Questo lavoro è stato supportato da un supplemento ARRA alla durata della vita / Tufts / Brown CFAR, P30AI042853-13S1, NIH P20GM103421, P01AA019072, R01HD072693, e K24HD080539 a BR. Questo lavoro è stato supportato anche da Fondo Durata pilota di ricerca (# 701- 5857), Rhode Island Medical Research Foundation Grant (# 20.133.969), e NIH COBRE URI / RIH Pilot Research Grant (P20GM104317) per ML. Ringraziamo James Myall e Vy Dang per un aiuto con la preparazione del manoscritto e la figura.

1. Colture cellulari e HIV-1 NOTA: Prima di iniziare gli esperimenti, si consiglia di verificare la vitalità delle cellule attraverso Trypan Blue colorazione 11 e la loro proliferazione attraverso un saggio di MTT 12. E &#39;inoltre fondamentale utilizzare media SILAC appena preparata. Varie linee cellulari possono essere usati, purché siano in fase attiva proliferativa, e sono suscettibili di HIV-1, o la condizione di test di scelta. In questo protocollo, usare la linea cellulare H9 come l&#39;esempio. <ol><li> Seed 2 x 10 6 cellule H9 in ciascun pallone di coltura cellulare. Grow un gruppo in 10 ml etichettati terreno RPMI 1640 contenente 10% siero bovino fetale dializzato (FBS), 100 mg / L 13 C 6 L-lisina e 100 mg / L 13 C 6 15 N 4 L-arginina. Grow l&#39;altro gruppo in 10 ml senza etichetta terreno RPMI 1640 con 10% FBS dializzato, 100 mg / L di L-lisina e 100 mg / L di L-arginina. </li><li>Far crescere le cellule per sei raddoppi a quel punto le proteine ​​delle cellule nel mezzo etichettato sono praticamente completamente etichettati (&gt; 99%) con aminoacidi pesanti. Aggiungere mezzi freschi o cambiare supporto regolarmente (ogni 1-3 giorni a seconda del tipo di cellula. Per cellule H9, cambiare media ogni 3 giorni). Alla fine di etichettatura, aumentare il volume di coltura (per es ~ 30 ml) per accogliere la crescita di nuove cellule. NOTA: Determinare cella esatto tempo di raddoppio all&#39;inizio dell&#39;esperimento tramite colorazione Trypan blu e conteggio delle cellule. </li><li> Infettare le cellule marcate con HIV-1 NL4-3 utilizzando standard di HIV-1 infezione protocollo 13 per 48 ore. Incubare le cellule con quantità appropriate di virus con una molteplicità di infezione (MOI) circa 0,3. Controllare HIV-1 da p24 quantificazione dei sovranatanti di coltura utilizzando un kit di p24 HIV-1 ELISA. Eseguire un test di immunofluorescenza 14 per confermare l&#39;infezione con successo delle cellule. Keep crescere le cellule non etichettati in media senza etichetta, senza HIV-1. </li><li> Raccogliere i supernatanti di entrambi i gruppi alla fine di infezione (passo 2.1). </li></ol> 2. Isolamento Exosome NOTA: Attraverso una serie di passaggi ultracentrifugazione, frazioni exosomal da sovranatanti sono arricchiti 15. Eseguire le seguenti operazioni a 4 ° C, con un rotore Ultracentrifuge che può raggiungere una velocità di almeno 100.000 x g. <ol><li> Raccogliere i surnatanti delle colture in 50 ml provette coniche, evitando le cellule. li centrifugare per 10 minuti a 300 xg per rimuovere le cellule rimanenti. </li><li> Raccogliere il surnatante in nuovi 50 ml provette coniche e centrifugare per 10 minuti a 2.000 xg per rimuovere le cellule morte. Trasferimento risultante supernatanti a tubi rotori compatibile commerciali che sono in grado di resistere ultracentrifugazione. </li><li> Assicurati di bilanciare i tubi ultracentrifuga. Centrifugare le provette per 30 min a 10.000 xg per rimuoveredetriti cellulari. </li><li> Raccogliere i surnatanti in tubi ultracentrifuga e centrifugare per 70 min a 100.000 x g. Eliminare il surnatante. </li><li> Risospendere il pellet ricco di exosome in 5 ml di PBS fresco. Trasferire le soluzioni a tubi ultracentrifuga freschi e centrifugare ancora per 70 min a 100.000 x g. </li><li> surnatanti scartare. Per memorizzare i exosomes a lungo termine, risospendere il pellet in 50 ml di PBS e conservare a -80 ° C. In alternativa, procedere direttamente alla estrazione di proteine ​​come illustrato di seguito. </li></ol> 3. Proteine ​​estrazione e preparazione <ol><li> Sciogliere isolati pellet exosomal a 100-200 ml RIPA lisi e l&#39;estrazione del buffer con cocktail inibitori della proteasi aggiunto. Il buffer è composta da 25 mM Tris-cloridrato, cloruro di sodio 150 mM, 1% NP-40, 1% sodio desossicolato, e 0,1% SDS (sodio dodecil solfato), a pH 7,6. I cocktail di inibitori della proteasi dovrebbero contenere una varietà di inibitori delle proteasi, come ad esempio l&#39;aprotinina, Bestatin, leupeptin, pepstatina A e EDTA (acido etilendiamminotetraacetico), che può inibire una gamma completa di proteasi. </li><li> Centrifugare le soluzioni disciolti per 10 minuti a 13.000 xg (4 ° C), e trasferire il surnatante eliminato per nuovi tubi da 1,5 ml microcentrifuga. </li><li> Quantificare concentrazione di proteine di ogni campione di esosomi con acido bicinconinico (BCA) o saggio Bradford 16. </li><li> Miscelare una quantità uguale di proteine ​​(2 mg) da campioni etichettati e senza etichetta ed eseguire la miscela uguale su un gel SDS-PAGE 4-20% a 120 mA / 200 V per 30 min. </li><li> Gel Stain con Coomassie blu seguito da decolorazione 17. </li><li> Utilizzando una lama di rasoio, tagliare la corsia campione dal gel. Poi tagliare la corsia di gel in 10-15 pezzi uguali. Mettere ogni pezzo in un 1,5 ml provetta fresco per un totale di 10-15 tubi. </li><li> Immergere cubi in 25 mM NH 4 HCO 3 nel 50% acetonitrile, vortice, e scartare surnatanti. Ripetere due volte. Asciuttocubi in un concentratore a vuoto 18, 19. </li><li> Reidratare cubi con 10 mM ditiotreitolo (DTT), vortex e centrifugare brevemente. Incubare a 56 ° C per 1 ora. surnatante degli scarti. </li><li> Immergere cubi di gel a 55 mm Iodoacetamide, vortice, ed effetti. Incubare a temperatura ambiente al buio per 45 min. surnatante degli scarti. </li><li> Immergere cubi in 25 mM NH 4 HCO 3, vortice, rotazione, e scartare il surnatante. </li><li> Immergere cubi in 25 mM NH 4 HCO 3 nel 50% acetonitrile, vortice, ed effetti. Ripetere 3.9 e 3.10. </li><li> Asciugare i cubi in un concentratore a vuoto. Aggiungere 25 ml di sequenziamento grado tripsina modificata in 25 mM NH 4 HCO 3, incubare a 4 ° C per 30 minuti, e scartare la soluzione in eccesso. Immergere cubi in 25 mM NH 4 HCO 3 senza tripsina, e incubare una notte a 37 ° C. </li><li> Brevemente girare cubetti verso il basso e trasferire il peptide risultante in piùct in una nuova provetta. Aggiungere 30 ml di acido formico al 5% in 50% acetonitrile ai cubi, vortex per 30 min, e rotazioni. Unire il surnatante con l&#39;estratto. Essiccare gli estratti peptide con un concentratore a vuoto a meno di 5 microlitri. </li><li> Invia campioni di impianto di base spettrometria di massa per l&#39;analisi LC-MS / MS. L&#39;analisi dei dati di MS, che può essere generato da un software open source, in genere include un numero di registrazione per ogni proteina identificata, etichettati rapporti / senza etichetta e il numero di peptidi unici identificati. </li></ol> 4. Western Blotting di verifica NOTA: Western blotting si raccomanda di verificare i risultati di spettrometria di massa. <ol><li> Seguire i protocolli Western Blotting standard e garantire che le quantità uguali di proteine ​​di ogni gruppo vengono caricati. Usare gli anticorpi contro le proteine di interesse (ad esempio A5 annexin, lattato deidrogenasi a catena B) individuati dalla SM. rilevamento Western blotting, insieme a densitometria analisi des, in grado di ribadire che l&#39;identificazione delle proteine ​​MS e quantificazione siano corretti. </li></ol> 5. Analisi dei dati proteomica NOTA: la valutazione della qualità dei dati, pre-trattamento dei dati, il calcolo e la determinazione di importanti candidati proteine ​​sono fatte separatamente per ciascuno Stato membro si replicano. Una volta che le analisi di cui sopra sono stati completati, i dati analizzati da repliche vengono confrontati e combinati 6, 20, 21. <ol><li> Valutare la qualità dei dati MS. <ol><li> In primo luogo, log2 trasformare la SILAC-MS etichettato / rapporti senza etichetta di proteine ​​quantificate in un foglio di calcolo. </li><li> Nella rappresentazione grafica scientifica e software statistico, i rapporti di gruppo in 40-100 bidoni rapporto e tracciare il numero di rapporti per scomparto per generare un istogramma. Un normale distribuzione di istogramma indica una buona qualità dei dati MS 6. Fare questo passo per tutta la MS replica. </li></ol></li><li>Per aumentare la fiducia e l&#39;accuratezza dei rapporti peptide MS, prendere in considerazione la rimozione di proteine ​​che hanno meno di due peptidi quantificati. </li><li> Per determinare i candidati di proteine in modo significativo l&#39;alto e verso il basso-regolato, utilizzare le seguenti operazioni per il calcolo soglie significative 22. <ol><li> Nella rappresentazione grafica scientifica e software statistico, generare una regressione non lineare o una curva-fit per i dati di distribuzione di frequenza. Questo passo produce valori che possono essere utilizzati per calcolare i valori di cutoff. Calcolare i valori di cut-off come mediana ± 1,96 σ per il 95% limiti di confidenza o 2,56 σ per il 99% limiti di confidenza. NOTA: I dettagli specifici del calcolo dei tagli può essere trovato alla Emmott et al. 22. </li><li> Selezionare i candidati proteina la cui rapporti sono o superiore a 1,96 (o 2,56) deviazioni standard sopra la mediana (significativamente sovra-espresso) o inferiore a 1,96 (o 2,56) deviazioni standard al di sotto della mediana (significativamente sotto-espressi). </li></ol></li><li> Confrontare le repliche dei candidati rilevanti sopra individuati per garantire la coerenza. Assicurarsi che soddisfano i seguenti criteri di passare questa fase di selezione finale: i candidati devono individuare in modo coerente in tutte le repliche; e repliche di un candidato devono essere coerenti nella loro direzione, del regolamento (preferibilmente almeno 2 su 3 repliche di un candidato dovrebbe essere o up-regolati o down-regolato). </li><li> Finalizzare i dati per i candidati che soddisfano i criteri di cui sopra unendo i dati replicati loro. </li></ol> 6. Bioinformatica verifica e caratterizzazione NOTA: esistenti informazioni genomiche e bioinformatica offre una ricchezza di informazioni per quasi ogni proteina. Data mining e analisi bioinformatica di tali informazioni possono aiutare ad acquisire una grande quantità di comprensione della proprietà e funzioni dei candidati significativi. questa process è di solito necessario per una corretta progettazione di esperimenti su valle. <ol><li> Usando i loro numeri GenBank adesione o gli ID UniProt, la ricerca di candidati contro database exosome correnti (Exocarta 23, Evpedia 24) per verificare che le proteine candidati sono stati precedentemente trovati in exosome. Questo passaggio aggiunge un livello di fiducia che i candidati sono infatti in esosomi. <ol><li> http://www.exocarta.org/ accesso, fai clic su &quot;Query&quot; e in ingresso sia il gene o una proteina nome / numero di adesione. Una pagina di riepilogo apparirà e le prove in exosome verrà mostrato, a condizione che se non vi è alcuna. </li></ol></li><li> Cerca i candidati contro l&#39;HIV-1 e la proteina umana Interazione Database 25. In alternativa, la ricerca di basi di dati specifici che possono fornire informazioni circa l&#39;interazione delle proteine ​​candidati e la condizione di test. <ol><li> Accedere al database a www.ncbi.nlm.nih.gov/RefSeq/HIVInterazioni. Sulla voce &#39;proteina nome di dominio&#39;, inserire i nomi dei candidati o dei numeri di adesione, e fare clic su Cerca. I risultati della ricerca possono fornire una conoscenza delle interazioni tra HIV-1 ei candidati della proteina, e suggerire che dei candidati potrebbe essere veramente HIV-1 associata. </li></ol></li><li> Importa GenBank numero o UniProt gli ID dei candidati in software di analisi GO (come FunRich, strumento di analisi di arricchimento funzionale 26) ed eseguire il software. <ol><li> Scaricare il software a http://www.funrich.org/. Dopo l&#39;installazione, aprire il software, in analisi di arricchimento, fare clic su &quot;Aggiungi Data Set&quot;, e caricare l&#39;elenco delle proteine ​​/ geni. Avanti, selezionare il tipo di grafico per visualizzare l&#39;analisi GO. NOTA: GO risultati delle analisi saranno visualizzati sotto forma di grafici a torta. Questo passo ci aiuta a ottenere una visione globale associato con i candidati nei settori della Processo biologico (BP), Cellular componente (CC), e funzione molecolare (MF). </li></ol></li><li> L&#39;accesso a DAVID http://david.ncifcrf.gov/ 27, fare clic su &quot;strumento di annotazione funzionale&quot; sul suo sito web. Inserire la lista dei candidati, selezionare l&#39;opzione &quot;Identifier&quot; del gene, come la &quot;UniProt ID&quot;, selezionare &quot;Lista Gene&quot; e di ricerca. I termini arricchiti GO, p-value, e altri parametri possono essere trovate cliccando la pagina &quot;Riepilogo risultati annotazione&quot; sotto la categoria &quot;Gene_Ontology&quot;. </li><li> Per studiare potenziali interazioni dei candidati, con altre proteine, usare apertamente accessibile database di STRING 28 per chiarire potenziali interazioni proteina-proteina e le possibili vie biochimiche. NOTA: GO analisi e annotazione funzionale (Sezione 6,3-6,4) può essere eseguita utilizzando il software più recente STRING. <ol><li> Accedere al database a http://string-db.org/. Inserire l&#39;ID di proteine ​​o la sequenza nelle s designatiscatola earch e selezionare le specie corretti per l&#39;analisi. Fai clic su &quot;Cerca&quot;. I risultati forniranno informazioni su entrambe le associazioni dirette (fisico) e indiretti (funzionale). I primi dieci partite noti per il candidato exosomal verranno visualizzati e devono essere considerati per la selezione significativa candidato. NOTA: Una grande quantità di informazioni associate con i candidati possono essere analizzati utilizzando la procedura descritta sopra. I candidati più significativi possono essere scelti per ulteriori analisi molecolari e biochimiche valle. </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4 (1), 44-57 (2009).</li><li>Szklarczyk, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STRING+database+in+2011:+functional+interaction+networks+of+proteins,+globally+integrated+and+scored.">The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.</a> Nucleic Acids Res. 39, 561-568 (2011).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , (2006).</li><li>Lobb, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> J Extracell Vesicles. 4, 27031 (2015).</li><li>Webber, J., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+pure+are+your+vesicles.">How pure are your vesicles.</a> J Extracell Vesicles. 2, (2013).</li><li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+exosomes+and+HIV-1:+purification+of+both+vesicles+from+cell-free+supernatants.">Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants.</a> J Immunol Methods. 338 (1-2), 21-30 (2008).</li><li>Chertova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+and+biochemical+analysis+of+purified+human+immunodeficiency+virus+type+1+produced+from+infected+monocyte-derived+macrophages.">Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.</a> J Virol. 80 (18), 9039-9052 (2006).</li><li>Nikolov, M., Schmidt, C., Urlaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry-based+proteomics:+an+overview.">Quantitative mass spectrometry-based proteomics: an overview.</a> Methods Mol Biol. 893, 85-100 (2012).</li></ol>

The authors have declared no conflict of interest.

Nelle procedure descritte in questo documento, abbiamo dimostrato l&#39;applicazione della tecnica SILAC per studiare l&#39;effetto di HIV-1 sul proteoma exosomal host. Inizialmente, le cellule infette non infettate e HIV-1 sono differenziale marcati con isotopi. I exosomes differenziale etichettati vengono quindi purificati prima di eseguire l&#39;estrazione di proteine. Avanti, cromatografia liquida-spettrometria di massa tandem è impiegato per analizzare il proteoma exosomal. Infine, i dati risultanti spettrometria ...

Molte malattie umane, tra cui le infezioni virali, sono spesso associate a processi cellulari distintivi che si svolgono dentro e intorno alle cellule colpite. Le proteine, spesso in qualità di effettori cellulari finale, mediare questi processi. L&#39;analisi delle proteine, spesso in grado di fornire informazioni preziose per l&#39;ambiente locale di cellule colpite e ci aiutano a comprendere il meccanismo alla base della patogenesi della malattia. Tra le varie tecniche di analisi delle proteine, la proteomica tiene particolarmente grande promessa. Come un potente strumento, su larga scala, proteomica possono fornire una comprensione sistemica di processi cellulari, in particolare nella zona della funzione e l&#39;interazione di proteine. Analizzando proteine ​​specifiche è resa più semplice grazie allo sviluppo di tecniche di marcatura, che consentono investigatori per monitorare l&#39;espressione di componenti cellulari, in particolare proteine, nel sito di indagine. Anche se molte analisi proteomica sono stati effettuati a livello cellularescala proteoma, caratterizzazioni proteomica su compartimenti subcellulari hanno dimostrato di essere particolarmente informativo 1. Questo è esemplificato bene negli studi di HIV-1. Esosomi, 30-100 vescicole di membrana nm secreti da una vasta gamma di tipi di cellule 2, 3, sono componenti critici di comunicazione intercellulare e di trasporto molecolare. Essi sono stati scoperti in precedenza a svolgere ruoli importanti nella HIV-1 in erba processo di 4, 5. Grazie alla combinazione di analisi proteomica con dissezione funzionale, abbiamo scoperto che esosomi rilasciati da HIV-1 le cellule infettate sono composti da un unico e quantitativamente diversi firma proteine e molecole regolatrici del porto che hanno un impatto proprietà cellulari su cellule vicine ricettive, tra cui l&#39;apoptosi cellulare e la proliferazione 6. I metodi sono descritti in questo protocollo,vale a dire SILAC (etichettatura degli isotopi stabili da aminoacidi nelle cellule in coltura) 7 sulla caratterizzazione proteomica di esosomi da HIV-1 le cellule infette. Approcci simili possono essere applicati per comprendere meglio altri compartimenti subcellulari durante patogenesi regolando stress sperimentale al compartimento o frazione di specifico interesse e di apportare modifiche necessarie alle procedure descritte. Dato il recente sviluppo di metodi di proteomica quantitativa, ci sono molti tra cui scegliere quando si seleziona il metodo più efficiente per un particolare esperimento. Tra questi ci sono il iTRAQ chimico-based (tag isobariche per la quantificazione relativa e assoluta) 8 e il libero etichetta MRM (monitoraggio reazione multipla) 9 tecniche. Entrambi i metodi sono strumenti potenti e sono buone scelte per le impostazioni specifiche. Per un laboratorio tipico lavorando principalmente con linee cellulari, tuttavia, questi due metodi hanno relatively costi più elevati e sono più tempo rispetto al metodo basato SILAC. SILAC è una tecnica di marcatura metabolica based che integra forme isotopiche non radioattivi di aminoacidi dal terreno di coltura in proteine ​​cellulari. In genere, gli esperimenti SILAC cominciano con due popolazioni di cellule, per esempio, infetti e non infetti. Ciascuno è differenzialmente etichettati nel suo ambiente isotopico specifico fino al raggiungimento della etichettatura. I exosomes etichettati di queste cellule vengono poi sottoposti ad estrazione delle proteine. Una volta estratti, le proteine exosomal marcate vengono analizzati utilizzando cromatografia liquida tandem massa spettroscopia 10. Infine, i risultati di spettrometria di massa e proteine ​​significativamente marcate sono sottoposti a statistica e bioinformatica analisi e verifica rigorosa biochimico. I nostri rapporti investigativi precedenti suggeriscono che le procedure SILAC / exosome sono più appropriati per linee cellulari di cellule primarie, come linee cellulari sono di solito in unstato proliferante attivo per l&#39;etichettatura isotopica efficiente,

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">H9 cell line</td>
			<td style="width:121px;">ATCC</td>
			<td style="width:111px;">HTB-176</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Trypan Blue</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">15250061</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MTT assay kit</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">V13154</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:227px;">Dialyzed fetal bovine serum (FBS)</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">26400044</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">SILAC Protein Quantitation Kit &#8211; RPMI 1640</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89982</td>
			<td style="width:168px;">DMEM version (89983)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">L-Arginine-HCl, 13C6, 15N4 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88434</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">L-Lysine-2HCl, 13C6 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88431</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">HIV-1NL4-3&#160;&#160;</td>
			<td style="width:121px;">NIH AIDS Reagent Program</td>
			<td style="width:111px;">2480</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Alliance HIV-1 p24 Antigen ELISA kit</td>
			<td style="width:121px;">PerkinElmer</td>
			<td style="width:111px;">NEK050001KT</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated super-speed centrifuge</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">22628045</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated ultracentrifuge</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">363118</td>
			<td style="width:168px;">Should be able to reach 100,000g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">14-432-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ultracentrifuge Tubes</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">326823</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SW 32 Ti Rotor</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">369694</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">RIPA buffer</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89900</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Protease Inhibitor Cocktails</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">&#160;78430</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">ThermoMixer&#160;</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">5384000020</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">BCA Protein Assay Kit&#160;</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">23250</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spectrophotometer</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:111px;">1702525</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SDS PAGE Gel apparatus</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">EI0001</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Novex 4-20% Tris-Glycine Mini Gels</td>
			<td style="width:121px;">Novex</td>
			<td style="width:111px;">XV04200PK20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Gel staining reagent</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:111px;">G1041</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sequencing Grade Modified Trypsin</td>
			<td style="width:121px;">Promega</td>
			<td style="width:111px;">V5111</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SpeedVac Concentrator</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">SPD131DDA</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Antibody to human annexin A5</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab14196</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Antibody to human lactate dehydrogenase B chain</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab53292</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:227px;">Graphing and Statistical Software</td>
			<td style="width:121px;">Systat&#160;</td>
			<td style="width:111px;">SigmaPlot&#160;</td>
			<td style="width:168px;">Or GraphPad Prism</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Quantitative proteomics software suite</td>
			<td style="width:121px;">Max Planck Institue of Biochemistry</td>
			<td style="width:111px;">Maxquant&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Software and databases</td>
			<td style="width:121px;">Various vendors</td>
			<td style="width:111px;"></td>
			<td style="width:168px;">Refer to main text for details</td>
		</tr>
	</tbody>
</table>

silac based proteomic characterization of exosomes from hiv-1 infected cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

La figura 1A è un diagramma di flusso che illustra la procedura di etichettatura SILAC 21. Al fine di purificare gli esosomi, i campioni devono essere centrifugati tramite centrifuga. Figura 1B mostra le fasi di purificazione exosome di ultracentrifugazione di serie 21. Una volta purificate, le esosomi sono oggetto di analisi sperimentale proteomica come indicato nella procedura. <p class="jove_content...

Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Qui, descriviamo un metodo di proteomica quantitativa utilizzando la tecnica di etichettatura degli isotopi stabili da aminoacidi nelle cellule in coltura (SILAC) per analizzare gli effetti di HIV-1 su proteomi exosomal host. Questo protocollo può essere facilmente adattato alle cellule in diverse condizioni di stress o infezione.

SILAC Based proteomica Caratterizzazione di Esosomi da HIV-1 le cellule infette

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

silac-based-proteomic-characterization-exosomes-from-hiv-1-infected

Research

JoVE Journal

Immunology and Infection

10.6K Views.  Brown University.  Qui, descriviamo un metodo di proteomica quantitativa utilizzando la tecnica di etichettatura degli isotopi stabili da aminoacidi nelle cellule in coltura (SILAC) per analizzare gli effetti di HIV-1 su proteomi exosomal host. Questo protocollo può essere facilmente adattato alle cellule in diverse condizioni di stress o infezione. 

Video: SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

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 viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Inferenza genotipica del virus HIV-1 Tropismo utilizzo basati sulla popolazione sequenziamento del 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 ...

Ricerca

Immunologia e infezioni

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

Preparazione e utilizzo di HIV-1 CD4 infetti primaria + T-Cells come cellule bersaglio nei test di cellule Natural Killer citotossici

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplificazione e quantificazione di HIV-1 RNA in soggetti affetti da HIV con una carica virale inferiore il limite di rilevamento da parte di Standard saggi clinici

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

In vitro Uncoating of HIV-1 Cores

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 composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

In uncoating vitro di HIV-1 Cores

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 ...

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

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

Nuovi strumenti per espandere cellule T regolatorie di individui HIV-1-infetti

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

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

Valutare l&#39;innato rilevamento dell&#39;infezione da HIV-1 CD4 infetti<sup&gt; +</sup&gt; Cellule T dalle cellule dendritiche plasmacitoidi Utilizzando un<em&gt; Ex vivo</em&gt; Sistema di co-coltura.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Valutazione conformazionale di HIV-1 trimeric busta glicoproteine ​​Utilizzando una base di Cell-ELISA Saggio

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

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. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

Isolamento di esosomi dal plasma di HIV-1 individui positivi

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. ...

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

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 essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

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.

Visualizzazione del virus HIV-1 gag rilegatura a Giant unilamellari vescicole (GUV) Membrane

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 ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.