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

<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

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.

<meta charset="utf8"></head><body>Auteur in de schijnwerpers: onderzoek naar de rol van ongevouwen eiwitrespons bij HIV-1-replicatie en infectiviteit

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

The scope of our research revolves around host pathogen interactions, with a specific focus on understanding the modulation of unfolded protein response pathways during HIV-1 one infection. This study addresses how ER stress and UPR activation markers are altered upon HIV-1 infection, as well as its impact on viral replication and infectivity in T cells. We, for the first time, through a comprehensive study, demonstrated that HIV-1 requires an optimum threshold of UPR activation for its efficient replication, and both induction of UPR beyond this threshold and innovation below this threshold, is deleterious for HIV-1 replication.Here we provide a set of comprehensive protocols to understand the role of UPR in HIV-1 replication, which was lacking in previous studies. This will provide a base for future researchers working on UPR in virus infection, and will help in addressing future challenges. Our study has highlighted several key points for further mechanistic studies to understand the involvement of specific viral proteins, and other host factors, that mediate the regulation of UPR during HIV-1 infection.Also, the involvement of UPR in regulating virion infectivity can be another interesting quest to be solved.

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

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

Abstract