scholarly journals Efficient inhibition of HIV using CRISPR/Cas13d nuclease system

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.

scholarly journals Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1850
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, non-overlapping 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.

scholarly journals Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1+and TIGIT+CD4 T Cells

2019 ◽  
Vol 93 (10) ◽  
Author(s):  
George N. Llewellyn ◽  
Eduardo Seclén ◽  
Stephen Wietgrefe ◽  
Siyu Liu ◽  
Morgan Chateau ◽  
...  
Keyword(s):  
T Cells ◽  
Mouse Model ◽  
Ex Vivo ◽  
Latent Reservoir ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

ABSTRACTCombination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used anex vivolatency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use theex vivolatency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishesin vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCEHIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body’s immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

scholarly journals Inhibition of the H3K27 demethylase UTX enhances the epigenetic silencing of HIV proviruses and induces HIV-1 DNA hypermethylation but fails to permanently block HIV reactivation

2021 ◽  
Author(s):  
Kien Nguyen ◽  
Jonathan Karn ◽  
Won Kyung ◽  
Curtis Dobrowolski ◽  
Meenakshi Shukla
Keyword(s):  
Dna Methylation ◽  
T Cells ◽  
Cell Receptor ◽  
Epigenetic Silencing ◽  
Dna Hypermethylation ◽  
Dual Inhibitor ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

One strategy for a functional cure of HIV-1 is block and lock, which seeks to permanently suppress the rebound of quiescent HIV-1 by epigenetic silencing. For the HIV LTR, both histone 3 lysine 27 tri-methylation (H3K27me3) and DNA methylation are associated with viral suppression, while H3K4 tri-methylation (H3K4me3) is correlated with viral expression. However, H3K27me3 is readily reversed upon activation of T-cells through the T-cell receptor. To suppress latent HIV-1 in a stable fashion, we depleted the expression or inhibited the activity of UTX/KDM6A, the major H3K27 demethylase, and investigated its impact on latent HIV-1 reactivation in T cells. Inhibition of UTX dramatically enhanced H3K27me3 levels at the HIV LTR and were associated with increased DNA methylation. In latently infected cells from patients, GSK-J4, which is a potent dual inhibitor of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A, effectively suppressed the reactivation of latent HIV-1 and induced DNA methylation at specific sites in the 5' LTR of latent HIV-1 by the enhanced recruitment of DNMT3A to HIV-1. Nonetheless, suppression of HIV-1 through epigenetic silencing required the continued treatment with GSK-J4 and was rapidly reversed after removal of the drug. Thus, epigenetic silencing by itself appears to be insufficient to permanently silence HIV-1 proviral transcription.

scholarly journals Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4+ T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer

2021 ◽  
Vol 12 ◽  
Author(s):  
Isobel Okoye ◽  
Lai Xu ◽  
Olaide Oyegbami ◽  
Shima Shahbaz ◽  
Desmond Pink ◽  
...  
Keyword(s):  
T Cells ◽  
Extracellular Vesicles ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Hiv Latency ◽  
Healthy Controls ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

HIV latency is a challenge to the success of antiretroviral therapy (ART). Hence patients may benefit from interventions that efficiently reactivate the latent virus to be eliminated by ARTs. Here we show that plasma extracellular vesicles (pEVs) can enhance HIV infection of activated CD4+ T cells and reactivate the virus in latently infected J-Lat 10.6 cells. Evaluation of the extravesicular miRNA cargo by a PCR array revealed that pEVs from HIV patients express miR-139-5p. Furthermore, we found that increased levels of miR-139-5p in J-Lat 10.6 cells incubated with pEVs corresponded with reduced expression of the transcription factor, FOXO1. pEV treatment also corresponded with increased miR-139-5p expression in stimulated PD1+ Jurkat cells, but with concomitant upregulation of FOXO1, Fos, Jun, PD-1 and PD-L1. However, J-Lat 10.6 cells incubated with miR-139-5p inhibitor-transfected pEVs from HIV ART-naïve and on-ART patients expressed reduced levels of miR-139-5p than cells treated with pEVs from healthy controls (HC). Collectively, our results indicate that pEV miR-139-5p belongs to a network of miRNAs that can promote cell activation, including latent HIV-infected cells by regulating the expression of FOXO1 and the PD1/PD-L1 promoters, Fos and Jun.

scholarly journals Potential Utility of Natural Killer Cells for Eliminating Cells Harboring Reactivated Latent HIV-1 Following the Removal of CD8+ T Cell-Mediated Pro-Latency Effect(s)

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1451
Author(s):  
Georges Khoury ◽  
Deanna A. Kulpa ◽  
Matthew S. Parsons
Keyword(s):  
T Cells ◽  
Natural Killer ◽  
Cd8 T Cells ◽  
People Living With Hiv ◽  
Latently Infected ◽  
Shock And Kill ◽  
Infected Cells ◽  
Latent Hiv ◽  
Living With Hiv ◽  
Hiv 1

An impediment to curing HIV-1 infection is the persistence of latently infected cells in ART-treated people living with HIV (PLWH). A key strategy for curing HIV-1 infection is to activate transcription and translation of latent virus using latency reversing agents (LRAs) and eliminate cells harboring reactivated virus via viral cytopathic effect or immune clearance. In this review, we provide an overview of available LRAs and their use in clinical trials. Furthermore, we describe recent data suggesting that CD8+ T cells promote HIV-1 latency in the context of ART, even in the presence of LRAs, which might at least partially explain the clinical inefficiency of previous “shock and kill” trials. Here, we propose a novel cure strategy called “unlock, shock, disarm, and kill”. The general premise of this strategy is to shut down the pro-latency function(s) of CD8+ T cells, use LRAs to reverse HIV-1 latency, counteract anti-apoptotic molecules, and engage natural killer (NK) cells to mediate the killing of cells harboring reactivated latent HIV-1.

scholarly journals CAGE-seq reveals that HIV-1 latent infection does not trigger unique cellular responses in a Jurkat T cell model.

2021 ◽  
Author(s):  
Hiroyuki Matsui ◽  
Kotaro Shirakawa ◽  
Yoshinobu Konishi ◽  
Shigeki Hirabayashi ◽  
Anamaria Daniela Sarca ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Mtor Pathway ◽  
Differentially Expressed ◽  
Transcriptional Responses ◽  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

The cure for HIV-1 is currently stalled by our inability to specifically identify and target latently infected cells. HIV-1 viral RNA/DNA or viral proteins are recognized by cellular mechanisms and induce interferon responses in virus producing cells, but changes in latently infected cells remain unknown. HIVGKO contains a GFP reporter under the HIV-1 promoter and an mKO2 reporter under the internal EF1α promoter. This viral construct enables direct identification of HIV-1 both productively and latently infected cells. In this study we aim to identify specific cellular transcriptional responses triggered by HIV-1 entry and integration using Cap Analysis of Gene Expression (CAGE). We deep sequenced CAGE tags in uninfected, latently and productively infected cells and compared their differentially expressed transcription start site (TSS) profiles. Virus producing cells had differentially expressed TSSs related to T-cell activation and apoptosis when compared to uninfected cells or latently infected cells. Surprisingly, latently infected cells had only 33 differentially expressed TSSs compared to uninfected cells. Among these, SPP1 and APOE were down-regulated in latently infected cells. SPP1 or APOE knockdown in Jurkat T cells increased susceptibility to HIVGKO infection, suggesting that they have anti-viral properties. Components of the PI3K/mTOR pathway, MLST8, 4EBP and RPS6, were significant TSSs in productively infected cells, and S6K phosphorylation was increased compared to latently infected cells, suggesting that mTOR pathway activity plays a role in establishing the latent reservoir. These findings indicate that HIV-1 entry and integration do not trigger unique transcriptional responses when infection becomes latent. Importance: Latent HIV-1 infection is established as early as the first viral exposure and remains the most important barrier in obtaining the cure for HIV-1 infection. Here, we used CAGE to compare the transcriptional landscape of latently infected cells with that of non-infected or productively infected cells. We found that latently infected cells and non-infected cells show quite similar transcriptional profiles. Our data suggest that T-cells cannot recognize incoming viral components nor the integrated HIV-1 genome when infection remains latent. These findings should guide future research into widening our approaches to identify and target latent HIV-1 infected cells.

scholarly journals Exonic Disruption Facilitates Antiviral CRISPR-Cas9 Activity for Multistrain HIV-1 Elimination

2021 ◽  
Author(s):  
Jonathan Herskovitz ◽  
Mahmudul Hasan ◽  
Milankumar Patel ◽  
Wilson R. Blomberg ◽  
Jacob D. Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
Consensus Sequence ◽  
Hiv Cure ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Antiretroviral Drug Resistance ◽  
Antiretroviral Drug ◽  
Latently Infected ◽  
Infected Cells ◽  
Hiv 1

AbstractA barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high mutation rate of HIV-1 gives rise to numerous circulating strains with increased capacity for immune evasion and antiretroviral drug resistance. To facilitate viral elimination while accounting for this diversity, we propose genetic inactivation of proviral DNA with CRISPR-spCas9. We designed a library of “mosaic gRNAs” against a HIV-1 consensus sequence constructed from 4004 clinical strains, targeting the viral transcriptional regulator tat. Testing in 7 HIV-1 transmitted founder strains led, on average, to viral reductions of 82% with tandem TatD and TatE (TatDE) treatment. No off-target cleavages were recorded. Lentiviral transduction of TatDE attenuated latency reversal by 94% in HIV-infected, transcriptionally silent ACH2 T cells. In all, TatDE guide RNAs successfully disrupted 5 separate HIV-1 exons (tat1-2/rev1-2/gp41) providing a pathway for CRISPR-directed HIV-1 cure therapies.Significance StatementOver 38 million individuals worldwide are infected with HIV-1, which necessitates lifelong dependence on antiretroviral therapy (ART) to prevent viral replication that leads to AIDS. Efforts to rid hosts of HIV-1 are limited by the virus’ abilities to integrate proviral DNA in nuclei, mutate their genomes, and lay dormant for decades during ART treatment. We developed mosaic guide RNAs, TatD and TatE, for CRISPR-Cas9 that recognize the majority of known HIV-1 strains and inactivate 94% of proviral DNA in latently infected cells. Tandem TatDE-CRISPR inactivation of 5 viral exons (tat1-2, rev1-2, and gp41), which blocked HIV-1 replication for 28 days in CD4+ T cells without unwanted editing to the host genome, may serve as a viable strategy for HIV cure.

scholarly journals The bromodomain and extraterminal domain inhibitor bromosporine synergistically reactivates latent HIV-1 in latently infected cells

Oncotarget ◽  
2017 ◽  
Vol 8 (55) ◽  
pp. 94104-94116 ◽  
Author(s):  
Hanyu Pan ◽  
Panpan Lu ◽  
Yinzhong Shen ◽  
Yanan Wang ◽  
Zhengtao Jiang ◽  
...  
Keyword(s):  
Latently Infected ◽  
Infected Cells ◽  
Latent Hiv ◽  
Hiv 1

A new small-molecule compound, Q308, silences latent HIV-1 provirus by suppressing Tat- and FACT-mediated transcription

2021 ◽  
Author(s):  
Chen-liang Zhou ◽  
Yi-fan Huang ◽  
Yi-bin Li ◽  
Tai-zhen Liang ◽  
Teng-yi Zheng ◽  
...  
Keyword(s):  
Small Molecule ◽  
Difficult Problem ◽  
Viral Reservoir ◽  
Functional Cure ◽  
Latently Infected ◽  
Small Molecule Compound ◽  
Infected Cells ◽  
Latent Hiv ◽  
Anti Hiv ◽  
Hiv 1

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.

scholarly journals Similar frequency and inducibility of intact HIV-1 proviruses in blood and lymph nodes

2020 ◽  
Author(s):  
Alyssa R Martin ◽  
Alexandra M Bender ◽  
Jada Hackman ◽  
Kyungyoon J Kwon ◽  
Briana A Lynch ◽  
...  
Keyword(s):  
T Cells ◽  
Lymph Nodes ◽  
Cd4 T Cells ◽  
Viral Rna ◽  
Latent Reservoir ◽  
Similar Frequency ◽  
Latently Infected ◽  
Infected Cells ◽  
Secondary Lymphoid Organs ◽  
Hiv 1

Abstract Background The HIV-1 latent reservoir (LR) in resting CD4 + T cells is a barrier to cure. LR measurements are commonly performed on blood samples and therefore may miss latently infected cells residing in tissues, including lymph nodes. Methods We determined the frequency of intact HIV-1 proviruses and proviral inducibility in matched peripheral blood (PB) and lymph node (LN) samples from ten HIV-1-infected patients on ART using the intact proviral DNA assay and a novel quantitative viral induction assay. Prominent viral sequences from induced viral RNA were characterized using a next-generation sequencing assay. Results The frequencies of CD4 + T cells with intact proviruses were not significantly different in PB vs LN (61vs104/10 6CD4 + cells), and were substantially lower than frequencies of CD4 + T cells with defective proviruses. The frequencies of CD4 + T cells induced to produce high levels of viral RNA were not significantly different in PB vs LN (4.3/10 6 vs 7.9/10 6), but were 14-fold lower than the frequencies of cells with intact proviruses. Sequencing of HIV-1 RNA from induced proviruses revealed comparable sequences in paired PB and LN samples. Conclusions These results further support the use of PB as an appropriate proxy for the HIV-1 LR in secondary lymphoid organs

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