Roles of Gag-RNA interactions in HIV-1 virus

Image

Single-molecule localization microscopy (SMLM) is useful for deciphering dynamic organizations of structures densely labeled by specific proteins in the cellular context with nanoscopic resolution not attainable by conventional imaging tools. Here we employed SMLM to investigate the mechanism by which the HIV-1 viral RNA (vRNA) mediates the assembly of thousands of Gag proteins into a virus particle at the plasma membrane. In contrast to the general notion that vRNA only triggers Gag assembly and is dispensable for subsequent assembly, we found that vRNA is indispensable throughout assembly, scaffolding the formation of assembly intermediates and maintaining their architectures via balancing of external forces acting on the assembly environment. These previously unidentified features may facilitate understanding of HIV-1 and, potentially, other retroviruses.

During HIV-1 assembly, the retroviral structural protein Gag forms an immature capsid, containing thousands of Gag molecules, at the plasma membrane (PM). Interactions between Gag nucleocapsid (NC) and viral RNA (vRNA) are thought to drive assembly, but the exact roles of these interactions have remained poorly understood. Since previous studies have shown that Gag dimer- or trimer-forming mutants (GagZiL) lacking an NC domain can form immature capsids independent of RNA binding, it is often hypothesized that vRNA drives Gag assembly by inducing Gag to form low-ordered multimers, but is dispensable for subsequent assembly. In this study, we examined the role of vRNA in HIV-1 assembly by characterizing the distribution and mobility of Gag and Gag NC mutants at the PM using photoactivated localization microscopy (PALM) and single-particle tracking PALM (spt-PALM). We showed that both Gag and GagZiL assembly involve a similar basic assembly unit, as expected. Unexpectedly, the two proteins underwent different subsequent assembly pathways, with Gag cluster density increasing asymptotically, while GagZiL cluster density increased linearly. Additionally, the directed movement of Gag, but not GagZiL, was maintained at a constant speed, suggesting that the two proteins experience different external driving forces. Assembly was abolished when Gag was rendered monomeric by NC deletion. Collectively, these results suggest that, beyond inducing Gag to form low-ordered multimer basic assembly units, vRNA is essential in scaffolding and maintaining the stability of the subsequent assembly process. This finding should advance the current understanding of HIV-1 and, potentially, other retroviruses.

Without NC (ΔNC-Gag), the Gag protein freely migrates at the PM without clustering. When Gag molecules are able to form basic assembly units (trimers) independent of interaction with vRNA through replacement of NC with an isoleucine zipper (GagZiL), Gag undergoes self-oligomerization to form high-density clusters that are highly immobile. Wild-type Gag interacts with vRNA to form clusters through a two-phase process, in which vRNA maintains the assembly environment by mediating a balance between forces that drive dispersion (as seen with ΔNC-Gag) and forces that drive high-density packing (as seen with GagZiL). Thus, contrary to the hypothesis that vRNA only functions to drive formation of low-order Gag multimers, our results reveal crucial organizational and dynamic dependencies of the subsequent assembly process on NC-vRNA interactions. We propose that these dependencies could serve as a quality-control system to allow the virus to ensure that only properly assembled particles are produced and disseminated into the extracellular milieu. Moreover, given that cellular RNAs can also mediate Gag assembly, we envision the methodologies and findings described in the study could lay foundations for future studies, deepening our understanding of retroviral assembly and production.

You can submit your research work through online at https://bit.ly/3k7QUiv  

Media contact:

Christina

Editorial Assistant

Mail Id: [email protected]

Journal of Cell Signaling.

Whatsapp number: + 1-504-608-2390