int J Antimicrob Agents</source>
<volume>23</volume>
<fpage>382</fpage>
<lpage>389</lpage>
<pub-id pub-id-type="pmid">15081088</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Sun1">
<label>34</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sun</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Finnegan</surname>
<given-names>CM</given-names>
</name>
<name>
<surname>Kish-Catalone</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Blumenthal</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Garzino-Demo</surname>
<given-names>P</given-names>
</name>
<etal></etal>
</person-group>
<year>2005</year>
<article-title>Human beta-defensins suppress human immunodeficiency virus infection: potential role in mucosal protection.</article-title>
<source>J Virol</source>
<volume>79</volume>
<fpage>14318</fpage>
<lpage>14329</lpage>
<pub-id pub-id-type="pmid">16254366</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-CarrielGomes1">
<label>35</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Carriel-Gomes</surname>
<given-names>MC</given-names>
</name>
<name>
<surname>Kratz</surname>
<given-names>JM</given-names>
</name>
<name>
<surname>Barracco</surname>
<given-names>MA</given-names>
</name>
<name>
<surname>Bachere</surname>
<given-names>E</given-names>
</name>
<name>
<surname>Barardi</surname>
<given-names>CR</given-names>
</name>
<etal></etal>
</person-group>
<year>2007</year>
<article-title>In vitro antiviral activity of antimicrobial peptides against herpes simplex virus 1, adenovirus, and rotavirus.</article-title>
<source>Mem Inst Oswaldo Cruz</source>
<volume>102</volume>
<fpage>469</fpage>
<lpage>472</lpage>
<pub-id pub-id-type="pmid">17612767</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Yan1">
<label>36</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Yan</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>He</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Wu</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Cai</surname>
<given-names>D</given-names>
</name>
<etal></etal>
</person-group>
<year>2011</year>
<article-title>A new natural alpha-helical peptide from the venom of the scorpion Heterometrus petersii kills HCV.</article-title>
<source>Peptides</source>
<volume>32</volume>
<fpage>11</fpage>
<lpage>19</lpage>
<pub-id pub-id-type="pmid">20950663</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Mohan1">
<label>37</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Mohan</surname>
<given-names>KV</given-names>
</name>
<name>
<surname>Rao</surname>
<given-names>SS</given-names>
</name>
<name>
<surname>Atreya</surname>
<given-names>CD</given-names>
</name>
</person-group>
<year>2010</year>
<article-title>Antiviral activity of selected antimicrobial peptides against vaccinia virus.</article-title>
<source>Antiviral Res</source>
<volume>86</volume>
<issue>3</issue>
<fpage>306</fpage>
<lpage>311</lpage>
<pub-id pub-id-type="pmid">20347875</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Li1">
<label>38</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>Q</given-names>
</name>
<name>
<surname>Zhao</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Zhou</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Hong</surname>
<given-names>W</given-names>
</name>
<etal></etal>
</person-group>
<year>2011</year>
<article-title>Virucidal activity of a scorpion venom peptide variant mucroporin-M1 against measles, SARS-CoV and influenza H5N1 viruses.</article-title>
<source>Peptides</source>
<volume>32</volume>
<fpage>1518</fpage>
<lpage>1525</lpage>
<pub-id pub-id-type="pmid">21620914</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Zeng1">
<label>39</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zeng</surname>
<given-names>XC</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>SX</given-names>
</name>
<name>
<surname>Zhu</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Zhu</surname>
<given-names>SY</given-names>
</name>
<name>
<surname>Li</surname>
<given-names>WX</given-names>
</name>
</person-group>
<year>2004</year>
<article-title>Identification and functional characterization of novel scorpion venom peptides with no disulfide bridge from Buthus martensii Karsch.</article-title>
<source>Peptides</source>
<volume>25</volume>
<fpage>143</fpage>
<lpage>150</lpage>
<pub-id pub-id-type="pmid">15062994</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Rost1">
<label>40</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Rost</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Sander</surname>
<given-names>C</given-names>
</name>
</person-group>
<year>1993</year>
<article-title>Prediction of protein secondary structure at better than 70% accuracy.</article-title>
<source>Journal of Molecular Biology</source>
<volume>232</volume>
<fpage>584</fpage>
<lpage>584</lpage>
<pub-id pub-id-type="pmid">8345525</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Cory1">
<label>41</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Cory</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Owen</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Barltrop</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Cory</surname>
<given-names>J</given-names>
</name>
</person-group>
<year>1991</year>
<article-title>Use of an aqueous soluble tetrazolium/formazan assay for cell growth assays in culture.</article-title>
<source>Cancer communications</source>
<volume>3</volume>
<fpage>207</fpage>
<pub-id pub-id-type="pmid">1867954</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Choudhry1">
<label>42</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Choudhry</surname>
<given-names>V</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>MY</given-names>
</name>
<name>
<surname>Sidorov</surname>
<given-names>IA</given-names>
</name>
<name>
<surname>Louis</surname>
<given-names>JM</given-names>
</name>
<name>
<surname>Harris</surname>
<given-names>I</given-names>
</name>
<etal></etal>
</person-group>
<year>2007</year>
<article-title>Cross-reactive HIV-1 neutralizing monoclonal antibodies selected by screening of an immune human phage library against an envelope glycoprotein (gp140) isolated from a patient (R2) with broadly HIV-1 neutralizing antibodies.</article-title>
<source>Virology</source>
<volume>363</volume>
<fpage>79</fpage>
<lpage>90</lpage>
<pub-id pub-id-type="pmid">17306322</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Liu1">
<label>43</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Liu</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Paxton</surname>
<given-names>WA</given-names>
</name>
<name>
<surname>Choe</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Ceradini</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Martin</surname>
<given-names>SR</given-names>
</name>
<etal></etal>
</person-group>
<year>1996</year>
<article-title>Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.</article-title>
<source>Cell</source>
<volume>86</volume>
<fpage>367</fpage>
<lpage>377</lpage>
<pub-id pub-id-type="pmid">8756719</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Arien1">
<label>44</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Arien</surname>
<given-names>KK</given-names>
</name>
<name>
<surname>Jespers</surname>
<given-names>V</given-names>
</name>
<name>
<surname>Vanham</surname>
<given-names>G</given-names>
</name>
</person-group>
<year>2011</year>
<article-title>HIV sexual transmission and microbicides.</article-title>
<source>Rev Med Virol</source>
<volume>21</volume>
<issue>2</issue>
<fpage>110</fpage>
<lpage>133</lpage>
<pub-id pub-id-type="pmid">21412935</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Li2">
<label>45</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Li</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Gao</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Mascola</surname>
<given-names>JR</given-names>
</name>
<name>
<surname>Stamatatos</surname>
<given-names>L</given-names>
</name>
<name>
<surname>Polonis</surname>
<given-names>VR</given-names>
</name>
<etal></etal>
</person-group>
<year>2005</year>
<article-title>Human Immunodeficiency Virus Type 1 env Clones from Acute and Early Subtype B Infections for Standardized Assessments of Vaccine-Elicited Neutralizing Antibodies.</article-title>
<source>Journal of Virology</source>
<volume>79</volume>
<fpage>10108</fpage>
<lpage>10125</lpage>
<pub-id pub-id-type="pmid">16051804</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Cole1">
<label>46</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Cole</surname>
<given-names>AL</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>OO</given-names>
</name>
<name>
<surname>Warren</surname>
<given-names>AD</given-names>
</name>
<name>
<surname>Waring</surname>
<given-names>AJ</given-names>
</name>
<name>
<surname>Lehrer</surname>
<given-names>RI</given-names>
</name>
<etal></etal>
</person-group>
<year>2006</year>
<article-title>HIV-1 adapts to a retrocyclin with cationic amino acid substitutions that reduce fusion efficiency of gp41.</article-title>
<source>The Journal of Immunology</source>
<volume>176</volume>
<fpage>6900</fpage>
<pub-id pub-id-type="pmid">16709850</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Brogden1">
<label>47</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Brogden</surname>
<given-names>KA</given-names>
</name>
</person-group>
<year>2005</year>
<article-title>Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?</article-title>
<source>Nat Rev Microbiol</source>
<volume>3</volume>
<fpage>238</fpage>
<lpage>250</lpage>
<pub-id pub-id-type="pmid">15703760</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Oren1">
<label>48</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Oren</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Shai</surname>
<given-names>Y</given-names>
</name>
</person-group>
<year>1998</year>
<article-title>Mode of action of linear amphipathic alpha-helical antimicrobial peptides.</article-title>
<source>Biopolymers</source>
<volume>47</volume>
<fpage>451</fpage>
<lpage>463</lpage>
<pub-id pub-id-type="pmid">10333737</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Ehrenstein1">
<label>49</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Ehrenstein</surname>
<given-names>G</given-names>
</name>
<name>
<surname>Lecar</surname>
<given-names>H</given-names>
</name>
</person-group>
<year>1977</year>
<article-title>Electrically gated ionic channels in lipid bilayers.</article-title>
<source>Quarterly reviews of biophysics</source>
<volume>10</volume>
<fpage>1</fpage>
<lpage>34</lpage>
<pub-id pub-id-type="pmid">327501</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Wei1">
<label>50</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wei</surname>
<given-names>X</given-names>
</name>
<name>
<surname>Decker</surname>
<given-names>JM</given-names>
</name>
<name>
<surname>Liu</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Zhang</surname>
<given-names>Z</given-names>
</name>
<name>
<surname>Arani</surname>
<given-names>RB</given-names>
</name>
<etal></etal>
</person-group>
<year>2002</year>
<article-title>Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy.</article-title>
<source>Antimicrob Agents Chemother</source>
<volume>46</volume>
<fpage>1896</fpage>
<lpage>1905</lpage>
<pub-id pub-id-type="pmid">12019106</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Platt1">
<label>51</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Platt</surname>
<given-names>EJ</given-names>
</name>
<name>
<surname>Wehrly</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Kuhmann</surname>
<given-names>SE</given-names>
</name>
<name>
<surname>Chesebro</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Kabat</surname>
<given-names>D</given-names>
</name>
</person-group>
<year>1998</year>
<article-title>Effects of CCR5 and CD4 cell surface concentrations on infections by macrophagetropic isolates of human immunodeficiency virus type 1.</article-title>
<source>J Virol</source>
<volume>72</volume>
<fpage>2855</fpage>
<lpage>2864</lpage>
<pub-id pub-id-type="pmid">9525605</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Wei2">
<label>52</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Wei</surname>
<given-names>X</given-names>
</name>
<name>
<surname>Decker</surname>
<given-names>JM</given-names>
</name>
<name>
<surname>Wang</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Hui</surname>
<given-names>H</given-names>
</name>
<name>
<surname>Kappes</surname>
<given-names>JC</given-names>
</name>
<etal></etal>
</person-group>
<year>2003</year>
<article-title>Antibody neutralization and escape by HIV-1.</article-title>
<source>Nature</source>
<volume>422</volume>
<fpage>307</fpage>
<lpage>312</lpage>
<pub-id pub-id-type="pmid">12646921</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Adachi1">
<label>53</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Adachi</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Gendelman</surname>
<given-names>HE</given-names>
</name>
<name>
<surname>Koenig</surname>
<given-names>S</given-names>
</name>
<name>
<surname>Folks</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Willey</surname>
<given-names>R</given-names>
</name>
<etal></etal>
</person-group>
<year>1986</year>
<article-title>Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone.</article-title>
<source>J Virol</source>
<volume>59</volume>
<fpage>284</fpage>
<lpage>291</lpage>
<pub-id pub-id-type="pmid">3016298</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Hamilton1">
<label>54</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Hamilton</surname>
<given-names>MA</given-names>
</name>
<name>
<surname>McKenzie-Pollock</surname>
<given-names>M</given-names>
</name>
<name>
<surname>Heath</surname>
<given-names>ME</given-names>
</name>
</person-group>
<year>1977</year>
<article-title>The treatment and prevention of chronic otorrhoea in the Northland health district.</article-title>
<source>N Z Med J</source>
<volume>86</volume>
<fpage>327</fpage>
<lpage>329</lpage>
<pub-id pub-id-type="pmid">272537</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Sakai1">
<label>55</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Sakai</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Ma</surname>
<given-names>XY</given-names>
</name>
<name>
<surname>Gordienko</surname>
<given-names>I</given-names>
</name>
<name>
<surname>Volsky</surname>
<given-names>DJ</given-names>
</name>
</person-group>
<year>1991</year>
<article-title>Recombinational analysis of a natural noncytopathic human immunodeficiency virus type 1 (HIV-1) isolate: role of the vif gene in HIV-1 infection kinetics and cytopathicity.</article-title>
<source>J Virol</source>
<volume>65</volume>
<fpage>5765</fpage>
<lpage>5773</lpage>
<pub-id pub-id-type="pmid">1920615</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Montefiori1">
<label>56</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Montefiori</surname>
<given-names>DC</given-names>
</name>
</person-group>
<year>2005</year>
<article-title>Evaluating neutralizing antibodies against HIV, SIV, and SHIV in luciferase reporter gene assays.</article-title>
<source>Curr Protoc Immunol Chapter 12: Unit 12</source>
<volume>11</volume>
</element-citation>
</ref>
<ref id="pone.0034947-Zhang1">
<label>57</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Zhang</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Yang</surname>
<given-names>J</given-names>
</name>
<name>
<surname>Bao</surname>
<given-names>R</given-names>
</name>
<name>
<surname>Chen</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Zhou</surname>
<given-names>D</given-names>
</name>
<etal></etal>
</person-group>
<year>2011</year>
<article-title>Unpolarized release of vaccinia virus and HIV antigen by colchicine treatment enhances intranasal HIV antigen expression and mucosal humoral responses.</article-title>
<source>PLoS ONE</source>
<volume>6</volume>
<fpage>e24296</fpage>
<pub-id pub-id-type="pmid">21935396</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Abdiche1">
<label>58</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Abdiche</surname>
<given-names>Y</given-names>
</name>
<name>
<surname>Malashock</surname>
<given-names>D</given-names>
</name>
<name>
<surname>Pinkerton</surname>
<given-names>A</given-names>
</name>
<name>
<surname>Pons</surname>
<given-names>J</given-names>
</name>
</person-group>
<year>2008</year>
<article-title>Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet.</article-title>
<source>Analytical Biochemistry</source>
<volume>377</volume>
<fpage>209</fpage>
<lpage>217</lpage>
<pub-id pub-id-type="pmid">18405656</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Do1">
<label>59</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Do</surname>
<given-names>T</given-names>
</name>
<name>
<surname>Ho</surname>
<given-names>F</given-names>
</name>
<name>
<surname>Heidecker</surname>
<given-names>B</given-names>
</name>
<name>
<surname>Witte</surname>
<given-names>K</given-names>
</name>
<name>
<surname>Chang</surname>
<given-names>L</given-names>
</name>
<etal></etal>
</person-group>
<year>2008</year>
<article-title>A rapid method for determining dynamic binding capacity of resins for the purification of proteins.</article-title>
<source>Protein Expr Purif</source>
<volume>60</volume>
<fpage>147</fpage>
<lpage>150</lpage>
<pub-id pub-id-type="pmid">18538581</pub-id>
</element-citation>
</ref>
<ref id="pone.0034947-Reed1">
<label>60</label>
<element-citation publication-type="journal">
<person-group person-group-type="author">
<name>
<surname>Reed</surname>
<given-names>LJ</given-names>
</name>
<name>
<surname>Muench</surname>
<given-names>H</given-names>
</name>
</person-group>
<year>1938</year>
<article-title>A simple method of estimating fifty per cent endpoints.</article-title>
<source>American Journal of Epidemiology</source>
<volume>27</volume>
<fpage>493</fpage>
</element-citation>
</ref>
</ref-list>
</back>
</pmc>
</record>

Pour manipuler ce document sous Unix (Dilib)

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/SrasV1/Data/Pmc/Corpus

HfdSelect -h $EXPLOR_STEP/biblio.hfd -nk 001376 | SxmlIndent | more

Ou

HfdSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd -nk 001376 | SxmlIndent | more

Pour mettre un lien sur cette page dans le réseau Wicri

{{Explor lien
   |wiki=    Sante
   |area=    SrasV1
   |flux=    Pmc
   |étape=   Corpus
   |type=    RBID
   |clé=     PMC:3334916
   |texte=   Anti-HIV-1 Activity of a New Scorpion Venom Peptide Derivative Kn2-7
}}

Pour générer des pages wiki

HfdIndexSelect -h $EXPLOR_AREA/Data/Pmc/Corpus/RBID.i   -Sk "pubmed:22536342" \
       | HfdSelect -Kh $EXPLOR_AREA/Data/Pmc/Corpus/biblio.hfd   \
       | NlmPubMed2Wicri -a SrasV1
Wicri

This area was generated with Dilib version V0.6.33.
Data generation: Tue Apr 28 14:49:16 2020. Site generation: Sat Mar 27 22:06:49 2021