An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation

Carin K Ingemarsdotter,Ziqi Long,Andrew M.L Lever,Julia C Kenyon,Jingwei Zeng

doi:10.1186/s12977-018-0407-4

Abstract

BackgroundNSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays.ResultsTreatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect.ConclusionsNSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594 the flexibility of SL3 appears to be a unique requirement for genome encapsidation and identifies this process as a highly specific drug target. This study is proof of principle that development of a new class of antiretroviral drugs that specifically target viral packaging by binding to the viral genomic RNA is achievable.

Highlights

NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag
Compounds that target the Gag-RNA interaction by binding to highly conserved sequences of the RNA instead of the cognate protein may provide far less scope for mutational escape. One such region that shows extremely limited variation is a 14 nt stretch just upstream of the Gag translation initiation site. This RNA folds into a stable helix known as SL3, which has been shown to be of importance during the viral packaging process, by binding the Gag protein in a highaffinity interaction [7, 8]
Phases of the viral lifecycle from entry through integration are unaffected by NSC NSC was previously observed to prevent Gag interacting with SL3 in vitro and to stabilize the RNA

Summary

Introduction

NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. The HIV-1 5′UTR is around 330 nucleotides (nt) long and contains a series of overlapping cis-acting signals that interact with viral and cellular factors to control the viral lifecycle It is far more highly conserved than the coding regions that follow it, with little or no variation observed between hundreds of sequenced clinical isolates in some sections [6]. Compounds that target the Gag-RNA interaction by binding to highly conserved sequences of the RNA instead of the cognate protein may provide far less scope for mutational escape. One such region that shows extremely limited variation is a 14 nt stretch just upstream of the Gag translation initiation site. This RNA folds into a stable helix known as SL3, which has been shown to be of importance during the viral packaging process, by binding the Gag protein in a highaffinity interaction [7, 8]

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Discussion

Conclusion