Abstract
During a proteolytically-driven maturation process, the orthoretroviral 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 near-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. RSV CA tubes are multi-layered, being formed by nested and concentric tubes of capsid hexamers. However the spacer peptide acts as a layering determinant during tube assembly. If only a minor fraction of CA-SP is present, multi-layered tube formation is blocked, and single-layered tubes predominate. This likely prevents formation of biologically aberrant multi-layered capsids in the virion. The generation of single-layered hexamer tubes facilitated 3D helical image reconstruction from cryo-electron microscopy data, revealing the basic tube architecture.
Highlights
To locally average the final reconstruction about the turrets on the tube surface, the optimal location of the 6-fold rotational symmetry axis was determined by a real space procedure
We show that incubation of RSV CA at physiological temperature, pH and neutral salt concentration results in the formation of tubes built from CA hexamers, the major component of the authentic capsid surface
Using this biologically relevant assembly protocol, we show that the spacer peptide that is transiently appended to the C-terminus of CA functions as a layering determinant during formation of the capsid hexamer array
Summary
To locally average the final reconstruction about the turrets on the tube surface, the optimal location of the 6-fold rotational symmetry axis was determined by a real space procedure. Sub-volumes were extracted, centered at each possible position within the angular and translational space defined by the helical repeat. For each sub-volume, a C6 symmetrized copy was created and correlated with the original sub-volume. From the resulting plot of cross-correlation versus centroid position, the location of symmetry axis was determined (Fig. S3), and the local C6 symmetry imposed on the reconstructi
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