HIV-1 Gag Extension: Conformational Changes Require Simultaneous Interaction with Membrane and Nucleic Acid

Siddhartha A.K Datta,Frank Heinrich,Sindhu Raghunandan,Susan Krueger,Joseph E Curtis,Alan Rein,Hirsh Nanda

doi:10.1016/j.jmb.2010.11.051

Abstract

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.

Highlights

Expression of a single retroviral protein, termed Gag, is sufficient for virus particle formation in mammalian cells.1; 2 Gag is a multi-domain protein always containing, from Nto C-terminus, a matrix (MA) domain, a capsid (CA) domain, and a nucleocapsid (NC) domain
The particles assembled from Gag are immature virions; in most retroviruses, including lentiviruses such as HIV-1, these particles form as Gag accumulates at the cytoplasmic face of the plasma membrane
The Gag proteins in these particles are extended rods, approximately 200 Å long.[4]. They are arranged as radii of the particles, with their N-termini in contact with the lipid bilayer that surrounds the particle and their C-termini projecting into the interior of the particle, presumably in contact with RNA

Summary

Introduction

Expression of a single retroviral protein, termed Gag, is sufficient for virus particle formation in mammalian cells.1; 2 Gag is a multi-domain protein always containing, from Nto C-terminus, a matrix (MA) domain, a capsid (CA) domain, and a nucleocapsid (NC) domain. Structural properties of recombinant Gag protein in solution were probed using hydrodynamic data and small-angle neutron scattering (SANS) together with molecular modeling.[5] This study used a Gag mutant, designated WM Gag, that was inhibited from dimerizing.

Results

Conclusion