Dataset Information

MS3D structural elucidation of the HIV-1 packaging signal.

ABSTRACT: The structure of HIV-1 Psi-RNA has been elucidated by a concerted approach combining structural probes with mass spectrometric detection (MS3D), which is not affected by the size and crystallization properties of target biomolecules. Distance constraints from bifunctional cross-linkers provided the information required for assembling an all-atom model from the high-resolution coordinates of separate domains by triangulating their reciprocal placement in 3D space. The resulting structure revealed a compact cloverleaf morphology stabilized by a long-range tertiary interaction between the GNRA tetraloop of stemloop 4 (SL4) and the upper stem of stemloop 1 (SL1). The preservation of discrete stemloop structures ruled out the possibility that major rearrangements might produce a putative supersite with enhanced affinity for the nucleocapsid (NC) domain of the viral Gag polyprotein, which would drive genome recognition and packaging. The steric situation of single-stranded regions exposed on the cloverleaf structure offered a valid explanation for the stoichiometry exhibited by full-length Psi-RNA in the presence of NC. The participation of SL4 in a putative GNRA loop-receptor interaction provided further indications of the plasticity of this region of genomic RNA, which can also anneal with upstream sequences to stabilize alternative conformations of the 5' untranslated region (5'-UTR). Considering the ability to sustain specific NC binding, the multifaceted activities supported by the SL4 sequence suggest a mechanism by which Gag could actively participate in regulating the vital functions mediated by 5'-UTR. Substantiated by the 3D structure of Psi-RNA, the central role played by SL4 in specific RNA-RNA and protein-RNA interactions advances this domain as a primary target for possible therapeutic intervention.

SUBMITTER: Yu ET

PROVIDER: S-EPMC2527897 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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MS3D structural elucidation of the HIV-1 packaging signal.

Yu Eizadora T ET Hawkins Arie A Eaton Julian J Fabris Daniele D

Proceedings of the National Academy of Sciences of the United States of America 20080819 34

The structure of HIV-1 Psi-RNA has been elucidated by a concerted approach combining structural probes with mass spectrometric detection (MS3D), which is not affected by the size and crystallization properties of target biomolecules. Distance constraints from bifunctional cross-linkers provided the information required for assembling an all-atom model from the high-resolution coordinates of separate domains by triangulating their reciprocal placement in 3D space. The resulting structure revealed ...[more]

PMID: 18713870

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Project description: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.

Project description:Alphaviruses are a group of small, enveloped viruses which are widely distributed on all continents. In infected cells, alphaviruses display remarkable specificity in RNA packaging by encapsidating only their genomic RNA while avoiding packaging of the more abundant viral subgenomic (SG), cellular messenger and transfer RNAs into released virions. In this work, we demonstrate that in spite of evolution in geographically isolated areas and accumulation of considerable diversity in the nonstructural and structural genes, many alphaviruses belonging to different serocomplexes harbor RNA packaging signals (PSs) which contain the same structural and functional elements. Their characteristic features are as follows. (i) Sindbis, eastern, western, and Venezuelan equine encephalitis and most likely many other alphaviruses, except those belonging to the Semliki Forest virus (SFV) clade, have PSs which can be recognized by the capsid proteins of heterologous alphaviruses. (ii) The PS consists of 4 to 6 stem-loop RNA structures bearing conserved GGG sequences located at the base of the loop. These short motifs are integral elements of the PS and can function even in the artificially designed PS. (iii) Mutagenesis of the entire PS or simply the GGG sequences has strong negative effects on viral genome packaging and leads to release of viral particles containing mostly SG RNAs. (iv) Packaging of RNA appears to be determined to some extent by the number of GGG-containing stem-loops, and more than one stem-loop is required for efficient RNA encapsidation. (v) Viruses of the SFV clade are the exception to the general rule. They contain PSs in the nsP2 gene, but their capsid protein retains the ability to use the nsP1-specific PS of other alphaviruses. These new discoveries regarding alphavirus PS structure and function provide an opportunity for the development of virus variants, which are irreversibly attenuated in terms of production of infectious virus but release high levels of genome-free virions.

Dataset Information

MS3D structural elucidation of the HIV-1 packaging signal.

Publications

MS3D structural elucidation of the HIV-1 packaging signal.

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