Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal

Alice Telesnitsky,Sapna Basappa,Alexis Waller,Emily Cannistraci,Hana Flores,Verna Van,Ugonna Mbaekwe,Mitali Sarkar,Pengfei Ding,Siarhei Kharytonchyk,Aaron Kidane,Christina Quasney,Heather M. Frank,Nansen Kuo,Canessa Swanson,Michael F. Summers,Ridhi Chaudhary

doi:10.1073/pnas.2008519117

Abstract

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5'-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5'-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles' heel to which HIV therapeutics may be targeted.

Highlights

A combination of mutagenesis, NMR, and RNA packaging experiments identified a region of the leader sufficient for packaging heterologous vectors into assembling virions [37], which has been shown by NMR to adopt a tandem three-way junction structure [30]
Studies suggested that the apical tetraloop of the Ψ-hairpin was the critical Gag/NC recognition element [48, 51], and its complex structure with NC revealed that two exposed guanosines from the “GGAG” loop are inserted into hydrophobic clefts on the surfaces of the two zinc finger domains of NC [39]
Subsequent studies showed that substitution of the GGAG loop by GCUA abolished NC binding but did not significantly affect RNA packaging [16, 52], and it soon became clear that regions other than the apical loop of the Ψ-hairpin, including sequences in the stem of that hairpin [52, 53], are important for competitive RNA packaging [6, 26, 37, 54]

Summary

Introduction

Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. A combination of mutagenesis, NMR, and RNA packaging experiments identified a region of the leader sufficient for packaging heterologous vectors into assembling virions (called the core encapsidation signal, ΨCES) [37], which has been shown by NMR to adopt a tandem three-way junction structure [30]. The full-length gRNA only accounts for 90% of all newly assembled progeny virions [12] This highly selective packaging process is mediated by specific interactions between a cis-acting element of the HIV-1 gRNA and the nucleocapsid (NC) domain of the major structural polyprotein Gag [13–15]

Methods

Results

Conclusion