RESUMEN
The latent HIV-1 reservoir is a major barrier to viral eradication. However, our understanding of how HIV-1 establishes latency is incomplete. Here, by performing a genome-wide CRISPR-Cas9 knockout library screen, we identify phosphatidylethanolamine-binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), as a novel gene inducing HIV latency. Depletion of PEBP1 leads to the reactivation of HIV-1 in multiple models of latency. Mechanistically, PEBP1 de-phosphorylates Raf1/ERK/IκB and IKK/IκB signaling pathways to sequestrate NF-κB in the cytoplasm, which transcriptionally inactivates HIV-1 to induce latency. Importantly, the induction of PEBP1 expression by the green tea compound epigallocatechin-3-gallate (EGCG) prevents latency reversal by inhibiting nuclear translocation of NF-κB, thereby suppressing HIV-1 transcription in primary CD4+ T cells isolated from patients receiving antiretroviral therapy (ART). These results suggest a critical role for PEBP1 in the regulation of upstream NF-κB signaling pathways governing HIV transcription. Targeting of this pathway could be an option to control HIV reservoirs in patients in the future.
Asunto(s)
Infecciones por VIH , VIH-1 , Linfocitos T CD4-Positivos/metabolismo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , VIH-1/genética , Humanos , FN-kappa B/genética , FN-kappa B/metabolismo , Proteínas de Unión a Fosfatidiletanolamina/genética , Latencia del Virus/genéticaRESUMEN
Adoptive cellular immunotherapy therapy using broadly neutralizing antibody-based chimeric antigen receptor-T cells (bNAb-based CAR-T) has shown great potency and safety for the functional cure of HIV. The efficacy of bNAb-based CAR-T cells could be compromised by adaptive resistance during HIV chronic infection according to the phenomenon that cellular exhaustion was observed in endogenous cytotoxic T-lymphocytes (CTLs) along with upregulated expression of PD-1. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells (PBMCs) and a 3BNC117-DNR CAR (3BD CAR) construct that enables the expression of PD-1 dominant negative receptor (DNR) and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV envelope glycoprotein (Env). Compared with HIV CAR expression alone, 3BD CAR-T cells displayed potent lytic and functional responses to Env-expressing cell lines and HIV-infected CD4+ T cells. Moreover, 3BD CAR-T cells can kill HIV-latently-infected cell lines, which are reactivated by the secretory cytokines of effector cells followed by contact with initial HIV-expressing fraction. Furthermore, bioluminescence imaging indicated that 3BD CAR-T cells displayed superior anti-HIV function in an HIV NCG mouse model of transplanting Env+/PD-L1+ cells (LEL6). These studies suggested that our proposed combinational strategy of HIV CAR-T therapy with PD-1 blockade therapy is feasible and potent, making it a promising therapeutic candidate for HIV functional cure.
RESUMEN
Highly active antiretroviral therapy (HAART) is very effective in suppressing human immunodeficiency virus type 1 (HIV1) replication. However, the treatment is required to be administered for the remainder of an individual's lifetime due to latent HIV1 reservoirs. The 'shockandkill' strategy, which involves using agents to reactivate latent HIV1 and subsequently killing latently infected cells in the presence of HAART, was recently proposed. Unfortunately, no agents have currently demonstrated an ability to reactivate latent HIV1 in vivo in the absence of toxicity. Therefore, the identification of novel latency activators is required. In order to identify a potential novel agent, the present study investigated the effect of quercetin on latent HIV1 reactivation using an established model of HIV1 latency. As a marker for reactivation of HIV1 in C11 Jurkat cells, the expression of green fluorescent protein, controlled by HIV1 long terminal repeat, was observed by fluorescence microscopy. The results of the present study demonstrated that quercetin effectively reactivated latent HIV1 gene expression alone, and led to synergistic reactivation when combined with prostratin or valproic acid. In addition, the present study provides evidence that quercetin may reactivate HIV1 expression by inducing nuclear factorκB nuclear translocation, and that the toxicity of quercetin is lower when compared with various additional activators of HIV1. Combined, the results of the present study indicate that quercetin may be an effective agent to disrupt HIV1 latency and may be useful in future eradication strategies.
Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , FN-kappa B/agonistas , Quercetina/farmacología , Latencia del Virus/efectos de los fármacos , Línea Celular , Humanos , Células Jurkat , Activación Viral/efectos de los fármacos , Activación Viral/genética , Replicación Viral/efectos de los fármacosRESUMEN
Highly active anti-retroviral therapy (HAART) cannot clear infected cells harboring HIV-1 proviral DNA from HIV-1-infected patients. We previously demonstrated that zinc-finger nucleases (ZFNs) can specifically and efficiently excise HIV-1 proviral DNA from latently infected human T cells by targeting long terminal repeats (LTRs), a novel and alternative antiretroviral strategy for eradicating HIV-1 infection. To prevent unwanted off-target effects from constantly expressed ZFNs, in this study, we engineered the expression of ZFNs under the control of HIV-1 LTR, by which ZFN expression can be activated by the HIV-1 (Trans-Activator of Transcription) Tat protein. Our results show that functional expression of ZFNs induced by Tat excise the integrated proviral DNA of HIV-NL4-3-eGFP in approximately 30% of the population of HIV-1-infected cells. The results from HIV-1-infected human primary T cells and latently infected T cells treated with the inducible ZFNs further validated that proviral DNA can be excised. Taken together, positively regulated expression of ZFNs in the presence of HIV-1 Tat may provide a safer and novel implementation of genome-editing technology for eradicating HIV-1 proviral DNA from infected host cells.
RESUMEN
Persistent latent reservoir in resting CD4+ T cells is a major obstacle in curing HIV-1 infection. Effective strategies for eradication of the HIV-1 reservoir are urgently needed. We report here for the first time that two BET inhibitors, RVX-208, which has entered phase II clinical trials for diverse cardiovascular disorders, and PFI-1, which has been widely studied in oncology, can reactivate HIV-1 from latency. RVX-208 and PFI-1 treatment alone or in combination with other latency reversing agents efficiently reactivated HIV-1 transcription through an up-regulation of P-TEFb by increasing CDK9 Thr-186 phosphorylation in latently infected Jurkat T cells in vitro. The two BET inhibitors also reactivated HIV-1 transcription in cART treated patient-derived resting CD4+ T cells ex vivo, without influence on global immune cell activation. Our findings, in combination with previous reports, further confirm that BET inhibitors are a group of leading compounds for combating HIV-1 latency for viral eradication.
Asunto(s)
Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Quinazolinonas/farmacología , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Antígenos CD4/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Quinasa 9 Dependiente de la Ciclina/metabolismo , Infecciones por VIH/tratamiento farmacológico , Humanos , Células Jurkat , Fosforilación , Quinazolinonas/química , Receptores CCR5/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
HIV-1 inserts its proviral DNA into the infected host cells, by which HIV proviral DNA can then be duplicated along with each cell division. Thus, provirus cannot be eradicated completely by current antiretroviral therapy. We have developed an innovative strategy to silence the HIV provirus by targeted DNA methylation on the HIV promoter region. We genetically engineered a chimeric DNA methyltransferase 1 composed of designed zinc-finger proteins to become ZF2 DNMT1. After transient transfection of the molecular clone encoding this chimeric protein into HIV-1 infected or latently infected cells, efficient suppression of HIV-1 expression by the methylation of CpG islands in 5'-LTR was observed and quantified. The effective suppression of HIV in latently infected cells by ZF2-DNMT1 is stable and can last through about 40 cell passages. Cytotoxic caused by ZF2-DNMT1 was only observed during cellular proliferation. Taken together, our results demonstrate the potential of this novel approach for anti-HIV-1 therapy.
RESUMEN
The long-lived latent HIV-1 reservoir is the major barrier for complete cure of Acquired Immune Deficiency Syndrome (AIDS). Here we report that a novel bromodomain and extraterminal domain (BET) inhibitor bromosporine which can broadly target BETs, is able to potently reactivate HIV-1 replication in different latency models alone and more powerful when combined with prostratin or TNF-α. Furthermore, the treatment with bromosporine induced HIV-1 full-length transcripts in resting CD4+ T cells from infected individuals with suppressive antiretroviral therapy (ART) ex vivo, with no obvious cytotoxicity or global activation of T cell. Finally, our data suggest that Tat plays a critical role in the bromosporine-mediated reactivation of latent HIV-1, which involved the increase of CDK9 T-loop phosphorylation. In summary, we found that the BET inhibitor bromosporine, alone or with other activators, might be a candidate for future HIV-1 eradication strategies.