Name: Het meten van endoplasmatisch reticulumstress en ongevouwen eiwitrespons in HIV-1-geïnfecteerde T-cellen en het analyseren van de rol ervan in HIV-1-replicatie (Video) | JoVE Journal
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Description: Leer hoe UPR-modulatie en ER-stress HIV-1-replicatie in T-cellen beïnvloeden met behulp van uitgebreide protocollen. Deze methode biedt inzicht in gastheer-pathogeen interacties.

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<meta charset="utf8"></head><body>Onderzoek naar ER-stress en UPR-activering tijdens HIV-1-infectie in T-cellen

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<table><tbody><tr><td>Acrylamamide</td><td>Biorad, USA</td><td>1610107</td><td></td></tr><tr><td>Agarose</td><td>G-Biosciences, USA</td><td>RC1013</td><td></td></tr><tr><td>Ammonium persulphate</td><td>Sigma-Aldrich, USA</td><td>A3678</td><td></td></tr><tr><td>anti-ATF4 antibody</td><td>Cell Signaling Technology, USA</td><td>11815</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-ATF6 antibody</td><td>Abcam, UK</td><td>ab122897</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-CHOP antibody</td><td>Cell Signaling Technology, USA</td><td>2897</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-eIF2α antibody</td><td>Santa Cruz Biotechnology, USA</td><td>sc-11386</td><td>Western blot detection Dilution-1:2000&nbsp;</td></tr><tr><td>anti-GADD34 antibody</td><td>Abcam, UK</td><td>ab236516</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-GAPDH antibody</td><td>Santa Cruz Biotechnology, USA</td><td>sc-32233</td><td>Western blot detection Dilution-1:3000&nbsp;</td></tr><tr><td>anti-HSPA5 antibody</td><td>Cell Signaling Technology, USA</td><td>3177</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-IRE1 antibody</td><td>Cell Signaling Technology, USA</td><td>3294</td><td>Western blot detection Dilution-1:2000</td></tr><tr><td>Anti-mouse HRP conjugate antibody&nbsp;</td><td>Biorad, USA</td><td>1706516</td><td>Western blot detection Dilution- 1:4000</td></tr><tr><td>anti-peIF2α antibody</td><td>Invitrogen, USA</td><td>44-728G</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-PERK antibody</td><td>Cell Signaling Technology, USA</td><td>5683</td><td>Western blot detection Dilution-1:2000</td></tr><tr><td>anti-pIRE1 antibody</td><td>Abcam, UK</td><td>ab243665</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>anti-pPERK antibody</td><td>Invitrogen, USA</td><td>PA5-40294</td><td>Western blot detection Dilution-1:2000</td></tr><tr><td>Anti-rabbit HRP conjugate antibody</td><td>Biorad, USA</td><td>1706515</td><td>Western blot detection Dilution- 1:4000</td></tr><tr><td>anti-XBP1 antibody</td><td>Abcam, UK</td><td>ab37152</td><td>Western blot detection Dilution-1:1000</td></tr><tr><td>Bench top high speed centrifuge</td><td>Eppendorf, USA</td><td>5804R</td><td>Rotor- F-45-30-11</td></tr><tr><td>Bench top low speed centrifuge</td><td>Eppendorf, USA</td><td>5702R</td><td>Rotor- A-4-38</td></tr><tr><td>Bis-Acrylamide</td><td>Biorad, USA</td><td>1610201</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>MP biomedicals, USA</td><td>160069</td><td></td></tr><tr><td>Bradford reagent</td><td>Biorad, USA</td><td>5000006</td><td></td></tr><tr><td>CalPhos mammalian Transfection kit</td><td>Clontech, Takara Bio, USA</td><td>631312</td><td>Virus stock preparation</td></tr><tr><td>CEM-GFP</td><td>NIH, AIDS Repository, USA</td><td>3655</td><td></td></tr><tr><td>Clarity ECL substrate</td><td>Biorad, USA</td><td>1705061</td><td>chemiluminescence detecting substrate</td></tr><tr><td>Clarity max ECL substrate</td><td>Biorad, USA</td><td>1705062</td><td>chemiluminescence detecting substrate</td></tr><tr><td>Confocal laser scanning microscope</td><td>Olympus, Japan</td><td>Model:FV3000</td><td></td></tr><tr><td>Cytospin centrifuge</td><td>Thermo Fisher Scientific, USA</td><td>ASHA78300003</td><td></td></tr><tr><td>DMEM</td><td>Invitrogen, USA</td><td>11995073</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich, USA</td><td>D2650</td><td></td></tr><tr><td>dNTPs</td><td>Promega, USA</td><td>U1515</td><td></td></tr><tr><td>DTT</td><td>Invitrogen, USA</td><td>R0861</td><td></td></tr><tr><td>EDTA</td><td>Invitrogen, USA</td><td>12635</td><td></td></tr><tr><td>EtBr</td><td>Invitrogen, USA</td><td>`15585011</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Invitrogen, USA</td><td>16000044</td><td></td></tr><tr><td>G418</td><td>Invitrogen, USA</td><td>11811023</td><td></td></tr><tr><td>Glutaraldehyde 25%</td><td>Sigma-Aldrich, USA</td><td>G6257</td><td>Infectivity assay</td></tr><tr><td>Glycine</td><td>Thermo Fisher Scientific, USA</td><td>Q24755</td><td></td></tr><tr><td>HEK-293T</td><td>NCCS, India</td><td></td><td></td></tr><tr><td>HIV-1 infectious Molecular Clone pNL4-3</td><td>NIH, AIDS Repository, USA</td><td>114</td><td></td></tr><tr><td>Inverted microscope</td><td>Nikon, Japan</td><td>Model: Eclipse Ti2</td><td></td></tr><tr><td>iTaq Universal SYBR Green Supermix</td><td>Biorad, USA</td><td>1715124</td><td></td></tr><tr><td>Jurkat J6</td><td>NCCS, India</td><td></td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma-Aldrich, USA</td><td>M8266</td><td>Infectivity assay</td></tr><tr><td>MMLV-RT&nbsp;</td><td>Invitrogen, USA</td><td>28025013</td><td></td></tr><tr><td>MTT reagent</td><td>Sigma-Aldrich, USA</td><td>M5655</td><td>Cell viability assay</td></tr><tr><td>N,N-dimethyl formamide</td><td>Fluka Chemika</td><td>40255</td><td>Infectivity assay</td></tr><tr><td>NaCl</td><td>Thermo Fisher Scientific, USA</td><td>Q27605</td><td></td></tr><tr><td>NaF</td><td>Sigma-Aldrich, USA</td><td>201154</td><td></td></tr><tr><td>NP40</td><td>Invitrogen, USA</td><td>85124</td><td></td></tr><tr><td>P24 antigen capture ELISA kit</td><td>ABL, USA</td><td>5421</td><td></td></tr><tr><td>PageRuler prestained protein ladder</td><td>Sci-fi Biologicals, India</td><td>PGPMT078</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich, USA</td><td>P6148</td><td></td></tr><tr><td>pEGFP-N1</td><td>Clontech, USA</td><td>632515</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen, USA</td><td>151140122</td><td></td></tr><tr><td>Phosphatase Inhibitor</td><td>Sigma-Aldrich, USA</td><td>4906837001</td><td></td></tr><tr><td>Phusion High-fidelity PCR mastermix with GC buffer</td><td>NEB,USA</td><td>M05532</td><td></td></tr><tr><td>pLKO.1-TRC</td><td>Addgene, USA</td><td>10878</td><td>Lentiviral cloning vector</td></tr><tr><td>pMD2.G</td><td>Addgene, USA</td><td>12259</td><td>VSV-G envelope vector</td></tr><tr><td>PMSF</td><td>Sigma-Aldrich, USA</td><td>P7626</td><td></td></tr><tr><td>Polyethylenimine (PEI)&nbsp;</td><td>Polysciences, Inc., USA</td><td>23966</td><td></td></tr><tr><td>Potassium ferricyanide</td><td>Sigma-Aldrich, USA</td><td>244023</td><td>Infectivity assay</td></tr><tr><td>Potassium ferrocyanide</td><td>Sigma-Aldrich, USA</td><td>P3289</td><td>Infectivity assay</td></tr><tr><td>Protease Inhibitor</td><td>Sigma-Aldrich, USA&nbsp;</td><td>5056489001</td><td></td></tr><tr><td>psPAX2</td><td>Addgene, USA</td><td>12260</td><td>Lentiviral packaging plasmid</td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich, USA</td><td>P8833</td><td>Selection of stable

het meten van endoplasmatisch reticulumstress en ongevouwen eiwitrespons in hiv-1-geïnfecteerde t-cellen en het analyseren van de rol ervan in hiv-1-replicatie

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication

Het meten van endoplasmatisch reticulumstress en ongevouwen eiwitrespons in HIV-1-geïnfecteerde T-cellen en het analyseren van de rol ervan in HIV-1-replicatie

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses ...

measuring-endoplasmic-reticulum-stress-unfolded-protein-response-hiv

Research

JoVE Journal

Immunology and Infection

Immunologie en Infectie

We hebben een lentivirale vector die, bezit naast de Tat-responsieve LTR, de Rev-respons element (RRE) die reporter genexpressie kan reguleren in een HIV-1-Tat en Rev-afhankelijke manier. De vector maakt de specifieke detectie van HIV-replicatie in levende cellen via de expressie van GFP.

Specifieke markering van de HIV-1 positieve cellen met behulp van een Rev-afhankelijke lentivirale vector uitdrukken van de Green Fluorescent Protein

Dit artikel beschrijft een methode om de vorming van een HIV-1 envelop geïnduceerde virologische synaps op glas ondersteund vlakke dubbellagen door totale interne reflectie fluorescentie (TIRF) microscopie visualiseren. De werkwijze kan ook worden gecombineerd met immunofluorescentiekleuring tot activering en herverdeling van signaalmoleculen die tijdens HIV-1-geïnduceerde envelop virologische synapsvorming detecteren.

Imaging van HIV-1 Envelope-geïnduceerde Virologische Synapse en signalering op synthetische lipidendubbellagen

We ontwikkelden een in vitro assay om de rol van immuunregulerende trajecten in de regulatie van cytokine secretie door HIV-specifieke CD4 T-cellen te onderzoeken.

In vitro onderzoek naar de impact van immunoregulatoire Pathways op HIV-specifieke CD4 T-cellen Effectorfunctiemanipulatie Evalueer

Unlike celvrij HIV-1 deeltjes geïnfecteerd CD4 + T-cellen effectief waargenomen door plasmacytoïde dendritische cellen (pDCs). Dit manuscript beschrijft een werkwijze waarbij perifere mononucleaire cellen (PBMC's) of geïsoleerde pDCs mede gekweekt met HIV-1 geïnfecteerde T-cellen aangeboren sensing evalueren pDCs zoals bepaald door afgifte van type I IFN.

Het beoordelen van de Aangeboren Sensing van HIV-1 geïnfecteerde CD4<sup&gt; +</sup&gt; T cellen door plasmacytoïde dendritische cellen gebruiken een<em&gt; Ex vivo</em&gt; Co-cultuur systeem.

Methoden om de werkzaamheid en toxiciteit van RNA-moleculen die zich richten op post-integratie stappen van de HIV-1 replicatiecyclus te evalueren, worden beschreven. Deze methoden zijn nuttig voor het screenen van nieuwe moleculen en het optimaliseren van de indeling van bestaande.

Evaluatie van de effectiviteit en toxiciteit van RNA&#39;s Targeting HIV-1 productie voor gebruik in Gene of Drug Therapy

Dit rapport beschrijft een in vitro test voor het meten van de human immunodeficiency virus type 1 (HIV-1) preintegratiecomplex (PIC)-geassocieerde integratie-activiteit in de cytoplasmatische extracten van acuut geïnfecteerde cellen. De integratie van PIC-geassocieerd HIV-1 DNA in heteroloog doel-DNA wordt gekwantificeerd door middel van een geneste real-time polymerasekettingreactiestrategie.

Meting van In Vitro Integratie activiteit van HIV-1 Preintegration Complexen

Beschreven is een methode om te kwantificeren van de expressie van genen die 96 en 18 oppervlakte eiwitten door afzonderlijke cellen ex vivo, waardoor voor de identificatie van differentially uitgedrukte genen en proteïnen in virus-geïnfecteerde cellen ten opzichte van niet-geïnfecteerde cellen. Passen we de aanpak van de studie SIV-geïnfecteerde CD4+ T cellen geïsoleerd van rhesus makaken.

Eencellige kwantificatie van mRNA en oppervlak eiwituitdrukking in Simian Immunodeficiëntie Virus-geïnfecteerde CD4+ T cellen geïsoleerd van Rhesus Makaken

Hier presenteren we een diepe rangschikken benadering waarmee een onbevooroordeelde bepaling van ontluikende 3'-termini en vogelgriepvirus profielen van single-stranded DNA moleculen. De belangrijkste toepassing is de karakterisatie van ontluikende retrovirale complementaire Ani (cDNAs), de tussenproducten die tijdens het proces van retrovirale omgekeerde transcriptie gegenereerd.

Bepalende 3'-Termini en sequenties van ontluikende Single-Stranded virale DNA moleculen tijdens HIV-1 Reverse transcriptie in geïnfecteerde cellen

Hier beschrijven we Rev immunoprecipitatie in de aanwezigheid van HIV-1 replicatie voor massaspectrometrie. De beschreven methoden kunnen worden gebruikt voor de identificatie van nucleollaire factoren die betrokken zijn bij de HIV-1-infectieuze cyclus en zijn van toepassing op andere ziekte modellen voor de karakterisering van onderbestudeerde trajecten.

Identificatie van Nucleollaire factoren tijdens de HIV-1-replicatie via Rev-immunoprecipitatie en massaspectrometrie

Hier presenteren we een protocol waarin afzonderlijke cellen worden gecontroleerd op acute gebeurtenissen en productieve HIV-1-infectie op een nanofluïdisch apparaat. Beeldvormingsgegevens definiëren virus-host receptorinteracties en signaalroutedynamiek. Dit is de eerste methode voor nanofluïdische longitudinale eencellige cultuur en beeldvorming met hoge doorvoer om signaalkinetiek en moleculaire interacties te bestuderen.

Eencellige karakterisering van calciuminstroom en HIV-1-infectie met behulp van een Multiparameter Optofluidic Platform

Leer hoe UPR-modulatie en ER-stress HIV-1-replicatie in T-cellen beïnvloeden met behulp van uitgebreide protocollen. Deze methode biedt inzicht in gastheer-pathogeen interacties.

Het meten van endoplasmatisch reticulumstress en ongevouwen eiwitrespons in HIV-1-geïnfecteerde T-cellen en het analyseren van de rol ervan in HIV-1-replicatie (Video) | JoVE Journal

Het meten van endoplasmatisch reticulumstress en ongevouwen eiwitrespons in HIV-1-geïnfecteerde T-cellen en het analyseren van de rol ervan in HIV-1-replicatie

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