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

• DOI: 10.1186/s12977-018-0407-4
• Publication Date: 2018-03-14T12:57:38Z
• Authors: Carin K. Ingemars...; Jingwei Zeng; Ziqi Long; Andrew M.L. Lever; Julia C. Kenyon
• Keywords: conformation; 0301 basic medicine; virus particle; virus assembly; Human immunodeficiency virus 1; HIV Infections; protein binding; virus entry; Western blotting; RNA encapsidation; Proviruses; genetics; Viral; RNA structure; Virus Release; 0303 health sciences; conformational transition; dimerization; Genome; virus DNA cell DNA interaction; antiretrovirus agent; Viral Load; provirus; unclassified drug; virology; 3. Good health; genomic RNA; real time polymerase chain reaction; Packaging; cytoplasm; 4 [(1 methyl 6 nitro 4(1h) quinolinylidene)amino] n [4 [(1 methyl 4(1h) pyridinylidene)amino]phenyl]benzamide; RNA, Viral; 5' untranslated region; Protein Binding; gag; 570; virus infectivity; Virus Integration; Gene Products, gag; nsc 260594; Genome, Viral; chemistry; Real-Time Polymerase Chain Reaction; Article; Genomic Instability; reverse transcription polymerase chain reaction; 03 medical and health sciences; virus RNA; Human immunodeficiency virus infection; Gene Products; Humans; controlled study; human; Gag protein; nonhuman; virion; Research; Virus Assembly; virus load; RC581-607; genomic instability; 540; enzyme linked immunosorbent assay; virus genome; physiology; virus release; Antiretroviral drugs; HIV-1; Nucleic Acid Conformation; Immunologic diseases. Allergy; 5' Untranslated Regions; metabolism

Background 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 () 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. Results Treatment 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. Conclusions NSC260594 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 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.<br/>This work was supported by grants from the Biomedical Research Centre (RCCT.EFPO to JK and AML) and the Medical Research Council (RCAG/565 to AML).<br/>