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

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 storage

1. HIV-1 virus stock preparation
   NOTE: It is advised to practice biosafety-related international guidelines as available in the World Health Organization (WHO) or Centers for Disease Control and Prevention (CDC) manual in all experiments involving HIV-1. Handling of the virus and any experiment with the live virus should only be done in an appropriate biosafety cabinet housed in at least a BSL2-level containment laboratory. Production of infectious HIV-1 particles using a Calcium phosphate mammalian transfection kit is described in this segment of the protocol.
   1. Seed HEK-293T cells in 90 mm dishes with 10 mL of complete (supplemented with 10% Fetal bovine serum and 1% penicillin-streptomycin) DMEM (Dulbecco's Modified Eagle Medium) in a class II type A2 biosafety cabinet and keep for approximately 12 h in a tissue culture incubator at 37 °C so that the confluence of the cells is between 50%-60%.
   2. Next day, make the transfection mix: Prepare Solution A containing 25 µg of pNL4.3 (a molecular clone of HIV-1) plasmid DNA in sterile buffer, 86.8 µL of 2 M CaCl_2, and the remaining sterile water to make the final volume 700 µL for each plate. Prepare Solution B containing 700 µL of 2x HEPES-buffered saline (HBS) for 1 plate. Then, adjust the calculation for the total number of plates.
   3. Now add Solution B to Solution A in a drop-wise manner while continuously vortexing Solution A. The total volume of the transfection mix now becomes 1.4 mL for one plate.
      NOTE: Precise drop-wise addition of Solution B to Solution A while continuous vortexing leads to the formation of perfect transfection complexes.
   4. Incubate this mixture at room temperature (RT) for about 20 min. Next, slowly add the mixture drop-wise to each plate containing fresh 9 mL of complete DMEM and transfer the plates to the CO₂ incubator. After 8-10 h, change the existing media with fresh 10 mL of complete DMEM.
   5. After 24 h post-media change, collect the supernatant (that contains the virus particles) from all the plates in conical tubes. Centrifuge at 600 x g for 5 min using a low-speed tabletop centrifuge (Table of Materials) to remove cell debris.
   6. Transfer the supernatant to polyallomer ultracentrifuge tubes and place them in the swingout rotor of an ultracentrifuge (Table of Materials). Check the tube holders' weights for balance and adjust with a sterile medium if needed.
   7. Ultracentrifuge at 1,41,000 x g for 2.5 h at 4 °C. After this, carefully take out the tubes and decant the supernatant slowly inside the biosafety cabinet.
   8. To the pellet (which is the concentrated virus now) in each tube, add 1 mL of incomplete RPMI (Roswell Park Memorial Institute) 1640 medium and do vigorous pipetting to dislodge the pellet.
      NOTE: The pellet after ultracentrifuge is almost invisible to the naked eye. So, it must be made sure to add RPMI 1640 in the middle of the tube so that the pellet is properly dislodged.
   9. Next, add 25 µL of 1 M HEPES pH 7.4 to the 1 mL of virus suspension. Aliquot 50 µL of the suspension to 1.5 mL centrifuge tubes and store them in a -80 °C freezer immediately for long-term storage.
2. Quantitation of virus concentration and virion infectivity
   NOTE: For calculating the infectivity of the virus, it is essential first to quantify its concentration, which is done by p24 Antigen Capture ELISA (according to the manufacturer's instructions and thus is not being described here; see Table of Materials). This process gives the virus' concentration in nanograms per microliter (ng/µL) of the stock, which is then used to identify the infective virion number in the stock through the following experiment in TZM-bl reporter cells²⁶.
   1. Count and seed 0.1 x 10⁶ TZM-bl cells in a 24 well plate using 500 µL of complete DMEM for each well and keep it in a cell culture incubator for 10-12 h.
   2. Make sure the cells are 50%-60% confluent before beginning with infection for virus quantitation. Change the existing media with 250 µL of fresh complete DMEM to each well.
      NOTE: Keep a positive control well to rule out any experimental fault.
   3. Calculate the amount of stock virus needed for infections of 1 ng, 0.1 ng, and 0.01 ng in duplicates. Add the appropriate amount of virus to the wells and keep the plate in the incubator at 37 °C for 4 h.
   4. Wash the cells twice with 500 µL of incomplete DMEM and then add 500 µL of complete DMEM.
   5. Proceed with the β-gal staining procedure after 36 h of media change.
      1. Discard the existing media and wash the cells twice with 1 mL of PBS. Fix the cells by adding 500 µL of the fixing solution (0.25% Glutaraldehyde in PBS) to each well and incubate at RT inside the biosafety cabinet for 5-7 min.
      2. Prepare the staining solution as given in Table 1. Keep it away from light as X-gal is light-sensitive.
      3. Wash the cells once with 1 mL of PBS. Overlay the cells with 500 µL of freshly prepared staining solution and incubate at 37 °C in the dark for 2-18 h.
      4. To stop the reaction afterward, rinse the cells with 500 µL of 3% dimethyl sulfoxide (DMSO) in PBS twice and then keep the cells in 500 µL of fresh PBS.
      5. Count the blue infected cells (as shown in Figure 1A) under the microscope in 10x magnification for 5 random fields. Take an average count of the 5 fields and multiply it with the field factor and dilution factor. This quantifies the infective virion particles in the virus stock.
         NOTE: For a 24 well plate, the field factor is 75.

Table 1: List of components required for β-gal staining in TZM-bl cells.

2. HIV-1 infection of T-cell lines

1. Thaw and culture an immortalized T-cell line. This study used CEM-GFP cell line cultured in complete RPMI 1640 medium supplemented with 10% Fetal bovine serum, 1% penicillin-streptomycin, and 500 µg/mL G418.
2. Count and take 2 million cells for each time point (Uninfected, 24 h, 48 h, 72 h, and 96 h). Harvest the uninfected cells immediately by centrifuging at 100 x g for 5 min and add cell lysis buffer (50 mM Tris-HCl pH 7.4, 0.12 M NaCl, 5 mM EDTA, 0.5% NP40, 0.5 mM NaF, 1 mM DTT), supplemented with protease inhibitor cocktail, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 1x phosphatase inhibitor (50 µL for 1-3 million cells) or lyse in Trizol reagent (1 mL for 1-3 million cells) for RNA isolation (see Table of Materials).
3. Wash the cells once with 1 mL of complete RPMI 1640 medium containing polybrene (5 µg/mL) at 100 x g for 5 min at RT. Resuspend the cells in 1 mL of complete medium.
   NOTE: Polybrene is a cationic polymer that is used to enhance retroviral infection in mammalian cells.
4. Resuspend the 8 million cells in 1 mL of polybrene containing complete medium. Now calculate the volume of viral stock required for 4 million virion particles, add the virus stock, and make up the total volume to 2 mL by adding complete media. This makes it an MOI of 0.5.
5. Keep the cells inside the CO₂ incubator at 37 °C for 4 h, with intermittent tapping every 30-45 min.
6. After 4 h, spin down the cells and discard the supernatant carefully inside the biosafety cabinet with proper disposal methods.
   NOTE: From this step onwards, centrifuge the cells at 100 x g for 5 min at RT.
7. Wash the cells twice with 1 mL of incomplete medium. Resuspend the cells in 8 mL of complete medium, making it 1 million cells/mL.
8. In a 6-well tissue culture plate, seed an equal number of cells in the required number of wells and keep the plate in a CO₂ incubator maintained at 37 °C.
9. For the next 4 days, harvest the cells every 24 h by adding cell lysis buffer. Also, collect the supernatant and immediately transfer it to the -80 °C freezer.
10. To check whether infection has happened, proceed for p24 antigen capture ELISA for all the time points. Make proper dilutions for the time points, i.e., a lower dilution for initial time points and higher for later ones ranging from 1:50 to 1:500. Plot the readings in a graph that depicts the infection progression (Figure 1B).

3. Thioflavin T staining to determine ER stress

1. Fix 1 million each uninfected and 0.5 MOI infected CEM-GFP cells (taken 72 h infected cells) with 200 µL of 4% paraformaldehyde in PBS for 20 min at RT.
2. Pellet down the cells at 100 x g for 5 min and treat with 500 µL of 0.1 M glycine for 5 min, followed by washing with 500 µL of PBS twice. Resuspend the cells in 100 µL of PBS.
3. Assemble the glass slides, filter cards, and sample chambers. Add the 100 µL of resuspended cells to the sample chambers and spin the cells at 1000 x g for 4 min to adhere the cells to the slides in a cytocentrifuge (Table of Materials).
   NOTE: There would not be enough cells immobilized on the slide following the cytocentrifuge centrifugation if the cell suspension is too diluted. The cells are likely to clump up and be poorly distributed after the cytocentrifuge, if the cell suspension is very concentrated.
4. Permeabilize the cells for 10 min using drops of 0.1% Triton-X-100 in PBS (enough to cover the area where the cells have adhered) and wash the cells twice by putting drop-wise PBS and wiping off the PBS with tissue by tilting the slide.
5. Incubate the cells with 10 µL of 5 µM Thioflavin T for 30 min.
   NOTE: Do not wash after incubation with ThT.
6. Mount the slides using 5 µL of mounting media (80% glycerol v/v, 20% PBS v/v, and 8 mg/mL 1,4-diazabicyclo[2.2.2]octane [DABCO]), put coverslip and seal the coverslip using nail polish. Leave the slides to dry.
   NOTE: At all these steps, make sure the cells do not dry up.
7. Take the confocal images of fixed cells with a 63x glycerol immersion lens on a confocal microscope (see Table of Materials). Adjust the following excitation and emission settings: GFP (Ex. 494 nm, Em. 519 nm) and ThT (Ex. 458 nm, Em. 480-520 nm).
   NOTE: Each fluorescent channel should be imaged sequentially, as opposed to simultaneously, to avoid channel overlap. GFP was taken as the indication of HIV-1 infection as CEM-GFP cells have GFP under the regulation of LTR promoter, which fires upon HIV-1 infection²⁵.
8. Convert the fluorescent images to 8-bit before quantifying by threshold analysis. Quantify the intensity of each cell manually using software like Image J (~50 cells)²⁷. Plot the graph with the intensity of each cell as points on the Y-axis against uninfected and infected criteria.
9. To demonstrate the effectiveness of this protocol, take a positive control, for example, Thapsigargin treatment (known ER stress inducer) in this case²⁸. Treat 1 million CEM-GFP cells, each with 1 µL of DMSO (vehicle control) and 100 nM of Thapsigargin for 12 h.
10. Harvest the cells and process for ThT staining as mentioned for infected samples (steps 3.1-3.8).
    NOTE: For these samples, GFP fluorescence is not required. Hence, only ThT fluorescence is captured in confocal microscopy.

4. Determining the activation and expression of various UPR markers

NOTE: To determine the expression of UPR markers two methods are used: Immunoblotting and RT-PCR. For immunoblotting, harvest the 0.5 MOI HIV-1 infected CEM-GFP cells at various time points post-infection (24 h, 48 h, 72 h, and 96 h post-infection) and resuspend the cell pellet in lysis buffer (as mentioned in step 2.2). Alternately, for RT-PCR, the cell pellets are lysed in Trizol reagent (1 mL for 1-3 million cells) (See Table of Materials). The cells resuspended in lysis buffer and Trizol reagent can be stored at -80 °C until further use.

1. Immunoblotting for analysis of UPR markers.
   1. Keep the resuspended cells in the lysis buffer on ice for 45 min with intermittent mixing using a vortex.
   2. Pellet down the cells at 18000 x g for 15 min using a benchtop high-speed centrifuge (see Table of Materials). Collect the supernatant in a fresh tube.
   3. Quantify the protein concentration in each sample using Bradford or a similar reagent.
   4. Take an equal concentration of protein for each sample and prepare the protein samples in Laemmli buffer (6x buffer: 250 mM Tris-Cl pH 6.8, 10% SDS, 30% Glycerol, and 0.02% Bromophenol blue; 5% β-Mercaptoethanol).
      NOTE: For immunoblotting for each UPR marker, a minimum of 60-80 µg of protein was used.
   5. Boil the protein samples at 96 °C for 5 min and give a short spin.
   6. Resolve the protein samples on a 10%-12% SDS-PAGE gel and transfer the proteins to a polyvinylidene difluoride (PVDF) membrane (see Table of Materials).
   7. Block with 5% non-fat dry milk or bovine serum albumin (BSA) for 1-2 h, wash twice with TBST (20 mM Tris pH7.4 and 0.137 M NaCl; 0.1% Tween 20), and probe with protein-specific primary antibodies against UPR markers (see Table of Materials) in a rocker, overnight at 4 °C. Next day after washing the blots twice with TBST, probe with respective secondary antibodies for 1.5 h.
   8. Wash the blots again with TBST and visualize the protein bands using a chemiluminescence-detecting substrate (see Table of Materials) in a western blot imaging instrument.
2. RT PCR
   1. Isolate the total RNA from the cells using Trizol reagent (see Table of Materials).
   2. Prepare the cDNA from equal concentration of RNA using Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT) (see Table of Materials).
   3. Analyze the modulation of the mRNA expression of HSPA5, spliced XBP1, ATF4, CHOP and GADD34 using qRT-PCR with gene-specific primers (Table 2). Briefly, prepare 10 µL reaction mixtures containing cDNA template (100 ng), 5 µL of SYBR Green dye (see Table of Materials), and 10 pmol of each gene-specific oligonucleotide primer pair. Adjust the volume using sterile H₂O.
   4. Run the mixtures on a real-time PCR machine using the following program: initial denaturation at 95 °C for 3 min and 40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s followed by the melt curve analysis. Calculate the fold change in the target gene expression relative to the housekeeping gene (for example β-Actin) as:
      Fold change = 2^-Δ(ΔCT)
      Where ΔCT = CT (target) − CT (house-keeping gene)
      and Δ(ΔCT) = ΔCT (treated) -ΔCT (control)
   5. To further confirm the splicing of XBP1, perform a semi-quantitative RT-PCR with uninfected and 72 h infected samples.
      ​NOTE: Splicing of the XBP-1 mRNA is studied by using RT-PCR across the splice site.
   6. Amplify the cDNA with XBP1 primers (Table 2) using a high-fidelity PCR master mix (see Table of Materials) under the following conditions: 1 cycle of 98 °C for 30 s, followed by 5 cycles of 98 °C for 15 s, 65^{*Δ-5 °C} for 30 s and 72 °C for 30 s, followed by 30 cycles of 98 °C for 15 s, 55 °C for 30 s and 72 °C for 30 s, followed by a final extension of 72 °C for 10 min and hold at 4 °C.
   7. Separate the amplicons on a 2.5% agarose gel containing 50 ng of ethidium bromide/mL and visualize in a gel doc instrument. The spliced XBP1 band is smaller by 26 nucleotides.

5. Knockdown of UPR markers and analysis of HIV-1 LTR-driven gene expression and virus production

1. Preparation of shRNA constructs for knockdown of UPR markers
   1. Create shRNA constructs using the pLKO.1-TRC vector backbone (lentiviral cloning vector)²⁹.
   2. Construct two shRNA constructs for each gene (IRE1, PERK, ATF6 and HSPA5). Sense and anti-sense oligonucleotides targeting the respective gene's mRNA are designed by RNAi Consortium (https://www.broadinstitute.org/rnai-consortium/rnai-consortium-shrna-library) (Table 3).
      NOTE: In a similar way, shRNA constructs can be made for other UPR markers.
   3. Anneal the primers (100 nM each of forward and reverse) at 95 °C followed by gradual cooling to RT.
   4. Then, ligate it into the Age1 and EcoRI sites of the pLKO.1 vector using a T4 DNA ligase (see Table of Materials). Confirm the sequence by DNA sequencing.
      NOTE: A shRNA construct targeting the LacZ gene is used as a non-targeting control.
2. Knockdown of PERK, ATF6, IRE1 and HSPA5 in HEK-293T cells, Luciferase assay and p24 ELISA
   1. Prepare a mix of shRNA construct (0.9 µg) and a transfection reagent like polyethylenimine (PEI) (3 µL of PEI for 1 µg of DNA) (see Table of Materials) in 50 µL of serum-free media by vortexing and incubate for 20 min.
   2. From a confluent HEK-293T cells containing flask, trypsinize and seed ~0.25 x 10⁶ cells along with the transfection mix in a 24 well plate, making the total volume of 125 µL and incubate for 6 h at 37 °C in a CO₂ incubator. This method is called reverse transfection where the cells are seeded along with the transfection mixture.
   3. After 6 h, add 125 µL of complete media to the wells and resuspend the cells for even seeding.
      NOTE: For knockdown experiments, reverse transfection (steps 5.2.1-5.2.3) gives better efficiency with both shRNAs and siRNAs.
   4. After 24 h, perform a second transfection. Prepare a mix of pNL4-3 (100 ng), pLTR-Luc (100 ng), and pEGFP-N1 (50 ng) (pNL4-3:pLTR-Luc:pEGFPN1-1:1:0.5) constructs with PEI (3 µL of PEI for 1 µg of DNA) in 50 µL of incomplete media by vortexing. Incubate the mix for 20 min at RT. Change the existing media with 250 µL of fresh incomplete media and add the mix to the cells.
      ​NOTE: A reporter vector for the HIV-1 LTR was developed by sub-cloning LTR from pU3RIII^30,31 into pGL3basic in the laboratory earlier³². GFP expression using pEGFP-N1 was utilized to normalize transfection efficiency³².
   5. Change the media after 6 h post-transfection with 500 µL of complete DMEM. Harvest the cells after 36 h for immunoblotting and luciferase assay.
   6. For luciferase assay, pellet the cells and resuspend in a commercially procured luciferase substrate (50 µL) (see Table of Materials). Add the resuspended cell lysate to an opaque 96 well plate and incubate for 10-15 min. Obtain the luciferase reading in a luminometer. Also measure GFP fluorescence in the same samples to normalize the luciferase reading in a microplate micromode plate reader (Ex: 494 nm, Em:519 nm).
   7. Plot the graph as the Luciferase unit/GFP on the Y-axis.
      NOTE: The knockdown of the respective UPR markers should be checked, and it can be done by either immunoblotting or qRT-PCR using gene-specific antibodies or primers, respectively.
   8. Collect the supernatants to process for p24 ELISA as mentioned above.
3. Creation of stable cells with knockdown of UPR markers and HIV-1 infection.
   1. Prepare the Lentivirus particles by transfecting HEK-293T cells seeded in a 6 well plate, with the respective shRNA (1 µg) constructs along with pMD2.G (VSV-G envelope vector) (0.75 µg), and psPAX2 (Lentiviral packaging plasmid) (0.25 µg) (see Table of Materials) using PEI in the ratio 1 µg of DNA: 3 µL of PEI. Prepare the mixture in 100 µL of incomplete DMEM and incubate for 20 min at RT. Add the mixture to 1.8 mL of complete media in the seeded HEK-293T cells.
      NOTE: Seed HEK-293T cells in a 6 well plate at least 12 h before transfection until they gain morphology and are approximately 60-70% confluent.
   2. After 24 h of transfection, add 1 mL of complete DMEM to each well.
   3. Collect the supernatants after 48 h, store at -80 °C freezer, and perform p24 ELISA to confirm the presence of the virus in these supernatants. Use this crude lentiviral supernatant for transducing J6 cells.
   4. Incubate 2 million cells in a 6 well plate with an equal volume of lentiviral supernatant (500 µL) for each control and gene-specific knockdown lentivirus and add fresh complete RPMI 1640 medium in the presence of polybrene (5 µg/mL) to make up the volume to 2 mL.
   5. After 24 h, add Puromycin (1 µg/mL) to each well to select stable cells. After adding Puromycin, pellet the cells every 24 h and add 2 mL of fresh media with Puromycin.
   6. Do this till there is no cell death (~1 week). The surviving cells are the stable cells with the desired gene knockdown.
      NOTE: The knockdown of respective genes can be checked at regular intervals and, finally, when only surviving cells are visible in Puromycin-containing media by using immunoblotting or qRT-PCR.
   7. Infect the LacZ and gene-specific knockdown cells with 0.5 MOI of HIV-1 as described in section 2 and harvest the cells 48 h post-infection.
   8. Collect the supernatant from each sample and process for p24-ELISA as described earlier and process the cells to check the knockdown level by immunoblotting.

6. Treatment with ER stress inducer and analyzing its effect on HIV-1 replication

NOTE: To determine the effect of overstimulation of UPR during HIV-1 replication, the pharmacological inducer molecule, Thapsigargin, can be used.

1. Determining the percent cell viability upon Thapsigargin treatment.
   NOTE: The cytotoxicity of Thapsigargin is determined by the MTT reagent (see Table of Materials).
   1. Seed 25,000-30,000 CEM-GFP cells per well in 96 well plate containing 100 µL of complete RPMI 1640. Add 100 nM Thapsigargin to the media and incubate at 37 °C in an incubator.
      NOTE: Cells treated with 1 µL of DMSO were taken as vehicle control.
   2. After 48 h, add 10 µL of MTT (5 mg/mL) in each well and incubate in the dark for 4 h for the formation of formazan crystals at 37 °C with 5% CO₂.
   3. After 4 h, dissolve the crystals using 100 µL of isopropanol to form a purple color and measure the absorbance at 570 nm using a spectrophotometer.
   4. Calculate the percent cell viability based on the equation given below
      % cell viability = (Control_OD570 – Treatment_OD570)/ Control_OD570 x 100
2. Pre-treatment of Thapsigargin and analysis of HIV-1 infection progression by p24 ELISA.
   1. Seed 1 million CEM-GFP cells in a 12 well plate containing 1 mL of complete RPMI 1640 and treat the cells with 100 nM of Thapsigargin or 1 µL of DMSO (vehicle control) for 12 h in a CO₂ incubator maintained at 37 °C. After 12 h, wash the cells with complete RPMI 1640 and infect with 0.5 MOI of HIV-1 as described earlier.
   2. Harvest the cells at different time points, such as 24 h, 48 h, and 72 h post-infection, for immunoblotting.
   3. Collect the supernatants from each sample and perform p24 ELISA as described earlier.
   4. Plot the graph with the p24 concentration in the sample as the Y-axis and the respective time points as the X-axis.
   5. Using immunoblotting, analyze the ER stress induction due to Thapsigargin treatment by measuring the level of any UPR markers. This study used HSPA5 as the marker.

7. TZM-bl β-gal infectivity assay to determine the effect of UPR on virion infectivity

NOTE: To understand the role of UPR in the virion infectivity, the supernatant from the knockdown as well as treatment with Thapsigargin, can be used for TZM-bl β-gal infectivity assay as described in section 1.2, based on the p24 concentration determined by p24 ELISA.

1. Infect TZM-bl cells with an equal concentration of p24 from each sample and perform β-gal staining. Count the blue infected cells under the microscope in 10x magnification for 5 random fields.
2. Take an average of the count of the 5 fields for each sample from the above steps.
3. Calculate the fold change as mentioned below:
   Fold change = Average number of blue cells in the test samples/Average number of blue cells in the control sample.