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A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

ABSTRACT: RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. 'XL-SHAPE' was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.

SUBMITTER: Kenyon JC

PROVIDER: S-EPMC4598872 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

Kenyon Julia C JC Prestwood Liam J LJ Lever Andrew M L AM

Scientific reports 20151009

RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein co ...[more]

PMID: 26449409

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Project description:The ability of the retroviral Gag protein of Rous sarcoma virus (RSV) to transiently traffic through the nucleus is well-established and has been implicated in genomic RNA (gRNA) packaging Although other retroviral Gag proteins (human immunodeficiency virus type 1, HIV-1; feline immunodeficiency virus, FIV; Mason-Pfizer monkey virus, MPMV; mouse mammary tumor virus, MMTV; murine leukemia virus, MLV; and prototype foamy virus, PFV) have also been observed in the nucleus, little is known about what, if any, role nuclear trafficking plays in those viruses. In the case of HIV-1, the Gag protein interacts in nucleoli with the regulatory protein Rev, which facilitates nuclear export of gRNA. Based on the knowledge that RSV Gag forms viral ribonucleoprotein (RNPs) complexes with unspliced viral RNA (USvRNA) in the nucleus, we hypothesized that the interaction of HIV-1 Gag with Rev could be mediated through vRNA to form HIV-1 RNPs. Using inducible HIV-1 proviral constructs, we visualized HIV-1 Gag and USvRNA in discrete foci in the nuclei of HeLa cells by confocal microscopy. Two-dimensional co-localization and RNA-immunoprecipitation of fractionated cells revealed that interaction of nuclear HIV-1 Gag with USvRNA was specific. Interestingly, treatment of cells with transcription inhibitors reduced the number of HIV-1 Gag and USvRNA nuclear foci, yet resulted in an increase in the degree of Gag co-localization with USvRNA, suggesting that Gag accumulates on newly synthesized viral transcripts. Three-dimensional imaging analysis revealed that HIV-1 Gag localized to the perichromatin space and associated with USvRNA and Rev in a tripartite RNP complex. To examine a more biologically relevant cell, latently infected CD4+ T cells were treated with prostratin to stimulate NF-κB mediated transcription, demonstrating striking localization of full-length Gag at HIV-1 transcriptional burst site, which was labelled with USvRNA-specific riboprobes. In addition, smaller HIV-1 RNPs were observed in the nuclei of these cells. These data suggest that HIV-1 Gag binds to unspliced viral transcripts produced at the proviral integration site, forming vRNPs in the nucleus.

Dataset Information

A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

Publications

A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

Similar Datasets

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