In vitroassembly of the Rous Sarcoma Virus capsid protein into hexamer tubes under physiological conditions

Abstract

During a proteolytically-driven maturation process, the ortho-retroviral capsid protein (CA) assembles to form the convex shell that surrounds the viral genome. In some orthoretroviruses, including Rous Sarcoma Virus (RSV), CA carries a short and hydrophobic spacer peptide (SP) at its C-terminus early in the maturation process, which is progressively removed as maturation proceeds. In this work, we show that RSV CA assembles in vitro at physiological temperatures, forming hexamer tubes that effectively model the mature capsid surface. Tube assembly is strongly influenced by electrostatic effects, and is a nucleated process that remains thermodynamically favored at lower temperatures, but is effectively arrested by the large Gibbs energy barrier associated with nucleation.

Structural Model of the Tubular Assembly of the Rous Sarcoma Virus Capsid Protein

“…To better understand the implications of these changes, an assembly model comprising seven hexamers on the tube surface was constructed by MDFF simulations that combine ssNMR constraints with cryo-EM data, as shown in Figures 5 and 6. Complementary to the torsion angles derived from ssNMR CS, the cryo-EM image reconstruction of an RSV CA tube at 24 Å resolution 60 determines the approximate locations of individual protein domains in the assembly, as shown in Figure 5. The pseudoatomic model of the RSV CA hexamer derived from crystallographic analysis (PDB entry 3TIR) was used as the initial structure in our MDFF simulations, and domain restraints on each NTD hexamer and CTD dimer were applied in the early stages of the simulations (see Supporting Information) to preserve the internal conformations of each domain as well as the NTD−NTD and CTD−CTD dimer interfaces.…”