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....

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.......

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

Name	Company	Catalog Number	Comments
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