Abstract
To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 A is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers.
Highlights
During human immunodeficiency virus 1 (HIV-1)1 assembly, the viral precursor Gag (PrGag) protein oligomerizes on cellular membranes and directs the budding of immature virus particles
In Vitro Assembly of MACANC Proteins—To investigate the influences that the HIV-1 Gag MA and NC domains might have on the assembly of capsid proteins, His-tagged MACANC proteins were expressed in bacteria
Much is known concerning the three-dimensional structures of the individual HIV-1 Gag protein structures (29 –37), considerably less is understood about how the Gag proteins or PrGag protein domains associate in immature and mature HIV virions
Summary
The similarity between our results and those observed in vivo prompted us to propose a model in which viral morphogenesis was accompanied by a shift from tightly packed hexamer rings to loosely packed rings, and we predicted that virions would have more capsid protein than was needed for mature core formation [2]. This prediction was borne out by the observation of frequent virions with multiple cores and the determination that HIV-1 appears to contain on the order of 5000 Gag proteins/particle, higher than previously expected [5]. Our results indicate that the presence of MA and NC domains is compatible with a tightly packed arrangement of PrGag proteins and support a model where PrGag processing yields a loose capsid core from a more crowded precursor arrangement
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