Elements in HIV-1 Gag contributing to virus particle assembly.

The Gag polyprotein is the building block of retroviral particles and its expression is sufficient for assembly in cells. In HIV-1, nucleic acid (NA) is required for recombinant Gag molecules to assemble in a defined system in vitro. Experiments performed by Barklis and co-workers suggested that NA contributes to assembly by promoting Gag oligomerization. Gag is composed of four main domains: the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains. We have recently shown that the SP1 linker, which lies between the CA and NC domains, assumes a helical structure at high, but not low, concentrations. We suggested that Gag oligomerization mediates assembly via an SP1-dependent conformational switch that exposes new interfaces for assembly. Although NA is required for assembly in vitro, deletion of NC, the main RNA-binding domain, does not eliminate particle formation in vivo; these particles lack NA. We hypothesized that alternative pathways that lead to Gag oligomerization or an increase in local Gag concentration, namely Gag-membrane or inter-protein interactions, rescue assembly in the absence of NC-RNA binding. We constructed mutants in which either Gag-membrane binding, the Gag dimer interface, or NC-RNA binding are disrupted. None of these mutants disables assembly. However, combined mutations in any two of these three classes render Gag completely unable to form virus-like particles. Thus, it seems, Gag utilizes at least three types of interactions to form oligomers and any two out of the three are sufficient for assembly.

Functional redundancy in HIV-1 viral particle assembly.

Expression of a retroviral Gag protein in mammalian cells leads to the assembly of virus particles. In vitro, recombinant Gag proteins are soluble but assemble into virus-like particles (VLPs) upon addition of nucleic acid. We have proposed that Gag undergoes a conformational change when it is at a high local concentration and that this change is an essential prerequisite for particle assembly; perhaps one way that this condition can be fulfilled is by the cooperative binding of Gag molecules to nucleic acid. We have now characterized the assembly in human cells of HIV-1 Gag molecules with a variety of defects, including (i) inability to bind to the plasma membrane, (ii) near-total inability of their capsid domains to engage in dimeric interaction, and (iii) drastically compromised ability to bind RNA. We find that Gag molecules with any one of these defects still retain some ability to assemble into roughly spherical objects with roughly correct radius of curvature. However, combination of any two of the defects completely destroys this capability. The results suggest that these three functions are somewhat redundant with respect to their contribution to particle assembly. We suggest that they are alternative mechanisms for the initial concentration of Gag molecules; under our experimental conditions, any two of the three is sufficient to lead to some semblance of correct assembly.

On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch?

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.