Our Science – Datta Website
Siddhartha A.K. Datta, Ph.D.
The main determinant in retroviral assembly is the Gag polyprotein. When expressed in cells, it is sufficient for the formation of virus-like particles (VLPs). In VLPs, Gag is an elongated molecule, arranged radially. We have analyzed the properties of recombinant HIV-1 Gag protein in some detail. The protein was found to exist in monomer-dimer equilibrium, using a previously described interface in the C-terminal domain of the capsid protein. A monomeric mutant of Gag was made, and characterized by a number of hydrodynamic and biophysical methods. The results, along with modelling studies, suggest that the molecule, in contrast to its structure in VLPs, adopts compact conformation(s) with the N- and C- termini in close proximity. In the presence of nucleic acid, the recombinant protein spontaneously assembles into VLPs. These VLPs are smaller than authentic VLPs produced in cells. However addition of inositol phosphates, co-factors which resemble lipid head groups, can correct for this defect. Interestingly, tight binding to hexakis inositol phosphate was found to depend on, and mapped to, both extremities of the protein. Since Gag molecules are extended rods in immature virus particles, they must undergo a drastic conformational change during particle assembly. Collaborative studies using neutron reflectivity have now identified conditions under which Gag can extend. This occurs when the matrix domain binds lipids and the nucleocapsid domain binds nucleic acid simultaneously.
The mechanism by which nucleic acid triggers particle assembly by the Gag protein is not known. We recently found that a very short region of Gag, termed 'SP1', undergoes a conformational change when it is at high concentration. We proposed that this region 'senses' the local Gag concentration, and that the increased Gag concentration resulting from cooperative binding to nucleic acid would thus lead to a change in Gag conformation, exposing new interfaces required for particle assembly. This hypothesis would explain the contribution of nucleic acid to particle assembly. Current studies are devoted to testing this hypothesis and exploring its implications.
This page was last updated on 3/31/2014.