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

Summary

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.

Abstract

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 eIF2α, 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.

Introduction

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'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 eIF2α, which leads to translational attenuation, lowering the protein load of the ER⁸. However, in the presence of phospho-eukaryotic initiation factor 2α (eIF2α), 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 pathways^9,10. 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 peIF2α 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 degradation^12,13. 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 genes^17,18. 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 markers^19,20. 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 eIF2α 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 β-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.

Protocol

NOTE: The cell lines used here are HEK-293T and Jurkat J6 (a CD4+T cell line), which were obtained from the Cell Repository, NCCS, Pune, India; TZM-bl, a HeLa derived cell line that has integrated copies of β-galactosidase and luciferase genes under the HIV-1 long terminal repeat (LTR) promoter24 and CEM-GFP (another CD4+ T reporter cell line)25 were obtained from the NIH AIDS Repository, USA. 1. HIV-1 virus stock preparation and stora…

Representative Results

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…

Discussion

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…

Materials

Acrylamamide	Biorad, USA	1610107
Agarose	G-Biosciences, USA	RC1013
Ammonium persulphate	Sigma-Aldrich, USA	A3678
anti-ATF4 antibody	Cell Signaling Technology, USA	11815	Western blot detection Dilution-1:1000
anti-ATF6 antibody	Abcam, UK	ab122897	Western blot detection Dilution-1:1000
anti-CHOP antibody	Cell Signaling Technology, USA	2897	Western blot detection Dilution-1:1000
anti-eIF2α antibody	Santa Cruz Biotechnology, USA	sc-11386	Western blot detection Dilution-1:2000
anti-GADD34 antibody	Abcam, UK	ab236516	Western blot detection Dilution-1:1000
anti-GAPDH antibody	Santa Cruz Biotechnology, USA	sc-32233	Western blot detection Dilution-1:3000
anti-HSPA5 antibody	Cell Signaling Technology, USA	3177	Western blot detection Dilution-1:1000
anti-IRE1 antibody	Cell Signaling Technology, USA	3294	Western blot detection Dilution-1:2000
Anti-mouse HRP conjugate antibody	Biorad, USA	1706516	Western blot detection Dilution- 1:4000
anti-peIF2α antibody	Invitrogen, USA	44-728G	Western blot detection Dilution-1:1000
anti-PERK antibody	Cell Signaling Technology, USA	5683	Western blot detection Dilution-1:2000
anti-pIRE1 antibody	Abcam, UK	ab243665	Western blot detection Dilution-1:1000
anti-pPERK antibody	Invitrogen, USA	PA5-40294	Western blot detection Dilution-1:2000
Anti-rabbit HRP conjugate antibody	Biorad, USA	1706515	Western blot detection Dilution- 1:4000
anti-XBP1 antibody	Abcam, UK	ab37152	Western blot detection Dilution-1:1000
Bench top high speed centrifuge	Eppendorf, USA	5804R	Rotor- F-45-30-11
Bench top low speed centrifuge	Eppendorf, USA	5702R	Rotor- A-4-38
Bis-Acrylamide	Biorad, USA	1610201
Bovine Serum Albumin (BSA)	MP biomedicals, USA	160069
Bradford reagent	Biorad, USA	5000006
CalPhos mammalian Transfection kit	Clontech, Takara Bio, USA	631312	Virus stock preparation
CEM-GFP	NIH, AIDS Repository, USA	3655
Clarity ECL substrate	Biorad, USA	1705061	chemiluminescence detecting substrate
Clarity max ECL substrate	Biorad, USA	1705062	chemiluminescence detecting substrate
Confocal laser scanning microscope	Olympus, Japan	Model:FV3000
Cytospin centrifuge	Thermo Fisher Scientific, USA	ASHA78300003
DMEM	Invitrogen, USA	11995073
DMSO	Sigma-Aldrich, USA	D2650
dNTPs	Promega, USA	U1515
DTT	Invitrogen, USA	R0861
EDTA	Invitrogen, USA	12635
EtBr	Invitrogen, USA	`15585011
Fetal Bovine Serum	Invitrogen, USA	16000044
G418	Invitrogen, USA	11811023
Glutaraldehyde 25%	Sigma-Aldrich, USA	G6257	Infectivity assay
Glycine	Thermo Fisher Scientific, USA	Q24755
HEK-293T	NCCS, India
HIV-1 infectious Molecular Clone pNL4-3	NIH, AIDS Repository, USA	114
Inverted microscope	Nikon, Japan	Model: Eclipse Ti2
iTaq Universal SYBR Green Supermix	Biorad, USA	1715124
Jurkat J6	NCCS, India
Magnesium chloride	Sigma-Aldrich, USA	M8266	Infectivity assay
MMLV-RT	Invitrogen, USA	28025013
MTT reagent	Sigma-Aldrich, USA	M5655	Cell viability assay
N,N-dimethyl formamide	Fluka Chemika	40255	Infectivity assay
NaCl	Thermo Fisher Scientific, USA	Q27605
NaF	Sigma-Aldrich, USA	201154
NP40	Invitrogen, USA	85124
P24 antigen capture ELISA kit	ABL, USA	5421
PageRuler prestained protein ladder	Sci-fi Biologicals, India	PGPMT078
Paraformaldehyde	Sigma-Aldrich, USA	P6148
pEGFP-N1	Clontech, USA	632515
Penicillin/Streptomycin	Invitrogen, USA	151140122
Phosphatase Inhibitor	Sigma-Aldrich, USA	4906837001
Phusion High-fidelity PCR mastermix with GC buffer	NEB,USA	M05532
pLKO.1-TRC	Addgene, USA	10878	Lentiviral cloning vector
pMD2.G	Addgene, USA	12259	VSV-G envelope vector
PMSF	Sigma-Aldrich, USA	P7626
Polyethylenimine (PEI)	Polysciences, Inc., USA	23966
Potassium ferricyanide	Sigma-Aldrich, USA	244023	Infectivity assay
Potassium ferrocyanide	Sigma-Aldrich, USA	P3289	Infectivity assay
Protease Inhibitor	Sigma-Aldrich, USA	5056489001
psPAX2	Addgene, USA	12260	Lentiviral packaging plasmid
Puromycin	Sigma-Aldrich, USA	P8833	Selection of stable cells
PVDF membrane	Biorad, USA	1620177
Random primers	Invitrogen, USA	48190011
RPMI 1640	Invitrogen, USA	22400105
SDS	Sigma-Aldrich, USA	L3771
Steady-Glo substrate	Promega, USA	E2510	Luciferase assay
T4 DNA ligase	Invitrogen, USA	15224017
TEMED	Invitrogen, USA	17919
Thapsigargin	Sigma-Aldrich, USA	T9033
Thioflavin T	Sigma-Aldrich, USA	596200
Tris	Thermo Fisher Scientific, USA	Q15965
Triton-X-100	Sigma-Aldrich, USA	T8787
Trizol	Invitrogen, USA	15596018
Tween 20	Sigma-Aldrich, USA	P1379
TZM-bl	NIH, AIDS Repository, USA	8129
Ultracentrifuge	Beckman Optima L90K, USA	330049	Rotor-SW28Ti
UltraPure X-gal	Invitrogen, USA	15520-018	Infectivity assay