The Molecular Mechanism Of HSF1 On Latent HIV-1 Reactivation

BACKGROUD:Combined anti-retroviral therapy (cART) effectively prolongs the lifetime of AIDS patients and limits the spread of the virus. However, the entire eradication of HIV infection is primarily hampered by latent HIV reservoir left post treatment. Since latent HIV is undetectable at protein levels, it escapes the monitoring of the host immune system. To overcome this situation, a promising strategy called "shock and kill" has been proposed for the therapy of latent HIV. The treatment aims to activate dormant proviruses in order to initiate the viral replication cycle (shock) in their host cells which are in parallel targeted by virus-induced cytotoxicity or cytotoxic immune cell (kill). Many latency reversing agents (LRAs) have been discovered, which are primarily divided into three categories:(i) epigenetic modification agents (histone deacetylase inhibitors (HDACis), histone methyltransferase inhibitors (HMTis) and DNA methyltransferase inhibitors (DNMTis)), (ii) cellular factor modulators (PKC and p-TEFb) and (iii) immune regulators (IL-2, IL-7, IL-15 and OKT3). Some of the LRAs, combined with cART, have been used in clinical trials to purge latent HIV, but the results were not satisfactory. One reason could be the consumption of CD4+T cells resulting from global T cell activation. Alternatively, it could be due to the forceful inflammatory response or absence of innate immune response (cytotoxic T lymphocyte). Indeed, besides romidepsin and panobinostat, which show good results in vivo, there are hardly any LRAs that can reduce HIV reservoirs.In recent years, several cellular and viral factors that are involved in retroviral latency have been identified. For instance, a lack of cellular transcription factors as well as the regulatory activity of the HIV-1 Tat protein limit the initiation and elongation of viral transcription in resting CD4+T cells and thus promote latency. However, our understanding of the molecular interactions and cellular mechanism that trigger latency or reactivate an integrated provirus remains shallow and fragmentary. In recent years, genome-wide siRNA screenings have identified a total of 842 host genes associated with HIV-1 infections, and a genome-wide mass spectrometry-based proteomic analysis has revealed 497 HIV-human protein-protein interactions involving 435 individual human proteins in HEK293 and Jurkat cells. Notably, only a small number of cellular factors have been described so far that influence the latency maintenance and reactivation stages; thus, it is likely that additional host proteins, exploited by the virus during these stages, remain to be identified. These factors might be engaged during transcriptional initiation and subsequent elongation processes.OBJECTIVES:In recent years, different cellular factors being involved in the development, maintaining and resolving of viral latency have been identified. Host key players include the transcription factor NF-κB and the heat shock protein heat shock protein 90 (HSP90) that have been reported to assist in controlling HIV reactivation from latency. Heat shock protein 40 (HSP40) and heat shock protein 70 (HSP70) have also been detected as interacting partners with viral proteins Nef and Tat respectively. Heat shock proteins (HSPs) are known as molecular chaperones extensively participating in the process of cellular homeostasis under stresses, in which their transcription factor heat shock factor 1 (HSF1) widely participate. Taken together, these data strongly support the hypothesis that HSF1 plays a direct role in HIV latency. HSF1 has been demonstrated to bind the HIV 5’-LTR and positively take a role in HIV vital activities further strengthening our hypothesis. In this project we therefore strive to provide a yet undescribed mechanistic and detailed understanding of HSF1 regulation of latent HIV reactivation. Particularly, we target on stresses mediated latent HIV reactivation, discusse the active form of HSF1 participating in latent HIV reactivation. Beyond that, we make efforts to investigate the possibility of HSF1 as target for latent HIV reactivation and to provide novel latent HIV activators and therapeutic strategies.METHODS:1. Flow cytometry (FCM)J-lat 10.6 is one of the most classical HIV latency cell model in which GFP is inserted as reporter of HIV, and it is widely used to screen LRAs. In our study, we regarded J-lat 10.6 cell line as our main research object. J-lat 10.6 cells were treated with various stress inducers such as salubrinal and carfilzomib, and GFP% obtained by FCM was calculated as indicator for latent HIV reactivation. Similarly, J-lat 10.6 cells were co-treated with cellular signaling pathway regulators agonists or antagonists and LRAs discovered in our study, and GFP% of J-lat 10.6 cells was detected by using FCM. In this way, we investigated the role of cellular signaling pathway regulators participating in latent HIV reactivation. In addition, human peripheral blood mononuclear cells (PBMCs) isolated from healthy personals were treated by LRAs, and activated T cell suface makers CD25 and CD69 were detected by FCM before surface staining. In this way, we evaluated the influence on T cell activation from LRAs.2. Cytotoxicity assayCCK-8 reagents were employed to inspect cell viability of J-lat 10.6, U1, ACH2, PBMCs after LRAs treatment or inhibitors co-treatment, and to provide evidence for objectively judging on the influence of compounds on latent HIV reactivation.3. Western Blot (WB)Nuclear protein were extracted from J-lat 10.6 cells after treatment with salubrinal, bortezomib, carfilzomib or other classical activators for 2 h through nuclear-cytoplasm separation extraction method, followed by analyzing the nuclear accumulation of various transcription regulation factors including transcription initiation factors NF-κB, NFAT, AP-1, HSF1, transcription elongation factors p-TEFb, bistone epigenetic modification enzymes p300. In this way, we assessed the promotion or inhibition influence on transcription initiation factors, transcription elongation factors or chromatin deconstruction factors induced by LRAs. Additionally, total protein were extracted after activators treatment or inhibitors co-treatment, and the expression level of HIV capsid protein p24, stress marker p-eIF2a (a subunit of eukaryotic translation initiation factor), HSP90, CDK9 was analyzed by WB to assess the influence on protein expression from activators or inhibitors.4. Quantitative PCR (qPCR)Total RNA was extracted from J-lat 10.6, U1, ACH2, PBMCs, primary CD4+T cells after treatment with activators for 12,24 or 48 h. RNA was transcribed to cDNA and followed by detection using corresponding primers for HIV genes, transcription factors related genes, cytokine genes and cell surface receptor genes. Corresponding Ct values were obtained by qPCR to analyze the relative increasing folds. In this way, we assessed the promotion impact on HIV genes (LTR, Gag, Tat, Vif, Vpr), transcription factors related genes(HSF1, RelA, NF-κB1, c-foc, ATF3, NFATcl and NFATc2), immune factors genes (TNF-α, IL-1β, IL-2, IL-6, IL-10, IL-12β, IL-13, IL-17a, IL-18) and inhibition impact on cell surface receptor genes (CD86, CD69, CD4, CXCR4). The data acquired by qPCR strengthen the results observed by WB.5. Enzyme-linked immune sorbent assay (ELISA)In order to determine the influence on the release of IL-1β and IL-18 from LRAs, culture supernates were collected to detect the concentrations by ELISA from J-lat 10.6 and Jurkat cells after treatment with carfilzomib for different times. In this way, we speculated the positive effect on the "kill" step from the promotion of innate immune respose induced by activators.6. Laser-scanning confocal microscopy (Confocal)The distribution of HSP90 and CDK9 in U1 cells treated by activators was determined by laser-scanning confocal microscopy, and the direct interaction between HSP90 and CDK9 was primarily estimated through Confocal.7. Co-immunoprecipitation (Co-IP)To investigate the mechanism underlying HSF1 mediated the latent HIV reactivation under stress, we employed Co-IP to analyze the cellular regulation factors direct interacted with transcription factor HSF1, such as the subunit of p-TEFb CDK9 and Cyclin T1, p300. We elaborated the promotion transcription function of HSF1 through analyzing the recruitment of HSF1 with p-TEFb, and the role of acetylated HSF1 in latent HIV reactivation through analyzing the recruitment of HSF1 with p300. As well, we detected the acetylation level of HSF1 by using IP. Beside these, Co-IP was employed to analyze the relationship between CDK9 and HSP90, and IP was employed to analyze the ubiquitylation of CDK9 stimulated by carfilzomib.8. Electrophoretic mobility shift assay (EMSA)Nuclear protein were extracted from J-lat 10.6 cells after treatment with salubrinal for 2 h through nuclear-cytoplasm separation extraction method, and was co-incubated with biotin-labeled probes of HSF1 or NF-κB, followed by analyzing the shifts through native PAGE. In this way, the binding of HSF1 or NF-κB stimulated by activators to HIV LTR in vitro was indentified.9. Chromatin immunoprecipitation (ChIP)To further investigate the binding of HSF1 or NF-κB to HIV LTR under natural state, ChIP was employed to analyze the amount of HSF1 or NF-κB binding to HIV LTR. HSF1 and RelA antibodies were used to do ChIP assays, and the relative amount of HSF1 or NF-κB binding to HIV LTR contained in the genome DNA was calculated through qPCR. In this way, we acquired the credible results of HSF1 or NF-κB binding to HIV LTR under the treatment with activators.10. Transcription activator-like effector nuclease (TALEN) knockoutTo further suvey the key role of HSF1 in HIV transcription, TALEN was employed to induce HSF1 knockout. TALENs arms were designed as L2×R3 combination targeting on HSF1 gene (NCBI gene ID:3297), and plasmids for the TALENs arms were constructed using the FAST TALEN Kit. After sequencing, five plasmids were transcfected into 293T cells. One pair of arms was selected as the most effective knockout group after 3-days puromycin screening and subsequent genomic PCR sequencing. Another round of screening was performed after a two-week monoclonal culture. The stable knockout cell line 293T-HSF1-/-(-4bp/-10bp) was acquired and identified by sequencing and WB.11. Plasmid transfectionPNL4-3-luci and pEZ-HSF1 were co-transfected into 293T-WT or 293T-HSF1-/-cells to investigate the influence of HSF overexpression or knockout on HIV transcription. Transfection reagents and DNA were co-incubated with cells for 24 h, and the cells were lysed then relative luciferase activity was detected using a Dual-Luciferase Reporter Assay System. The relative activity was used to asses the influence of HSF1 on HIV transcription.12. Data analysisHistograms and graphs in this study were plotted through using Prism 5.0 software. And all WB bands were processed by using Adobe Photoshop CS 5.0 software. All data acquired by FCM were processed by using FlowJo 7.6 software. Fluoresence images were analyzed by Olympus FV10-ASW1.7 Viewer. And data statistics were analyzed by using GraphPad Prism 5.0 software, and the error bars were presented as mean±SD based on at least three independent experiments, the p values were defined as*p<0.05,**p<0.01 and***p<0.001.RESULTS:1. Heat shock stress, innate immune stress, oxidative stress and endoplasmic reticulum stress were discovered to reactivate latent HIV in varying degree after screening by FCM. Also, p-eIF2α dephosphorylatase GADD34 inhibitor salubrinal was found to induce stress response as well as latent HIV reactivation. This result was observed on various cell models including J-lat 10.6, U1, ACH2 and primary CD4+T cells isolated from HIV+ patients. These together demonstrated that stress reactivated latent HIV was universal. In further, we found salubrinal induced latent HIV reactivation was without T cell activation.2. Through further investigation on stress mediated latent HIV reactivation,we found HSF1 played main role, and HSF1 downstream effectors such as HSP27, HSP70 and HSP90 widely participated in the latent HIV reactivation process. Through revealing the molecular mechanism underlying HSF1 mediated latent HIV reactivation, HSF1 was demonstrated to bind on HIV LTR via EMSA and ChIP assays and HSF1 was demonstrated to recruit p-TEFb to promote transcription elongation via co-IP assays. Additionally, HSF1 was demonstrated to recruit p300 to acetylate itself, which was considered as one of the important mechanism underlying latent HIV reactivation.3. Targeting on endoplasmic reticulum stress-induced latent HIV reactivation, we found proteasome inhibitors carfilzomib effectively reactivated latent HIV at namole concentrations with low cytotoxicity. What worth to be mentioned is that carfilzomib reactivated latent HIV without global T cell activation and at the same time selectively promoted the release of innate immune cytokines IL-1β and IL-18. Also, carfilzomib effectively enhanced the latent HIV reactivation induced by classical LRAs such as prostratin, SAHA and JQ1 in primary CD4+T cells from suppressive HIV+ patients. Through researching on the latent HIV reactivation mediated by proteasome inhibitors, we strenghthen the conclusion that HSF1 playing important role in latent HIV reactivation.4. In the process of doing research on HSF1 downstream effector HSP90, we found HSP90 inhibitors 17-DMAG and NVP-AUY922 not only upregulated the expression of HSP90 and also induced latent HIV reactivation in J-lat 10.6 cells. Moreover, HSP90 inhibitors were found to synergied with many LRAs to reactivate latent HIV. Similarly, p-TEFb was found to take important part in proteasome inhibitors-induced latent HIV reactivation. HSP90 binding to CDK9 to protect it from ubiquitylated degradation was also demonstrated by co-IP assays.5. Through exploring the universality of HSF1 mediating latent HIV reactivation, HSF1 active form p-HSF1(Ser320) and HSP70, HSP90 participated in classical LRAs dilazep, SAHA, JQ1, prostratin, TNF-α besides stress inducers was detected. Subsequently,293T-HSF1-/-(-4bp/-10bp) cell line was obtained via TALEN knockout. Through pNL4-3-luc transfection in activated-HSF1 overexpressed or absenced cells, we found the knockout of HSF1 significantly impaired HIV transcription while conditional ovexpression of HSF1 improved HIV transcription. Consequently, we consider that HSF1 plays key role in HIV transcription, suggesting its potential to be target for drug screen. According to it, a series of LRAs were acquired in our study.CONCLUSSION:In this study, stress inducers salubrinal and carfilzomib were discovered to reactivate latent HIV without global T cell activation. Particularly, proteasome inhibitor carfilzomib selectively promoted the release of innate immune cytokines. Stress-induced latent HIV reactivation was demonstrated to be mediated by HSF1, and the underlying mechamism was that HSF1 transforms to p-HSF1 (Ser320) and p-HSF1 translocates into the nucleus to recruit p300 following transforms to acetylated HSF1; active HSF1 binds to HIV LTR and recruits p-TEFb to promote transcription elongation; HSF1 downstream factor HSP90 binds to CDK9 to protect it from degradation thus giving positive feedback to HSF1.Finally, HSF1 was demonstrated to be indispensable in the HIV transcription, suggesting it potential as target for drug development.