Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay
The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a...
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| Veröffentlicht in: | PloS one 2011-11, Vol.6 (11), p.e27234 |
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| description | The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity. |
| doi_str_mv | 10.1371/journal.pone.0027234 |
| format | Article |
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Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0027234</identifier><identifier>PMID: 22073298</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Aberration ; Alanine ; Animals ; Antiviral activity ; Antiviral agents ; Assaying ; Assembly ; Baculovirus ; Betulinic acid ; Biochemistry, Molecular Biology ; Biological effects ; Biology ; Budding ; Cell culture ; Cell growth ; Cell Line ; Crosslinking ; Cytochrome ; Cytotoxicity ; Derivatives ; Electrophoresis, Polyacrylamide Gel ; Enzymatic activity ; Fusion protein ; Gag protein ; Glycine ; HIV ; HIV-1 ; HIV-1 - physiology ; Human immunodeficiency virus ; Immunology ; Intermediates ; Life Sciences ; Luciferase ; Lysine ; Materials Science ; Medicine ; Melanoma ; Microscopy, Electron ; Morphology ; Mutation ; N-Terminus ; Nanoparticles ; Packaging ; Particulates ; Pathology ; Proteins ; Saccharomyces cerevisiae ; Spodoptera ; Structural Biology ; Toxicity ; Trimers ; Virion ; Virion - physiology ; Virion - ultrastructure ; Virions ; Virus-like particles ; Viruses ; vpr Gene Products, Human Immunodeficiency Virus ; vpr Gene Products, Human Immunodeficiency Virus - metabolism ; Vpr protein</subject><ispartof>PloS one, 2011-11, Vol.6 (11), p.e27234</ispartof><rights>COPYRIGHT 2011 Public Library of Science</rights><rights>2011 Gonzalez et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: https://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>Gonzalez et al. 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c725t-4cf6d18fd1f52f46d7bf1fcd73eebfeed51cc06acfce712a8d4718d8d30a70103</citedby><orcidid>0000-0003-4805-7316 ; 0000-0001-7792-8307 ; 0000-0001-8484-1381</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207847/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3207847/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,2100,2926,23864,27922,27923,53789,53791,79370,79371</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/22073298$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-02122413$$DView record in HAL$$Hfree_for_read</backlink></links><search><contributor>Major, Eugene Oliver</contributor><creatorcontrib>Gonzalez, Gaëlle</creatorcontrib><creatorcontrib>DaFonseca, Sandrina</creatorcontrib><creatorcontrib>Errazuriz, Elisabeth</creatorcontrib><creatorcontrib>Coric, Pascale</creatorcontrib><creatorcontrib>Souquet, Florence</creatorcontrib><creatorcontrib>Turcaud, Serge</creatorcontrib><creatorcontrib>Boulanger, Pierre</creatorcontrib><creatorcontrib>Bouaziz, Serge</creatorcontrib><creatorcontrib>Hong, Saw See</creatorcontrib><title>Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.</description><subject>Aberration</subject><subject>Alanine</subject><subject>Animals</subject><subject>Antiviral activity</subject><subject>Antiviral agents</subject><subject>Assaying</subject><subject>Assembly</subject><subject>Baculovirus</subject><subject>Betulinic acid</subject><subject>Biochemistry, Molecular Biology</subject><subject>Biological effects</subject><subject>Biology</subject><subject>Budding</subject><subject>Cell culture</subject><subject>Cell growth</subject><subject>Cell Line</subject><subject>Crosslinking</subject><subject>Cytochrome</subject><subject>Cytotoxicity</subject><subject>Derivatives</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Enzymatic activity</subject><subject>Fusion protein</subject><subject>Gag protein</subject><subject>Glycine</subject><subject>HIV</subject><subject>HIV-1</subject><subject>HIV-1 - physiology</subject><subject>Human immunodeficiency virus</subject><subject>Immunology</subject><subject>Intermediates</subject><subject>Life Sciences</subject><subject>Luciferase</subject><subject>Lysine</subject><subject>Materials Science</subject><subject>Medicine</subject><subject>Melanoma</subject><subject>Microscopy, Electron</subject><subject>Morphology</subject><subject>Mutation</subject><subject>N-Terminus</subject><subject>Nanoparticles</subject><subject>Packaging</subject><subject>Particulates</subject><subject>Pathology</subject><subject>Proteins</subject><subject>Saccharomyces cerevisiae</subject><subject>Spodoptera</subject><subject>Structural Biology</subject><subject>Toxicity</subject><subject>Trimers</subject><subject>Virion</subject><subject>Virion - physiology</subject><subject>Virion - ultrastructure</subject><subject>Virions</subject><subject>Virus-like particles</subject><subject>Viruses</subject><subject>vpr Gene Products, Human Immunodeficiency Virus</subject><subject>vpr Gene Products, Human Immunodeficiency Virus - 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Academic</collection><collection>ProQuest Engineering Collection</collection><collection>ProQuest Biological Science Collection</collection><collection>Agricultural Science Database</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biological Science Database</collection><collection>Engineering Database</collection><collection>Nursing & Allied Health Premium</collection><collection>Advanced Technologies & Aerospace Database</collection><collection>ProQuest Advanced Technologies & Aerospace Collection</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Environmental Science Database</collection><collection>Materials Science Collection</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Engineering Collection</collection><collection>Environmental Science Collection</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>Hyper Article en Ligne (HAL)</collection><collection>Hyper Article en Ligne (HAL) (Open Access)</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gonzalez, Gaëlle</au><au>DaFonseca, Sandrina</au><au>Errazuriz, Elisabeth</au><au>Coric, Pascale</au><au>Souquet, Florence</au><au>Turcaud, Serge</au><au>Boulanger, Pierre</au><au>Bouaziz, Serge</au><au>Hong, Saw See</au><au>Major, Eugene Oliver</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2011-11-03</date><risdate>2011</risdate><volume>6</volume><issue>11</issue><spage>e27234</spage><pages>e27234-</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>22073298</pmid><doi>10.1371/journal.pone.0027234</doi><tpages>e27234</tpages><orcidid>https://orcid.org/0000-0003-4805-7316</orcidid><orcidid>https://orcid.org/0000-0001-7792-8307</orcidid><orcidid>https://orcid.org/0000-0001-8484-1381</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2011-11, Vol.6 (11), p.e27234 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1310776108 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Public Library of Science (PLoS); EZB-FREE-00999 freely available EZB journals; PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Aberration Alanine Animals Antiviral activity Antiviral agents Assaying Assembly Baculovirus Betulinic acid Biochemistry, Molecular Biology Biological effects Biology Budding Cell culture Cell growth Cell Line Crosslinking Cytochrome Cytotoxicity Derivatives Electrophoresis, Polyacrylamide Gel Enzymatic activity Fusion protein Gag protein Glycine HIV HIV-1 HIV-1 - physiology Human immunodeficiency virus Immunology Intermediates Life Sciences Luciferase Lysine Materials Science Medicine Melanoma Microscopy, Electron Morphology Mutation N-Terminus Nanoparticles Packaging Particulates Pathology Proteins Saccharomyces cerevisiae Spodoptera Structural Biology Toxicity Trimers Virion Virion - physiology Virion - ultrastructure Virions Virus-like particles Viruses vpr Gene Products, Human Immunodeficiency Virus vpr Gene Products, Human Immunodeficiency Virus - metabolism Vpr protein |
| title | Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative luciferase-Vpr packaging-based assay |
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