Interactions between HIV-1 Gag Molecules in Solution: An Inositol Phosphate-mediated Switch

Abstract

Retrovirus particle assembly is mediated by the Gag protein. Gag is a multi-domain protein containing discrete domains connected by flexible linkers. When recombinant HIV-1 Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) is mixed with nucleic acid, it assembles into virus-like particles (VLPs) in a fully defined system in vitro. However, this assembly is defective in that the radius of curvature of the VLPs is far smaller than that of authentic immature virions. This defect can be corrected to varying degrees by addition of inositol phosphates to the assembly reaction. We have now explored the binding of inositol hexakisphosphate (IP6) to Gag and its effects upon the interactions between Gag protein molecules in solution. Our data indicate that basic regions at both ends of the protein contribute to IP6 binding. Gag is in monomer-dimer equilibrium in solution, and mutation of the previously described dimer interface within its capsid domain drastically reduces Gag dimerization. In contrast, when IP6 is added, Gag is in monomer-trimer rather than monomer-dimer equilibrium. The Gag protein with a mutation at the dimer interface also remains almost exclusively monomeric in IP6; thus the "dimer interface" is essential for the trimeric interaction in IP6. We discuss possible explanations for these results, including a change in conformation within the capsid domain induced by the binding of IP6 to other domains within the protein. The participation of both ends of Gag in IP6 interaction suggests that Gag is folded over in solution, with its ends near each other in three-dimensional space; direct support for this conclusion is provided in a companion manuscript. As Gag is an extended rod in immature virions, this apparent proximity of the ends in solution implies that it undergoes a major conformational change during particle assembly.

Domain-swapped dimerization of the HIV-1 capsid C-terminal domain

“…Intriguingly, the size distribution of the particles formed by the ⌬177 Gag in vitro is similar to the one observed in vivo for a Gag mutant with impaired CA-CTD dimerization (31). Moreover, the size of the virus-like particles formed by the p6-deleted Gag was shown to be influenced by inositol phosphates (32), and the C-terminal domain of the CA seems to be involved in the inositol phosphate binding (33). Therefore, it is possible that the CA-CTDmediated interactions may affect the curvature and size of the assembled particle.…”

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

“…Free HIV-1 Gag protein monomers have a relatively weak tendency to dimerize, with a K d (dissociation constant) of ϳ10 Ϫ5 M (22). In contrast, Gag-Z proteins form dimers with significantly higher affinity (19).…”

“…All in vitro studies were with derivatives of ⌬16-99 ⌬p6 Gag protein. Recombinant protein was purified for in vitro assembly as described previously (20,22). Assembly experiments were performed by adding various amounts of Saccharomyces cerevisiae tRNA (Ambion) to 1 mg/ml of purified protein in assembly buffer (0.1 M NaCl-0.02 M Tris [pH 8.0]-5 mM ␤-mercaptoethanol).…”

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

“…Interestingly, Gag lacking the p6 domain and the N-terminal myristoyl moiety (Gag⌬myr⌬p6) in the presence of nucleic acid assembles into 30-nm-diameter spherical par-ticles (9). And the presence of inositol hexakisphosphate (IP6) is required to form VLPs of the correct 100-to-150-nm-diameter size (10,13,14). The interaction of MA with IP6 presumably mimics the interaction of Gag with the phosphoinositides found in the plasma membrane (14)(15)(16)(17).…”