Protein Delivery of Cell-Penetrating Zinc-Finger Activators Stimulates Latent HIV-1-Infected Cells

Eliminating the latent HIV reservoir remains a difficult problem for creating an HIV functional cure or achieving remission. The “block-and-lock” strategy aims to steadily suppress transcription of the viral reservoir and lock the HIV promoter in deep latency using latency-promoting agents (LPAs). However, to date, most of the investigated LPA candidates are not available for clinical trials, and some of them exhibit immune-related adverse reactions. The discovery and development of new, active, and safe LPA candidates for an HIV cure are necessary to eliminate residual HIV-1 viremia through the “block-and-lock” strategy. In this study, we demonstrated that a new small-molecule compound, Q308, silenced the HIV-1 provirus by inhibiting Tat-mediated gene transcription and selectively downregulating the expression levels of the facilitated chromatin transcription (FACT) complex. Strikingly, Q308 induced the preferential apoptosis in HIV-1 latently infected cells, indicating that Q308 may reduce the size of the viral reservoir and thus further prevent viral rebound. These findings highlight that Q308 is a novel and safe anti-HIV-1 inhibitor candidate for a functional cure.

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A CRISPR/Cas9 screen identifies the histone demethylase MINA53 as a novel HIV-1 latency-promoting gene (LPG)

Nucleic Acids Research ◽

10.1093/nar/gkz493 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7333-7347 ◽

Cited By ~ 8

Author(s):

Huachao Huang ◽

Weili Kong ◽

Maxime Jean ◽

Guillaume Fiches ◽

Dawei Zhou ◽

...

Keyword(s):

Local Level ◽

Histone Demethylase ◽

Host Factors ◽

Epigenetic Mark ◽

Combination Antiretroviral Therapy ◽

Downstream Effectors ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

AbstractAlthough combination antiretroviral therapy is potent to block active replication of HIV-1 in AIDS patients, HIV-1 persists as transcriptionally inactive proviruses in infected cells. These HIV-1 latent reservoirs remain a major obstacle for clearance of HIV-1. Investigation of host factors regulating HIV-1 latency is critical for developing novel antiretroviral reagents to eliminate HIV-1 latent reservoirs. From our recently accomplished CRISPR/Cas9 sgRNA screens, we identified that the histone demethylase, MINA53, is potentially a novel HIV-1 latency-promoting gene (LPG). We next validated MINA53’s function in maintenance of HIV-1 latency by depleting MINA53 using the alternative RNAi approach. We further identified that in vitro MINA53 preferentially demethylates the histone substrate, H3K36me3 and that in cells MINA53 depletion by RNAi also increases the local level of H3K36me3 at LTR. The effort to map the downstream effectors unraveled that H3K36me3 has the cross-talk with another epigenetic mark H4K16ac, mediated by KAT8 that recognizes the methylated H3K36 and acetylated H4K16. Removing the MINA53-mediated latency mechanisms could benefit the reversal of post-integrated latent HIV-1 proviruses for purging of reservoir cells. We further demonstrated that a pan jumonji histone demethylase inhibitor, JIB-04, inhibits MINA53-mediated demethylation of H3K36me3, and JIB-04 synergizes with other latency-reversing agents (LRAs) to reactivate latent HIV-1.

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Epigenetic compound screening uncovers small molecules for re-activation of latent HIV-1

10.1101/2020.08.21.262311 ◽

2020 ◽

Author(s):

Ariane Zutz ◽

Lin Chen ◽

Franziska Sippl ◽

Christian Schölz

Keyword(s):

High Throughput Screening ◽

Model Systems ◽

Reporter System ◽

Compound Screening ◽

Infected Cells ◽

Low Toxicity ◽

Immunological Destruction ◽

New Treatment ◽

Latent Hiv ◽

Hiv 1

AbstractDuring infection with the human immunodeficiency virus type 1 (HIV-1), latent reservoirs are established, which circumvent full eradication of the virus by antiretroviral therapy (ART) and are the source for viral rebound after cessation of therapy. As these reservoirs are phenotypically undistinguishable from infected cells, current strategies aim to reactivate these reservoirs, followed by pharmaceutical and immunological destruction of the cells.Here, we employed a simple and convenient cell-based reporter system, which enables sample handling under biosafety level (BSL)-1 conditions, to screen for compounds that were able to reactivate latent HIV-1. The assay showed a high dynamic signal range and reproducibility with an average Z-factor of 0.77, classifying the system as robust. The assay was used for high-throughput screening (HTS) of an epigenetic compound library in combination with titration and cell-toxicity studies and revealed several potential new latency reversing agents (LRAs). Further validation in well-known latency model systems verified earlier studies and identified two novel compounds with very high reactivation efficiency and low toxicity. Both drugs, namely N-hydroxy-4-(2-[(2-hydroxyethyl)(phenyl)amino]-2-oxoethyl)benzamide (HPOB) and 2',3'-difluoro-[1,1'-biphenyl]-4-carboxylic acid, 2-butylhydrazide (SR-4370), showed comparable performances to other already known LRAs, did not activate CD4+ T-cells or caused changes in the composition of PBMCs as shown by flow cytometry analyses.Both compounds may represent an effective new treatment possibility for revocation of latency in HIV-1 infected individuals.

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Potential of Radiation-Induced Cellular Stress for Reactivation of Latent HIV-1 and Killing of Infected Cells

AIDS Research and Human Retroviruses ◽

10.1089/aid.2016.0006 ◽

2016 ◽

Vol 32 (2) ◽

pp. 120-124 ◽

Cited By ~ 9

Author(s):

Sergey Iordanskiy ◽

Fatah Kashanchi

Keyword(s):

Cellular Stress ◽

Radiation Induced ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

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Prevention of Activation of HIV-1 by Antiviral Agents in OM-10.1 Cells

Antiviral Chemistry and Chemotherapy ◽

10.1177/095632029300400107 ◽

1993 ◽

Vol 4 (1) ◽

pp. 55-63 ◽

Cited By ~ 10

Author(s):

P. M. Feorino ◽

S. T. Butera ◽

T. M. Folks ◽

R. F. Schinazi

Keyword(s):

Antiviral Agents ◽

Interferon Γ ◽

Reverse Transcriptase Activity ◽

Chronic Infections ◽

New Therapeutic Approaches ◽

Cellular Expression ◽

Infected Cells ◽

Latent Hiv ◽

Factor Α ◽

Hiv 1

The development of a reliable and simple system for evaluating compounds that could prevent activation of latent HIV would allow us to devise new therapeutic approaches. These compounds could eventually be used in combination with drugs that are effective against acute and chronic infections. The OM-10.1 cell line is a chronically infected clone which remains CD4+ until HIV-1 activation with tumour necrosis factor-α. A variety of compounds are known to have antiviral properties against either acutely or chronically infected cells were evaluated for their ability to inhibit HIV induced expression in these cells. We also examined the effect of several compounds that interact with biochemical pathways that may interfere with or enhance the reactivation process. These included nucleoside analogues, cytokines, steroidal and nonsteroidal anti-inflammatory agents, polyoxometalates, a TAT inhibitor, various natural products (including nerve growth factor, N-acetyl-L-cysteine, taxol, and interferons), TIBO, porphyrins, and various oligomers. CD4 cellular expression and supernatant reverse transcriptase activity were quantitated as markers of induced viral expression. Among the 58 compounds evaluated, 3′-fluoro-3′-deoxythymidine (FLT), interferon γ, Ro 5–3335 (a TAT inhibitor) and desferrioxamine were modest and selective inhibitors of HIV-1 activation.

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Dilazep synergistically reactivates latent HIV-1 in latently infected cells

Molecular Biology Reports ◽

10.1007/s11033-014-3662-z ◽

2014 ◽

Vol 41 (11) ◽

pp. 7697-7704 ◽

Cited By ~ 4

Author(s):

Hanxian Zeng ◽

Sijie Liu ◽

Pengfei Wang ◽

Xiying Qu ◽

Haiyan Ji ◽

...

Keyword(s):

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

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CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

10.1101/2020.09.25.308734 ◽

2020 ◽

Author(s):

Qiankun Wang ◽

Hongbo Gao ◽

Kolin M. Clark ◽

Pengfei Tang ◽

Gray H. Harlan ◽

...

Keyword(s):

Rapid Evolution ◽

Mutation Rates ◽

Host Immune System ◽

Virus Reactivation ◽

Viral Protease ◽

Viral Budding ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1 ◽

Recognition Receptor

AbstractHIV-1 has high mutation rates and exists as mutant swarms in the host. Rapid evolution of HIV-1 allows the virus to outpace host immune system, leading to viral persistence. Novel approaches to target immutable components are needed to clear HIV-1 infection. Here we report a pattern-recognition receptor CARD8 that senses enzymatic activity of the HIV-1 protease, which is indispensable for the virus. All subtypes of HIV-1 can be sensed by CARD8 despite substantial viral diversity. HIV-1 evades CARD8 sensing because the viral protease remains inactive in infected cells prior to viral budding. Induction of premature intracellular activation of the viral protease triggers CARD8 inflammasome-mediated pyroptosis of HIV-1-infected cells. This strategy leads to clearance of latent HIV-1 in patient CD4+ T cells after virus reactivation. Taken together, our study identifies CARD8 as an inflammasome sensor of HIV-1 that holds promise as a strategy for clearance of persistent HIV-1 infection.

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Efficient inhibition of HIV using CRISPR/Cas13d nuclease system

10.1101/2021.07.21.453273 ◽

2021 ◽

Author(s):

Hoang Nguyen ◽

Hannah Wilson ◽

Sahana Jayakumar ◽

Viraj Kulkarni ◽

Smita Kulkarni

Keyword(s):

T Cells ◽

Alternative Treatment ◽

Treatment Modality ◽

Rna Viruses ◽

Guide Rnas ◽

Rna Targeting ◽

Latently Infected ◽

Infected Cells ◽

Latent Hiv ◽

Hiv 1

Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13 mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.

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