Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies.

Sarah C Keane,Yun Bai,Jennifer A Doudna,James H Hurley,Lars-Anders Carlson

doi:10.7554/elife.14663

Abstract

HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5' untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another.

Highlights

Packaging the correct genetic material is a key event in the assembly of an infectious virus
We showed that giant unilamellar vesicles (GUVs)-bound Gag clusters mimic the behavior of cellular HIV-1 budding sites in their lipid requirements for assembly, nucleic acid- and ESCRT protein binding
ribonucleic acid (RNA) were labeled with the fluorophore Alexa488 on random guanosines, at measured average labeling efficiencies of ~1.1–1.2 fluorophores per RNA molecule. 100 nM Gag-ATTO594 was premixed with 0.5 nM fluorescent RNA, and added to GUVs which were imaged by confocal microscopy starting 10 min after addition of protein and RNA

Summary

Introduction

Packaging the correct genetic material is a key event in the assembly of an infectious virus. In the case of HIV-1, both the recognition of the genomic RNA, its targeting to the plasma membrane, and assembly of a membrane-enveloped virus around it is carried out by the viral protein Gag (Rein et al, 2011; Sundquist and Krausslich, 2012; Freed, 2015). Full-length genomic RNA through interactions with its 5’ untranslated region (5’UTR), which is different in spliced and unspliced constructs (Lu et al, 2011b; Kuzembayeva et al, 2014). In addition to sequences that mediate the packaging of the genomic RNA, the 5’UTR has sequence elements related to transcription processivity (TAR), binding of tRNALys-3 as a primer for reverse transcription (PBS), and genome dimerization (DIS) (Johnson and Telesnitsky, 2010; Lu et al, 2011b; Kuzembayeva et al, 2014)

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