Effect of transcription inhibition and generation of suppressive viral non-coding RNAs

Daniel O Pinto,Chen Zeng,Michelle L Pleet,Catherine Demarino,Mohammed Saifuddin,Fatah Kashanchi,Tristan A Scott,Maria Cowen,Marc S Weinberg,Robert A Barclay,Dmytro Kovalskyy,Thy T Vo,James Erickson

doi:10.1186/s12977-019-0475-0

Abstract

BackgroundHIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require life-long adherence at high expense. In chronic cART-treated patients with undetectable viral titers, cell-associated viral RNA is still detectable, pointing to low-level viral transcriptional leakiness. To date, there are no FDA-approved drugs against HIV-1 transcription. We have previously shown that F07#13, a third generation Tat peptide mimetic with competitive activity against Cdk9/T1-Tat binding sites, inhibits HIV-1 transcription in vitro and in vivo.ResultsHere, we demonstrate that increasing concentrations of F07#13 (0.01, 0.1, 1 µM) cause a decrease in Tat levels in a dose-dependent manner by inhibiting the Cdk9/T1-Tat complex formation and subsequent ubiquitin-mediated Tat sequestration and degradation. Our data indicate that complexes I and IV contain distinct patterns of ubiquitinated Tat and that transcriptional inhibition induced by F07#13 causes an overall reduction in Tat levels. This reduction may be triggered by F07#13 but ultimately is mediated by TAR-gag viral RNAs that bind suppressive transcription factors (similar to 7SK, NRON, HOTAIR, and Xist lncRNAs) to enhance transcriptional gene silencing and latency. These RNAs complex with PRC2, Sin3A, and Cul4B, resulting in epigenetic modifications. Finally, we observed an F07#13-mediated decrease of viral burden by targeting the R region of the long terminal repeat (HIV-1 promoter region, LTR), promoting both paused polymerases and increased efficiency of CRISPR/Cas9 editing in infected cells. This implies that gene editing may be best performed under a repressed transcriptional state.ConclusionsCollectively, our results indicate that F07#13, which can terminate RNA Polymerase II at distinct sites, can generate scaffold RNAs, which may assemble into specific sets of “RNA Machines” that contribute to gene regulation. It remains to be seen whether these effects can also be seen in various clades that have varying promoter strength, mutant LTRs, and in patient samples.

Highlights

human immunodeficiency virus-1 (HIV-1) patients receiving combination antiretroviral therapy survive infection but require lifelong adherence at high expense
We explored the effect of F07#13 on Tat levels and found that Tat was present in detectable amounts; when F07#13 (0.01, 0.1, 1 μM) was added to cells 24 h post-transfection with Flag-Tat101 or 89.6 and incubated for 48 h with F07#13, the levels of Tat decreased with increasing concentration of F07#13 (Fig. 1b)
Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology against transactivating response element (TAR), we found that 2 guide RNA (gRNA) were effective in binding to their target sites and editing the HIV-1 genome in three cell lines infected with LAI strains

Summary

Introduction

HIV-1 patients receiving combination antiretroviral therapy (cART) survive infection but require lifelong adherence at high expense. There are multiple complications associated with chronic infection, such as HIV-1 associated neurocognitive disorders (HAND), which encompasses neurocognitive impairment in about 50% of patients despite the use of combination antiretroviral therapy (cART) [23, 26, 30, 34]. This chronic state, especially under cART, promotes a viral state of latency that may be represented by low level manifestations of viral products [7, 18]. It is critical to understand the basic mechanisms of pathogenesis and discover new treatments to combat the virus

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