In vitro assembly of the Rous Sarcoma Virus capsid protein into hexamer tubes at physiological temperature.

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.

A structural model for the generation of continuous curvature on the surface of a retroviral capsid.

The genome of a retrovirus is surrounded by a convex protein shell, or capsid, that helps facilitate infection. The major part of the capsid surface is formed by interlocking capsid protein (CA) hexamers. We report electron and X-ray crystallographic analysis of a variety of specimens assembled in vitro from Rous sarcoma virus (RSV) CA. These specimens all contain CA hexamers arranged in planar layers, modeling the authentic capsid surface. The specimens differ only in the number of layers incorporated and in the disposition of each layer with respect to its neighbor. The body of each hexamer, formed by the N-terminal domain of CA, is connected to neighboring hexamers through C-terminal domain dimerization. The resulting layer structure is very malleable due to inter-domain flexibility. A helix-capping hydrogen bond between the two domains of RSV CA creates a pivot point, which is central to controlling their relative movement. A similar mechanism for the governance of inter-domain motion was recently described for the human immunodeficiency virus type 1 (HIV-1) capsid, although there is negligible sequence identity between RSV and HIV-1 CA in the region of contact, and the amino acids involved in creating the pivot are not conserved. Our observations allow development of a physically realistic model for the way neighboring hexamers can tilt out of plane, deforming the hexamer layer and generating the continuously curved surfaces that are a feature of all retroviral capsids.

Proton-linked dimerization of a retroviral capsid protein initiates capsid assembly.

In mature retroviral particles, the capsid protein (CA) forms a shell encasing the viral replication complex. Human immunodeficiency virus (HIV) CA dimerizes in solution, through its C-terminal domain (CTD), and this interaction is important for capsid assembly. In contrast, other retroviral capsid proteins, including that of Rous sarcoma virus (RSV), do not dimerize with measurable affinity. Here we show, using X-ray crystallography and other biophysical methods, that acidification causes RSV CA to dimerize in a fashion analogous to HIV CA, and that this drives capsid assembly in vitro. A pair of aspartic acid residues, located within the CTD dimer interface, explains why dimerization is linked to proton binding. Our results show that despite overarching structural similarities, the intermolecular forces responsible for forming and stabilizing the retroviral capsid differ markedly across retroviral genera. Our data further suggest that proton binding may regulate RSV capsid assembly, or modulate stability of the assembled capsid.

A study of the foodborne pathogens: Campylobacter, Listeria and Yersinia, in faeces from slaughter-age cattle and sheep in Australia.

In a study of faeces from 475 slaughter-age cattle and sheep from 19 herds or flocks, Campylobacter species (C. jejuni and C. coli) were cultured from all production systems studied and from 73.7 per cent (14/19) of herds or flocks. Within individual properties there was a higher prevalence in cattle than in sheep, with Campylobacter being most commonly isolated from feedlot cattle. The median prevalences and ranges were: for dairy cattle, six per cent (0-24%), feedlot beef cattle, 58 per cent (12-92%) pasture beef cattle, two per cent (0-52%), mutton sheep, 0 per cent (0-4%) and prime lambs eight per cent. Listeria ivanovii was cultured from one dairy cow but Yersinia enterocolitica was not cultured from any animal. Campylobacter is the leading bacterial causative agent of acute diarrhoea in humans in many industrialised countries. While the role of cattle and sheep in producing human campylobacteriosis either directly or via contaminated food, remains to be epidemiologically clarified, this study suggests that the production system, particularly for cattle, may be an important consideration.