Abstract

A growing number of studies indicate that mRNAs and long ncRNAs can affect protein populations by assembling dynamic ribonucleoprotein (RNP) granules. These phase-separated molecular ‘sponges’, stabilized by quinary (transient and weak) interactions, control proteins involved in numerous biological functions. Retroviruses such as HIV-1 form by self-assembly when their genomic RNA (gRNA) traps Gag and GagPol polyprotein precursors. Infectivity requires extracellular budding of the particle followed by maturation, an ordered processing of ∼2400 Gag and ∼120 GagPol by the viral protease (PR). This leads to a condensed gRNA-NCp7 nucleocapsid and a CAp24-self-assembled capsid surrounding the RNP. The choreography by which all of these components dynamically interact during virus maturation is one of the missing milestones to fully depict the HIV life cycle. Here, we describe how HIV-1 has evolved a dynamic RNP granule with successive weak–strong–moderate quinary NC-gRNA networks during the sequential processing of the GagNC domain. We also reveal two palindromic RNA-binding triads on NC, KxxFxxQ and QxxFxxK, that provide quinary NC-gRNA interactions. Consequently, the nucleocapsid complex appears properly aggregated for capsid reassembly and reverse transcription, mandatory processes for viral infectivity. We show that PR is sequestered within this RNP and drives its maturation/condensation within minutes, this process being most effective at the end of budding. We anticipate such findings will stimulate further investigations of quinary interactions and emergent mechanisms in crowded environments throughout the wide and growing array of RNP granules.

Highlights

  • Biomolecular condensates (BCs) are membraneless, intracellular assemblies formed by the phenomenon of liquid–liquid phase separation (LLPS) [1,2,3,4,5]

  • We describe in this study HIV-1 nucleocapsid maturation as a dynamic RNA granule processing phenomenon, involving differential RNA binding activities of the NC domain that are dependent on processing state

  • Weak NC-RNA contacts fit with the concept of quinary interactions [28] that lead to genomic RNA (gRNA) condensation in the context of RNA-directed phase separation [25]

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Summary

Introduction

Biomolecular condensates (BCs) are membraneless, intracellular assemblies formed by the phenomenon of liquid–liquid phase separation (LLPS) [1,2,3,4,5]. The slow in vitro kinetics of Gag proteolysis supports a general scheme for PR to be auto-processed during the completion of budding, driving viral maturation within free, released particles in a computed time-scale close to 30 min [81] This model is, inconsistent with many observations from electron microscopy that show (i) a huge majority of free but freshly released particles in a mature form containing condensed RNP [82], (ii) both capsid and budding defects in the presence of PR inhibitors [83], and (iii) budding and maturation defects for critical NC mutants, whereas Western blots from cell extracts detect PR-processed Gag products [82]. Reconstituted systems that model non-sequence-specific binding on a large scale, together with molecular dynamics simulations and RNP-modulated enzyme-substrate reaction kinetics theory, allow us (i) to detail the quinary effects and their variations engaged in this dynamic process and (ii) to focus on PR action in such a quinary interaction context

Materials and Methods
NP Complex Assembly and Electrophoretic Mobility Shift Assay
Dynamic Light Scattering
AFM Imaging
Proteolysis Assays
Electron Microscopy of HIV-1 Particles
Molecular Dynamics Simulations and Analysis
Cleavage of NCp15 to NCp9 and NCp7 Underpins Weak-Strong-Moderate Quinary
Discussion and Conclusions
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