Abstract
In HIV, the polyprotein precursor Gag orchestrates the formation of the viral capsid. In the current view of this viral assembly, Gag forms low-order oligomers that bind to the viral genomic RNA triggering the formation of high-ordered ribonucleoprotein complexes. However, this assembly model was established using biochemical or imaging methods that do not describe the cellular location hosting Gag–gRNA complex nor distinguish gRNA packaging in single particles. Here, we studied the intracellular localization of these complexes by electron microscopy and monitored the distances between the two partners by morphometric analysis of gold beads specifically labeling Gag and gRNA. We found that formation of these viral clusters occurred shortly after the nuclear export of the gRNA. During their transport to the plasma membrane, the distance between Gag and gRNA decreases together with an increase of gRNA packaging. Point mutations in the zinc finger patterns of the nucleocapsid domain of Gag caused an increase in the distance between Gag and gRNA as well as a sharp decrease of gRNA packaged into virions. Finally, we show that removal of stem loop 1 of the 5′-untranslated region does not interfere with gRNA packaging, whereas combined with the removal of stem loop 3 is sufficient to decrease but not abolish Gag-gRNA cluster formation and gRNA packaging. In conclusion, this morphometric analysis of Gag-gRNA cluster formation sheds new light on HIV-1 assembly that can be used to describe at nanoscale resolution other viral assembly steps involving RNA or protein–protein interactions.
Highlights
Production of HIV-1 particles by infected cells is a complex process coordinated by Gag and GagPol polyprotein precursors that promote the specific encapsidation of the psi containing genomic RNA
These results show that cytoplasmic Gag–genomic RNA (gRNA) complexes traffic toward the plasma membrane (PM) as a function of time and the quantitative analysis of our transmission electronic microscopy (TEM) images reveal that this spatio-temporal distribution is concomitant with a condensation of the nucleoprotein complex that in turn is essential for a specific gRNA packaging
To monitor the colocalization of HIV-1 Gag and gRNA at high resolution, we used TEM on HeLa cells transfected by pNL4.3-MS2-Δenv and pMCP-enhanced GFP (eGFP)-NLS (Fig. 1A)
Summary
Received for publication, December 3, 2021 Published, Papers in Press, December 17, 2021, https://doi.org/10.1016/j.jbc.2021.101500 Stéphanie Durand1, Florian Seigneuret1, Julien Burlaud-Gaillard2, Roxane Lemoine3, Marc-Florent Tassi1 , Alain Moreau1 , Marylène Mougel4 , Philippe Roingeard1,2 , Clovis Tauber5, and Hugues de Rocquigny1,* From the 1Morphogenesis and Antigenicity of HIV and Hepatitis Viruses, Inserm – U1259 MAVIVH, Bretonneau Hospital, Tours Cedex 1, France; 2Microscopy IBiSA Platform, PPF ASB, University of Tours and CHRU of Tours, Tours Cedex 1, France; 3B Cell Ressources Platform, EA4245 “Transplantation, Immunology and Inflammation”, University of Tours, Tours Cedex 1, France; 4Équipe R2D2 Retroviral RNA Dynamics and Delivery, IRIM, CNRS UMR9004, University of Montpellier, Montpellier, France; 5UMR U1253 iBrain, Inserm, University of Tours, Tours Cedex 1, France
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