We thank the National Centre for Cell Science, Department of Biotechnology, Government of India, for intramural support. AT and AD are grateful for the Ph.D. research support received from the National Centre for Cell Science, Department of Biotechnology, Government of India. DM is thankful for the JC Bose National Fellowship from SERB, Government of India.

Studying ER Stress and UPR Activation During HIV-1 Infection in T-Cells

The authors have no conflicts of interest to declare.

The scope of the present protocol includes (i) the handling of HIV-1 virus stocks and the measurement of the virus concentration and virion infectivity, (ii) Infection of T-cells with HIV-1 and assessing its effect on ER stress and different markers of UPR, (iii) Effect of knockdown of UPR markers and their effect on HIV-1 LTR driven gene activity, virus production and virion infectivity and (iv) Overstimulating the UPR using pharmacological molecule and analyzing its effect on HIV-1 replication. Using the present set of...

Acquired immunodeficiency syndrome (AIDS) is characterized by a gradual reduction in the number of CD4+ T-lymphocytes, which leads to the progressive failure of immune response. Human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS. It is an enveloped, positive sense, single-stranded RNA virus with two copies of RNA per virion and belongs to the retroviridae family. Production of high concentrations of viral proteins within the host cell places excessive stress on the protein folding machinery of the cell1. ER is the first compartment in the secretory pathway of eukaryotic cells. It is in charge of producing, altering, and delivering proteins to the secretory pathway and the extracellular space target sites. Proteins undergo numerous post-translational changes and fold into their natural conformation in the ER, including asparagine-linked glycosylation and the creation of intra- and intermolecular disulfide bonds2. Therefore, high concentrations of proteins are present in the ER lumen and these are very prone to aggregation and misfolding. Various physiological conditions, such as heat shock, microbial, or viral infections, which demand enhanced protein synthesis or protein mutation, lead to ER stress due to increased protein accumulation in the ER, thereby disturbing the ER lumen homeostasis. The ER stress activates a network of highly conserved adaptive signal transduction pathways, the Unfolded Protein Response (UPR)3. UPR is employed to bring back the normal ER physiological condition by aligning its unfolded protein burden and folding capacity. This is brought upon by increasing the ER size and ER-resident molecular chaperones and foldases, resulting in an elevation of the ER&#39;s folding ability. UPR also decreases the protein load of the ER through global protein synthesis attenuation at the translational level and increases clearance of unfolded proteins from the ER by upregulating ER-associated degradation (ERAD)4,5.
ER stress is sensed by three ER-resident transmembrane proteins: Protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), Activating transcription factor 6 (ATF6), and Inositol-requiring enzyme type 1 (IRE1). All these effectors are kept inactive by binding to the chaperone Heat shock protein family A (Hsp70) member 5 (HSPA5), also known as binding protein (BiP)/78-kDa glucose-regulated protein (GRP78). Upon ER stress and accumulation of unfolded/misfolded proteins, HSPA5 dissociates and leads to activation of these effectors, which then activate a series of downstream targets that help in resolving the ER stress and, in extreme conditions, promote cell death6. Upon dissociation from HSPA5, PERK autophosphorylates, and its kinase activity is activated7. Its kinase activity phosphorylates eIF2&#945;, which leads to translational attenuation, lowering the protein load of the ER8. However, in the presence of phospho-eukaryotic initiation factor 2&#945; (eIF2&#945;), non-translated open reading frames on specific mRNAs become preferentially translated, such as ATF4, regulating stress-induced genes. ATF4 and C/EBP homologous protein (CHOP) are transcription factors that regulate stress-induced genes and regulate apoptosis and cell death pathways9,10. One of the targets of ATF4 and CHOP is the growth arrest and DNA damage-inducible protein (GADD34), which, along with protein phosphatase 1 dephosphorylate peIF2&#945; and acts as a feedback regulator for translational attenuation11. Under ER stress, ATF6 dissociates from HSPA5, and its Golgi-localization signal is exposed, leading to its translocation to Golgi apparatus. In the Golgi apparatus, ATF6 is cleaved by site-1 protease (S1P) and site-2 protease (S2P) to release the cleaved form of ATF6 (ATF6 P50). ATF6 p50 is then translocated to the nucleus, where it induces the expression of genes involved in protein folding, maturation, and secretion as well as protein degradation12,13. During ER stress, IRE1 dissociates from HSPA5, multimerizes, and auto-phosphorylates14. Phosphorylation of IRE1 activates its RNase domain, specifically mediating the splicing of 26 nucleotides from the central part of the mRNA of X-box binding protein 1 (XBP1)15,16. This generates a novel C-terminus conferring transactivation function, generating functional XBP1s protein, a potent transcription factor controlling several ER stress-induced genes17,18. The combined activity of these transcription factors switches on genetic programs aimed at restoring ER homeostasis.
There are various methods to detect ER stress and UPR. These include the conventional methods of analyzing the UPR markers19,20. Various non-conventional methods include measuring the redox state of the UPR and calcium distribution in the ER lumen as well as assessing the ER structure. Electron microscopy may be used to see how much the ER lumen enlarges in response to ER stress in cells and tissues. However, this method is time-consuming and depends on the availability of an electron microscope, which may not be available to every research group. Also, measuring the calcium flux and the redox state of the ER is challenging due to the availability of reagents. Moreover, the readout from these experiments is very sensitive and might be affected by other factors of cellular metabolism.
A powerful and simple technique for monitoring the UPR outputs is to measure the activation of the different signaling pathways of the UPR and has been used for decades in various stress scenarios. These conventional methods to measure UPR activation are economical, feasible, and provide the information in less time as compared to other known methods. These include immunoblotting to measure the expression of UPR markers at the protein level, such as phosphorylation of IRE1, PERK, and eIF2&#945; and cleavage of ATF6 by measuring the P50 form of ATF6 and protein expression of other markers such as HSPA5, spliced XBP1, ATF4, CHOP and GADD34 as well as RT-PCR to determine the mRNA levels as well as splicing of XBP1 mRNA.
This article describes a validated and reliable set of protocols to monitor ER stress and UPR activation in HIV-1 infected cells and to determine the functional relevance of UPR in HIV-1 replication and infectivity. The protocols utilize easily available as well as economical reagents and provide convincing information about the UPR outputs. ER stress is the result of the accumulation of unfolded/misfolded proteins, which are prone to forming protein aggregates21. We hereby describe a method to detect these protein aggregates in HIV-1-infected cells. Thioflavin T staining is a relatively new method being used to detect and quantify these protein aggregates22. Beriault and Werstuck described this technique to detect and quantify protein aggregates and, hence ER stress levels in live cells. It has been demonstrated that the small fluorescent molecule thioflavin T (ThT) binds selectively to protein aggregates, especially amyloid fibrils.
In this article, we describe the use of ThT to detect and quantify ER stress in HIV-1 infected cells and correlate it to the conventional method of monitoring UPR by measuring the activation of different signaling pathways of UPR.
Since, there is also a lack of comprehensive information regarding the role of UPR during HIV-1 infection, we provide a set of protocols to understand the role of UPR in HIV-1 replication and virion infectivity. These protocols include the lentivirus mediated knockdown of UPR markers as well as treatment with pharmacological ER stress inducers. This article also shows the types of read-out which can be used to measure the HIV-1 gene expression, viral production as well as the infectivity of the produced virions, such as long terminal repeat (LTR)-based luciferase assay, p24 enzyme-linked immunosorbent assay (ELISA) and &#946;-gal reporter staining assay respectively.
Using the majority of these protocols, we have recently reported the functional implication of HIV-1 infection on UPR in T-cells23, and the results of that article suggest the reliability of the methods described here. Thus, this article provides a set of methods for comprehensive information regarding the interplay of HIV-1 with ER stress and UPR activation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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-eIF2&#945; antibody</td>
			<td>Santa Cruz Biotechnology, USA</td>
			<td>sc-11386</td>
			<td>Western blot detection Dilution-1:2000&#160;</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&#160;</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&#160;</td>
			<td>Biorad, USA</td>
			<td>1706516</td>
			<td>Western blot detection Dilution- 1:4000</td>
		</tr>
		<tr>
			<td>anti-peIF2&#945; 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&#160;</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)&#160;</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&#160;</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 cells</td>
		</tr>
		<tr>
			<td>PVDF membrane</td>
			<td>Biorad, USA</td>
			<td>1620177</td>
			<td></td>
		</tr>
		<tr>
			<td>Random primers</td>
			<td>Invitrogen, USA</td>
			<td>48190011</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Invitrogen, USA</td>
			<td>22400105</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Sigma-Aldrich, USA</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Steady-Glo substrate</td>
			<td>Promega, USA</td>
			<td>E2510</td>
			<td>Luciferase assay</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen, USA</td>
			<td>15224017</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Invitrogen, USA</td>
			<td>17919</td>
			<td></td>
		</tr>
		<tr>
			<td>Thapsigargin</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T9033</td>
			<td></td>
		</tr>
		<tr>
			<td>Thioflavin T</td>
			<td>Sigma-Aldrich, USA</td>
			<td>596200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>Q15965</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma-Aldrich, USA</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen, USA</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma-Aldrich, USA</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>TZM-bl</td>
			<td>NIH, AIDS Repository, USA</td>
			<td>8129</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Optima L90K, USA</td>
			<td>330049</td>
			<td>Rotor-SW28Ti</td>
		</tr>
		<tr>
			<td>UltraPure X-gal</td>
			<td>Invitrogen, USA</td>
			<td>15520-018</td>
			<td>Infectivity assay</td>
		</tr>
	</tbody>
</table>

measuring endoplasmic reticulum stress and unfolded protein response in hiv-1 infected t-cells and analyzing its role in hiv-1 replication

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. 
Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2&#945;, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

In this work, we have described a detailed protocol to study in vitro ER stress and UPR activation upon HIV-1 infection in T-cells (Figure 2). This study also describes methods to analyze the functional relevance of UPR in HIV-1 replication and virion infectivity (Figure 3).
To this purpose, we analyzed the ER stress caused by HIV-1 infection by observing the protein aggregates inside the cell by staining with Thioflavin T. A...

Author Spotlight: Exploring the Role of Unfolded Protein Response in HIV-1 Replication and Infectivity

Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

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

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

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Research

JoVE Journal

Immunology and Infection

1.1K Views.  SP Pune University Campus. Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

Video: Author Spotlight: Exploring the Role of Unfolded Protein Response in HIV-1 Replication and Infectivity

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.

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 ...

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.

 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 ...

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.

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 ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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.

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.

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

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

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 conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

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

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

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

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.

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.

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.

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 ...