Name: visualization of hiv-1 gag binding to giant unilamellar vesicle (guv) membranes
Uploaded: 2016-07-28T14:00:00
Description: Watch this Scientific Journal Video about Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes at JoVE.com

Vi vil gerne takke Mohammad Saleem, Jing Wu og Krishnan Raghunathan for nyttige diskussioner. Vi takker også Priya Begani for hjælp under optagelserne. Dette arbejde er støttet af National Institutes of Health giver R01 AI071727 (til AO) og R01 GM110052 (til SLV).

Dag 1: Angivelse af Gag proteiner Brug af Wheat Germ Lysat-baserede in vitro-transskription-translation System 1. Fremstilling af HIV-1 Gag <ol><li> Fjern alle reagenserne ifølge kommercielle hvedekim CECF kit fra frysere (hvedekim lysater opbevares ved -80 ºC andre reagenser ved -20 ºC). Tø dem på is og blandes komponenterne som vist i tabel 1. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> <...

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Den GUV bindingsassay som beskrevet ovenfor giver et godt alternativ i scenarier, hvor andre protein-lipid interaktion assays har deres begrænsninger. Dette assay giver os mulighed for at undersøge interaktioner mellem myristoyleret fuldlængde Gag og sure lipider med nativ længde acylkæder i lipiddobbeltlaget kontekst og gøre det uden langvarig flotation centrifugering gennem high-density saccharosegradienter eller andre post-bindende forarbejdning af Gag-lipid komplekser. En anden fordel er, at opførslen af Gag ...

Humant immundefektvirus type 1 (HIV-1) er et kappebærende virus, der samler på og knopper fra plasmamembranen (PM) i de fleste celletyper. Samlingen af HIV-1 viruspartikler er drevet af 55 kDa viralt kerneprotein kaldet Pr55 Gag (Gag). Gag syntetiseres som en precursor polyprotein består af fire store strukturelle domæner, nemlig matrix, capsid, nucleocapsid og p6, samt to spacer peptider SP1 og SP2. Under montering, matricen (MA) domæne er ansvarlig for målretning af Gag til forsamlingen site, capsid (CA) domæne medierer Gag-Gag interaktioner, nucleocapsid (NC) domæne rekrutterer viralt genomisk RNA, og p6 rekrutter vært faktorer at støtten viruspartikel spaltning fra plasmamembranen. Gag undergår også en co-translationel modifikation ved tilsætning af en 14-carbon fedtsyre eller myristat-delen ved dens N-terminus. Membran binding af Gag er en væsentlig forudsætning for den virale samling, da mutants der er defekt i membranen binding ikke producere viruspartikler. Vi og andre har vist, at membranen binding af Gag medieres af tosidede signaler i MA domæne: det N-terminale myristat-del, der medierer hydrofobe interaktioner med lipiddobbeltlaget og en klynge af basiske rester i MA domæne betegnes som stærkt basiske region ( HBR), der interagerer med sure lipider på PM 1-4. Studier af Gag-membran interaktioner under anvendelse ektopisk ekspression af polyphosphoinositide 5-phosphatase IV (5ptaseIV), et enzym, der katalyserer hydrolysen af PM-specifik surt phospholipid phosphatidylinositol- (4,5) -bisphosphate [PI (4,5) P2] til phosphatidylinositol-4-phosphat, i celler antydede, at Gag-PM lokalisering medieres af PI (4,5) P2 3,5. Men in vitro-undersøgelser med sigte på at forstå mere mekanistiske detaljer Gag-PI (4,5) P 2 interaktioner har vist sig at være en udfordring for en række årsager. TilEksempelvis har oprensning af fuld længde myristoylerede HIV-1 Gag til biokemiske eksperimenter været teknisk vanskeligt i det mindste delvis på grund af tendensen til, Gag at aggregere under oprensning. Derfor trunkerede former af HIV-1 gag, såsom Myr-MA eller Myr-MA-CA, eller den ikke-myristoylerede formular, er blevet hyppigt anvendt til undersøgelser, der kræver Gag oprensning (f.eks kernemagnetisk resonans, protein footprinting, og overfladen plasmonresonans 6-9). Alternativt koblet in vitro-transkription-translationsreaktioner er blevet anvendt til at producere fuld-længde myristoylerede HIV-1 Gag i andre biokemiske undersøgelser 1,2. Typisk i dette system, er en Gag-kodning plasmid transskriberet og oversat med eukaryote cellelysater (f.eks kanin reticulocytlysater), som er blottet for enhver cellemembraner og messenger RNA&#39;er men indeholder maskiner til transskription og translation. Efter reaktionen er cellelysater indeholdende Gag blandet med migmbranes til analyse af GAG-interaktioner med lipider. Foruden den lette fremstilling fuld længde myristoylerede Gag, fremgangsmåder, der anvender in vitro-transskription translationssystem har en fordel, at Gag-syntese og efterfølgende membran bindingsreaktioner forekommer i en &quot;eukaryot cytosol-lignende&quot; miljø, der kan bedre repræsentere fysiologiske betingelser. Denne egenskab har bidraget til de undersøgelser, der viste, at RNA-molekyler bundet til MA domænet regulere Gag binding til sure lipider i et konkurrencepræget måde 1,2,10-12. Da den samlede mængde af Gag-proteiner opnået ved disse cellelysater er ikke høje, metabolisk mærkning af proteiner med radiomærkede aminosyrer er nødvendig for deres detektion. Afhængig af fremgangsmåden til måling af Gag-lipid interaktioner, har en bred vifte af membranpræparater blevet anvendt. Hver af disse metoder har sine styrker og begrænsninger. De fleste NMR-baserede assays kræver anvendelsen af ​​lipider med korte acylkæder, der er vandopløselige (f.eks C4- og C8-PI (4,5) P2) 6,8. Mens NMR metoder til at teste binding af Gag til de lipider, der har lange acylkæder findes i cellerne der udvikles, er de blevet brugt kun med myristoyleret eller nonmyristoylated MA hidtil 8,13. Alternativt er liposomer fremstillet ud fra lipider, som har native længde acylkæder blevet anvendt i biokemiske fremgangsmåder, såsom liposom flotation eller fluorescerende liposom bead bindingsassays 2,3,10,14-16. Men liposomer anvendt i disse assays har små diametre, og dermed deres membraner har stejle og positive krumninger. I modsætning hertil i den tidlige fase af partikelsamling i HIV-1-inficerede celler, Gag binder til PM, som er næsten plane om omfanget af Gag, og efterfølgende inducerer negativ krumning under knopskydning. Derfor kan liposommembraner med stejle og positiv krumning ikke være ideel lipiddobbeltlag for at studere Gag-lipid interaktioner. Som for liposom Flotation analyser, en anden potentiel advarsel er, at eksponeringen af ​​Gag-lipidkomplekser til hypertonisk saccharosegradient under centrifugering kan påvirke den eksperimentelle resultat. At afhjælpe disse begrænsninger og tilvejebringe en komplementær eksperimentelle system, har assays for Gag-binding til giant unilamellare vesikler (GUVs) blevet udviklet i de senere år. GUVs er enkelte lipiddobbeltlagsvesikler hvis diametre udvides til flere snese mikrometer. Således krumning disse membraner ligner PM på omfanget af Gag. På grund af sin størrelse, som muliggør visuel inspektion under optiske mikroskoper, membran binding af fluorescerende mærkede eller mærkede Gag-proteiner til disse vesikler efter blanding kan let bestemmes uden efterfølgende bearbejdning af Gag-lipidkomplekser. Vi her beskriver en protokol for at studere HIV-1 Gag membran binding ved hjælp GUVs opnået fra en electroformation metode. Forskellige metoder såsom blid hydrering, gel-assisteret hydrering, microfluidic jetting, og electroformation 17-22 er blevet anvendt til at opnå GUVs. For protokollen beskrevet her, er den electroformation anvendte primært på grund af dens effektivitet ved dannelse GUVs med sure lipider og dens relative anvendelse uden behov for dyre opsætninger. Da visualisering af Gag nødvendiggør en fluorescerende reporter, er gult fluorescerende protein (YFP) genetisk tilsat til C-terminalen af ​​Gag (Gag-YFP). Gag-YFP proteiner opnås ved in vitro-transskription og oversættelse reaktioner i hvedekim lysater baseret på den løbende udveksling-kontinuerlig strøm (CECF) teknologi. I denne teknologi er både fjernelse af inhiberende biprodukter af de reaktioner og levering af reaktionssubstrater og energikomponenter opnået i et dialyse-mekanisme. For disse reaktioner, er et plasmid, der koder Gag-YFP under kontrol af en T7-promotor anvendes. Notatet som vist tidligere, hvedekim lysater understøtter myristoylering uden yderligere komponenter 23,24. Ved hjælp af denne metode, har det været muligt at opnå tilstrækkelige mængder af fuld længde myristoylerede Gag-YFP til visualisering af Gag på GUV membraner 24. Her beskriver vi den protokol, hvormed HIV-1 Gag-binding til PI (4,5) P2-holdige GUV membraner kan undersøges uden langvarig efterfølgende bearbejdning efter bindingsreaktioner og foreslår, at denne metode supplerer allerede eksisterende Gag-membran bindingsassays og kan være udvides til yderligere at forstå HIV-1 Gag-membran-binding.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digital Multimeter</td>
			<td>Meterman</td>
			<td>30XR</td>
			<td>A generic multimeter that can measure resistance and volts will serve the purpose.</td>
		</tr>
		<tr>
			<td>Function Generator</td>
			<td>Instek</td>
			<td>GFG-8216A</td>
			<td>A generic function generator that is capable of generating a sine wave at 10hz and 1V is sufficient.</td>
		</tr>
		<tr>
			<td>ITO coated glass slides</td>
			<td>Delta Technologies, Loveland, CO</td>
			<td>CG-90IN</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Hoefer</td>
			<td></td>
			<td>Any incubator that can accurately maintain temperature will be sufficient</td>
		</tr>
		<tr>
			<td>Vacuum chamber</td>
			<td>Nalgene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermomixer R</td>
			<td>Eppendorf</td>
			<td>21516-166</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Hamilton</td>
			<td>80400</td>
			<td>Gauge 22S, Syringe number 702</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Sylgard elastomer base kit, Dow-Corning</td>
			<td>Sylgard, 184</td>
			<td></td>
		</tr>
		<tr>
			<td>RTS 100 Wheat Germ CECF Kit</td>
			<td>BiotechRabbit, 
			Berlin, Germany</td>
			<td>BR1401001</td>
			<td></td>
		</tr>
		<tr>
			<td>DiD</td>
			<td>Life Technologies, Carlsbad, CA&#160;</td>
			<td>D7757</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti Polar Lipids, Alabaster, AL&#160;</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>POPS</td>
			<td>Avanti Polar Lipids</td>
			<td>840034C</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholesterol</td>
			<td>Avanti Polar Lipids</td>
			<td>700041P</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain-PI(4,5)P2&#160;</td>
			<td>Avanti Polar Lipids</td>
			<td>840046X</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of hiv-1 gag binding to giant unilamellar vesicle (guv) membranes

Den strukturelle protein af HIV-1, Pr55 Gag (eller Gag), binder til plasmamembranen i celler under virus samleprocessen. Membran binding af Gag er et vigtigt skridt for dannelse virus partikel, da en defekt i Gag membran bindende resultater i alvorlig svækkelse af viral produktion partikel. For at få mekanistiske detaljer om Gag-lipidmembran interaktioner, in vitro-metoder baseret på NMR, protein footprinting, overfladeplasmonresonans, liposom flotation centrifugering eller fluorescens lipid kuglebinding blevet udviklet hidtil. Men hver af disse in vitro-metoder har sine begrænsninger. For at overvinde nogle af disse begrænsninger og tilvejebringe en komplementær tilgang til de tidligere etablerede metoder, udviklede vi et in vitro assay, hvor interaktioner mellem HIV-1 Gag og lipidmembraner finder sted i en &quot;celle-lignende&quot; miljø. I dette assay Gag binding til lipidmembraner visuelt analyseret under anvendelse YFP-tagged Gag syntetiseret i en hvedekim-baserede in vitro translation system, og GUVs udarbejdet af en electroformation teknik. Her beskriver vi baggrunden og de protokoller, for at opnå Myristoylated fuld længde Gag proteiner og GUV membraner, der er nødvendige for analysen og opdage Gag-GUV binding ved mikroskopi.

<html> Ved hjælp af ovennævnte protokol, vi udarbejdet GUVs sammensat af POPC + POPS + Chol (molforholdet: 4,66: 2,33: 3). Denne sammensætning blev valgt til ca. afspejle PS og kolesterol koncentrationer af PM. Robust og effektiv binding af Gag blev kun observeret når hjerne-PI (4,5) P2 blev medtaget i POPC + POPS + Chol blanding (POPC + POPS + Chol + hjerne-PI (4,5) P2 [molforhold : 4: 2: 3: 1] i dette eksempel) (figur 4, sammenlign paneler B og...

Watch this Scientific Journal Video about Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes at JoVE.com

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

Visualisering af HIV-1 Gag binding til Giant unilamellare vesikel (GUV) Membraner

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Research

JoVE Journal

Immunology and Infection

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating af HIV-1 kerner

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

En<em&gt; In vitro</em&gt; Samtidig infektion model for at studere<em&gt; Plasmodium falciparum</em&gt;-HIV-1 Interactions i humane primære Monocytafledte immunceller

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

Nye værktøjer til at udvide regulatoriske T celler fra HIV-1-inficerede personer

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

Et restriktionsenzym Baseret Kloning metode til at vurdere den<em&gt; In vitro</em&gt; Replikationskapacitet af HIV-1 subtype C Gag-MJ4 Kimære Vira

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

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HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Isolering af Exosomer fra plasma af HIV-1 Positive Personer

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

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måling af<em&gt; In vitro</em&gt; Integration aktivitet af HIV-1 Preintegration komplekser

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...