thioflavin t staining for assessing endoplasmic reticulum stress in cem-gfp cells

Studying ER Stress and UPR Activation During HIV-1 Infection in T-Cells

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.

Author Spotlight: Exploring the Role of Unfolded Protein Response in HIV-1 Replication and Infectivity

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

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.

Thioflavin T Staining for Assessing Endoplasmic Reticulum Stress in CEM-GFP 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 100g for five minutes and treat the pellet with 500 microliters of 0.1 monoglycine for five minutes. After washing the cells twice with PBS and re-suspending 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 1000g 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.

thioflavin-t-staining-for-assessing-endoplasmic-reticulum-stress-cem

Research

JoVE Journal

Immunology and Infection

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 eIF2&#945;, 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.

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.

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

Quantitation of HIV-1 Infection in TZM-bl Cells and Infection in CEM-GFP T-Cell Line

To begin, seed 0.1 x 10 to the power of six 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 two 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 six-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.

Determining Activation and Expression of Unfolded Protein Response Markers in HIV-1 Infected CEM-GFP Cells

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 XBP1, separate the amplicons into a 2.5%agarose gel containing 50 nanograms of ethidium bromide per milliliter, and visualize in a gel documentation instrument.

shRNA-Mediated Gene Knockdown in HEK-293T and Jurkat J6 Cells, Treatment with ER Stress Inducer, and Analyzing its Effect on HIV-1 Replication

After trypsinization, seed approximately 0.25 times 10 to the power of six 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 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 six-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 six-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 MTTSA, 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 ELIZA showed the effect on virus production.