A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

Abstract

The conformational changes within single HIV-1 Gag molecules that occur during assembly into immature viruses are poorly understood. Using an in vitro assembly assay, it has been proposed that HIV-1 Gag undergoes a conformational transition from a compact conformation in solution to an extended rod-like conformation in virus-like particles (VLPs). Here we used singlemolecule Förster resonance energy transfer (smFRET) to test this model by directly probing the conformation of single HIV-1 Gag molecules. We demonstrate that monomeric HIV-1 Gag lacking the p6 domain and the N-terminal myristoyl moiety is found in solution predominantly in a compact conformation. Gag in this conformation, and in the presence of nucleic acid, assembles into 30-nm-diameter particles. However, with the addition of inositol hexakisphosphate, Gag adopts a linear conformation and assembles into full-sized ϳ100-to-150-nm-diameter VLPs. Parallel fluorescence correlation spectroscopy measurements show that this conformational transition occurs early in the assembly process when Gag oligomers are small, perhaps as early as upon dimerization. Thus, smFRET measurements confirm that HIV-1 Gag transitions from a compact to a linear conformation during the formation of VLPs. Our results are consistent with a model whereby binding of HIV-1 Gag to phosphoinositides at the plasma membrane stabilizes an extended conformation and promotes oligomerization into the radially aligned immature capsid.

Role of the Nucleocapsid Domain in HIV-1 Gag Oligomerization and Trafficking to the Plasma Membrane: A Fluorescence Lifetime Imaging Microscopy Investigation

“…2c and d), compact Gag oligomers can already form in the cytoplasm and do not undergo important structural changes at the PM. However, our data do not allow discriminating between the bent and extended conformations of Gag proteins [3,53], since closely packed oligomers may likely form with both conformations.…”

The HIV-1 Nucleocapsid Protein Recruits Negatively Charged Lipids To Ensure Its Optimal Binding to Lipid Membranes

“…This hypothesis is supported by the multivalent nature of NC, which allows it to bind at the same time two nucleic acid molecules (110). These differences between the relative stabilities of the different complexes, together with the ability of NC and NC/nucleic acid complexes to interact with negatively charged lipid membranes, lead us to revisit the HIV-1 assembly model, previously proposed by several groups (55,61,70,111) (for reviews, see references 59, 60, and 63). In this revised model, we propose that the NC domain of Gag participates in a transient manner in the binding of Gag to the PM, allowing the recruitment of negatively charged lipids (Fig.…”

“…5, step 1), which is facilitated by the globular or bent conformation of Gag (36,55,59,61,62,69,70,112). At this stage, the binding of the gRNA to GagMA is thought to be mediated by the HBR sequence of Gag (61,64), while the myristyl group is still buried in the GagMA core.…”

“…Phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ] and probably other phosphoinositides (64,65), which are essentially found on the cytoplasmic leaflet of the PM, can outcompete gRNA (61,66) and promote the anchoring of the myristoylated MA domain to the PM (62,67). The PI(4,5)P 2 -induced release of GagMA from the gRNA is also thought to promote Gag trimerization and a structural switch of Gag to its rodshaped conformation that is observed in virus particles (68)(69)(70).…”

HIV-1 Gag as an Antiviral Target: Development of Assembly and Maturation Inhibitors

“…This observation suggested that Gag is initially folded and more compact, and upon membrane interactions forms an extended conformation necessary for oligomerization. Single-molecule FRET experiments supported this model, again suggesting that there is a conformational switch to an extended conformation that may be triggered by MA interactions with phosphoinositides at the plasma membrane, promoting subsequent oligomerization [58]. These in vitro and single molecule experiments are consistent with cell-based experiments in which three components appear essential to successful particle assembly: membrane interactions (including a role for myristoylation), NC-RNA interactions, and direct Gag-Gag interactions [59–60].…”