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

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.Electronic supplementary materialThe online version of this article (10.1186/s12977-018-0407-4) contains supplementary material, which is available to authorized users.

Coronavirus genomic RNA packaging

“…The discovery that a packaging-defective MHV mutant was markedly suppressed by host innate immunity suggests an attractive pathway toward live-attenuated vaccine design (Athmer et al, 2018). The PS itself may also become a candidate for therapeutic intervention as RNA structures are being increasingly developed as small-molecule druggable targets (Anokhina et al, 2019;Ingemarsdotter et al, 2018). Additionally, more precise elucidation of packaging-specific oligomeric interactions could uncover molecular targets that would hinder the escape of drug-resistant viral mutants (Tanner et al, 2014).…”

NMR Studies of Retroviral Genome Packaging

“…Multiple small molecules were identified to inhibit NC-Ψ interactions [ 387 , 388 ]. Among them, a quinolinium derivative NSC260594 (NSC, Figure 19 A), was shown to be a specific HIV-1 RNA packaging inhibitor [ 389 ] and exhibited potent antiviral activity [ 388 , 389 ]. NSC treatment caused a similar packaging defect as that of the Δp1 mutation—a 19 nt deletion that historically led to the identification of the Ψ-hairpin as a major packaging determinant; Figure 19 C [ 228 , 389 ].…”

“…Among them, a quinolinium derivative NSC260594 (NSC, Figure 19 A), was shown to be a specific HIV-1 RNA packaging inhibitor [ 389 ] and exhibited potent antiviral activity [ 388 , 389 ]. NSC treatment caused a similar packaging defect as that of the Δp1 mutation—a 19 nt deletion that historically led to the identification of the Ψ-hairpin as a major packaging determinant; Figure 19 C [ 228 , 389 ]. NSC was initially identified as an inhibitor of the interaction between Gag and a 20-nt construct containing the upper stem and the apical tetraloop of the Ψ-hairpin [ 388 ].…”

Development of a Novel Competitive qRT-PCR Assay to Measure Relative Lentiviral Packaging Efficiency

“…ΔP1 is an HIV-1 mutant with a 19-bp deletion in the Ψ region whose packaging efficiency has been measured previously by different methodologies to be less than 2% of that of the WT virus. 43 , 44 The ΔP1-pCCL-EGFP transfer vector was created and modified to contain the BglG insert. The ΔP1-pCCL-EGFP-BglG plasmid was used for co-transfections with WT-pCCL-EGFP-MS2.…”