Conformation of the HIV-1 Gag Protein in Solution

Abstract

A single multi-domain viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. We have purified the human immunodeficiency virus type 1 (HIV-1) Gag protein (lacking myristate at its N terminus and the p6 domain at its C terminus) from bacteria. This protein is capable of assembly into virus-like particles in a defined in vitro system. We have reported that it is in monomer-dimer equilibrium in solution, and have described a mutant Gag protein that remains monomeric at high concentrations in solution. We report that the mutant protein retains several properties of wild-type Gag. This mutant enabled us to analyze solutions of monomeric protein. Hydrodynamic studies on the mutant protein showed that it is highly asymmetric, with a frictional ratio of 1.66. Small-angle neutron scattering (SANS) experiments confirmed its asymmetry and yielded an R(g) value of 34 A. Atomic-level structures of individual domains within Gag have previously been determined, but these domains are connected in Gag by flexible linkers. We constructed a series of models of the mutant Gag protein based on these domain structures, and tested each model computationally for its agreement with the experimental hydrodynamic and SANS data. The only models consistent with the data were those in which Gag was folded over, with its N-terminal matrix domain near its C-terminal nucleocapsid domain in three-dimensional space. Since Gag is a rod-shaped molecule in the assembled immature virion, these findings imply that Gag undergoes a major conformational change upon virus assembly.

Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

“…These results are in agreement with chemical cross-linking studies that showed that Gag is predominantly dimeric in the cytoplasm and does not form higher order oligomers until it reaches the plasma membrane (7). Gag trimerization in the presence of inositol phosphate derivatives has been reported previously (8). However, no evidence for the existence of a ΔGag trimer or higher order structures (e.g., hexamer comprising a trimer of dimers) was found by analytical ultracentrifugation either in the presence of inositol hexakisphosphate (K dimer = 35 ± 5 μM) or nucleic acids (K dimer = 14-18 μM) (Table S1 and Fig.…”

“…A previous hydrodynamic and neutron scattering study of a monomeric variant of Gag, obtained by a double mutation (W316A and M317A) at the dimerization interface, claimed that MA and NC are close to one another in three-dimensional space (8). The current RDC data, however, are inconsistent with this proposal because the values of D NH a and η for MA and the two zinc knuckles of NC are very different from one another (Table S2).…”

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

“…These spheres are not as uniform and are significantly larger than the particles reported for the wild-type p6-deleted Gag constructs (5,27,29). 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).…”

In vitro expression of the HIV-2 genomic RNA is controlled by three distinct internal ribosome entry segments that are regulated by the HIV protease and the Gag polyprotein

“…The recombinant HIV-1 Gag protein was prepared as described (Datta et al 2007) and solubilized in a buffer containing 20 mM Tris HCl pH 7.4, 0.5 M NaCl, 5 mM DTT, 1 mM PMSF, and a protease inhibitors cocktail (Roche Inc.). These conditions are taken in consideration for translation experiments performed in the presence of this protein to maintain equivalent levels of salt, pH, DTT, and antiprotease in each reaction tube.…”