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The HIV-1 Gag precursor protein, Pr55(Gag), is a multi-domain polyprotein that drives HIV-1 assembly. The morphological features of HIV-1 suggested Pr55(Gag) assumes a variety of different conformations during virion assembly and maturation, yet structural determination of HIV-1 Pr55(Gag) has not been possible due to an inability to express and to isolate large amounts of full-length recombinant Pr55(Gag) for biophysical and biochemical analyses. This challenge is further complicated by HIV-1 Gag's natural propensity to multimerize for the formation of viral particle (with ∼2500 Gag molecules per virion), and this has led Pr55(Gag) to aggregate and be expressed as inclusion bodies in a number of in vitro protein expression systems. This study reported the production of a recombinant form of HIV-1 Pr55(Gag) using a bacterial heterologous expression system. Recombinant HIV-1 Pr55(Gag) was expressed with a C-terminal His×6 tag, and purified using a combination of immobilized metal affinity chromatography and size exclusion chromatography. This procedure resulted in the production of milligram quantities of high purity HIV-1 Pr55(Gag) that has a mobility that resembles a trimer in solution using size exclusion chromatography analysis. The high quantity and purity of the full length HIV Gag will be suitable for structural and functional studies to further understand the process of viral assembly, maturation and the development of inhibitors to interfere with the process.

The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.

The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.