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 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. To begin seed 0.1 times 10 to the power of 6 TZM-bl cells in a 24-well plate using 500 microliters of complete DMEM for each well and place it in a cell culture incubator for 10 to 12 hours. From the vial containing the HIV-1 virus transfer an appropriate amount of the virus to the wells and incubate at 37 degrees Celsius for four hours.
Wash the cells twice with one milliliter of PBS before fixing them in 500 microliters of 0.25%glutaraldehyde solution. Incubate the cells at room temperature for five to seven minutes. After the PBS wash, overlay the cells with 500 microliters of freshly prepared staining solution and incubate at 37 degrees Celsius in the dark for 2 to 18 hours.
Under the microscope at 10X magnification count the blue infected cells for five random fields. Resuspend the 8 million CEM-GFP cells in one milliliter of polybrene-containing complete RPMI 1640 medium. Add the virus stock and make up the volume to two milliliters with complete medium.
Place the cells in a 37 degrees Celsius carbon dioxide incubator for four hours with intermittent tapping every 30 to 45 minutes. Culture an equal number of cells in a 6-well culture plate at 37 degrees Celsius. Every 24 hours after centrifugation collect the supernatant from pelleted cells and add cell lysis buffer into the pelleted cells.
Fix 1 million each uninfected and 0.5 multiplicity of infection HIV-1 infected CEM-GFP cells with 200 microliters of 4%paraformaldehyde in PBS for 20 minutes. Centrifuge the cells at 100 G for five minutes and treat the palate with 500 microliters of 0.1 molar glycine for five minutes. After washing the cells twice with PBS and resuspending them in 100 microliters of PBS, add the 100 microliters of the cell suspension to the sample chambers.
Spin the cells in a cytocentrifuge at 1, 000 G for four minutes to adhere the cells to the slides. For permeabilization add a few drops of 0.1%Triton X-100 to the cells. After 10 minutes dropwise add PBS to wash the cells.
Incubate the cells with 10 microliters of five micromolar thioflavin T for 30 minutes, then add five microliters of mounting media and put a cover slip. Under a 63X glycerol immersion lens capture confocal images of fixed cells. Endoplasmic reticulum or ER stress caused by HIV-1 infection was analyzed by observing protein aggregates inside the cell using thioflavin T staining.
Enhanced thioflavin T intensity suggested more protein aggregates and increased ER stress. To begin resuspend 0.5 multiplicity of infection HIV-1 infected CEM-GFP cells in the lysis buffer on ice for 45 minutes with intermittent vortexing. After centrifugation, collect the supernatant in a fresh tube.
Boil equal concentrations of protein samples in Laemmli buffer at 96 degrees Celsius for five minutes. Resolve the samples on a 10 to 12%SDS PAGE gel. Transfer the gel onto a PVDF membrane.
Block the membrane with 5%BSA for one to two hours, then wash the membrane twice with TBST and probe with protein-specific primary antibodies on a rocker overnight at four degrees Celsius. The next day, probe the blot with secondary antibodies for 1.5 hours. After washing with TBST, add a chemiluminescence detecting substrate to visualize the protein bands.
To analyze mRNA expression prepare 10 microliter reaction mixtures containing CDNA template, five microliters of SYBR Green dye, and 10 picomoles of each gene-specific oligonucleotide primer pair. Run the mixtures on a real-time PCR machine. To analyze the splicing of XBP-1 separate the amplicons into a 2.5%agarose gel containing 50 nanograms of ethidium bromide per milliliter and visualize in a gel documentation instrument.
After trypsinization, seed approximately 0.25 times 10 to the power of 6 HEK 293T cells with the transfection mix in a 24 well plate and incubate for six hours at 37 degrees Celsius. Add 125 microliters of complete DMEM medium to the wells and resuspend the cells for even seeding. After 24 hours, replace the existing medium with 250 microliters of incomplete DMEM medium.
Add the freshly prepared second transfection mix and incubate for 36 hours. After centrifugation resuspend the cells in a commercially procured luciferase substrate. Add the resuspended cell lysate to an opaque 96 well plate and incubate for 10 to 15 minutes.
In a luminometer obtain the luciferase reading. Measure the GFP fluorescence in the same samples to normalize the luciferase reading in a microplate micromode plate reader. Following 24 hours of HEK-293T cells transfection, add one milliliter of complete DMEM to each well of a 6-well plate and collect the lentiviral supernatant after 48 hours.
Incubate 2 million Jurkat J6 cells suspended in polybrene containing complete RPMI medium in a 6-well plate with an equal volume of lentiviral supernatant for each control and gene-specific knockdown lentivirus. Add puromycin after 24 hours to each well to select stable cells. Pellet the cells every 24 hours and add two milliliters of fresh complete RPMI 1640 medium with puromycin.
Treat CEM-GFP cells with 100 nanomolar of thapsigargin for 12 hours at 37 degrees Celsius. After washing the cells with RPMI 1640 medium, infect with 0.5 multiplicity of infection of HIV-1. Collect the supernatants from each sample for p24 ELISA.
The cytotoxicity of thapsigargin was analyzed using an NTT assay showing non-significant cytotoxicity at the desired concentration. Immunoblotting for HSPA5 analyzed the drug's effect on ER stress. Pretreatment with 100 nanomolar thapsigargin for 12 hours induced HSPA5 expression at all time points.
P24 ELISA showed the effect on virus production.
