Plate tectonics of virus shell assembly and reorganization in phage φ8, a distant relative of mammalian reoviruses

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity

The structure and function of normal and mutated collagen IX

Abstract Collagen IX belongs to the superfamily of collagenous proteins and is present on the surface of the heterotypic collagen fibrils that are predominantly composed of collagen II, and also collagen XI. The major sites of expression of collagen IX include the articular cartilage, intervertebral disc, inner ear and the vitreous body of the eye. Previous reports have indicated that mutations in the genes encoding the three polypeptide chains of collagen IX may lead to intervertebral disc disease and multiple epiphyseal dysplasia, a chondrodysplasia characterized by early onset osteoarthritis. These observations and results from genetically modified mouse lines suggest that collagen IX is crucial in the maintenance of the long-term integrity of tissues. However, the structure-function relationship as well as detailed information concerning the functional roles of this protein has remained unclear. Recombinant human collagen IX was obtained using an insect cell expression system. Besides full-length molecules, five truncated variants of collagen IX were produced to examine chain association and trimerization. Contrary to previous observations, it was shown that the COL1 and NC1 domains are not essential for trimerization. Instead, they seem to play an important role in the specificity of chain selection. The results also suggest that the N-terminal domains, NC3 or COL3, are required for complete folding and stabilization of collagen IX molecules, implicating cooperativity between different domains in the folding process. Collagen IX was found to mediate cell adhesion and bind efficiently to collagen receptor integrins α1β1, α2β1, α10β1 and α11β1. The binding was found to represent a novel type of mechanism, and the binding site of the integrin I domain was located at the N-terminal end of the COL3 domain in collagen IX. The obtained results suggest that the FACITs may play an important role as mediators of cell adhesion to collagen fibrils. Antibodies binding to human recombinant collagen IX were measured among 53 patients with seropositive rheumatoid arthritis (RA). These autoantibodies were significantly elevated among the RA patients when compared to the controls, suggesting that autoantibodies to collagen IX show diagnostic potential in early RA. However, no association was found between the antibody levels and outcome

Electron cryomicroscopy comparison of the architectures of the enveloped bacteriophages phi6 and phi8.

The enveloped dsRNA bacteriophages phi6 and phi8 are the two most distantly related members of the Cystoviridae family. Their structure and function are similar to that of the Reoviridae but their assembly can be conveniently studied in vitro. Electron cryomicroscopy and three-dimensional icosahedral reconstruction were used to determine the structures of the phi6 virion (14 A resolution), phi8 virion (18 A resolution), and phi8 core (8.5 A resolution). Spikes protrude 2 nm from the membrane bilayer in phi6 and 7 nm in phi8. In the phi6 nucleocapsid, 600 copies of P8 and 72 copies of P4 interact with the membrane, whereas in phi8 it is only P4 and 60 copies of a minor protein. The major polymerase complex protein P1 forms a dodecahedral shell from 60 asymmetric dimers in both viruses, but the alpha-helical fold has apparently diverged. These structural differences reflect the different host ranges and entry and assembly mechanisms of the two viruses

Trimerization of collagen IX alpha-chains does not require the presence of the COL1 and NC1 domains.

Collagen IX is a heterotrimer of three alpha-chains, which consists of three COL domains (collagenous domains) (COL1-COL3) and four NC domains (non-collagenous domains) (NC1-NC4), numbered from the C-terminus. Although collagen IX chains have been shown to associate via their C-terminal NC1 domains and form a triple helix starting from the COL1 domain, it is not known whether chain association can occur at other sites and whether other collagenous and non-collagenous regions are involved. To address this question, we prepared five constructs, two long variants (beginning at the NC4 domain) and three short variants (beginning at the COL2 domain), all ending at the NC2 domain (or NC2 replaced by NC1), to study association and selection of collagen IX alpha-chains. Both long variants were able to associate with NC1 or NC2 at the C-terminus and form various disulfide-bonded trimers, but the specificity of chain selection was diminished compared with full-length chains. Trimers of the long variant ending at NC2 were shown to be triple helical by CD. Short variants were not able to assemble into disulfide-bonded trimers even in the presence of both conserved cysteine residues from the COL1-NC1 junction. Our results demonstrate that collagen IX alpha-chains can associate in the absence of COL1 and NC1 domains to form a triple helix, but the COL2-NC2 region alone is not sufficient for trimerization. The results suggest that folding of collagen IX is a co-operative process involving multiple COL and NC domains and that the COL1-NC1 region is important for chain specificity

Serum antibodies against intact human collagen IX are elevated at onset of rheumatoid arthritis but are not related to development of erosions.

OBJECTIVE: To measure the presence of autoantibodies binding to intact human recombinant collagen IX and assess their usefulness as a diagnostic marker and an indicator of disease activity in rheumatoid arthritis (RA). METHODS: Recombinant human full-length collagen IX (rCIX) was produced in a baculovirus expression system and purified for use in ELISA developed to detect antibodies to native and denatured collagen IX. Fifty-three patients with recent-onset rheumatoid factor-seropositive RA were analyzed for the presence of rCIX antibodies of the IgG type at the time of initial diagnosis and after 3, 6, 12, and 24 months of followup. The RA sera were accompanied by 30 controls. Associations were determined between patients\u27 antibody titers, development of erosions in the hands and feet, and various clinical and laboratory markers. RESULTS: Serum antibody levels among patients with RA at time of diagnosis were 1.78 times higher against native rCIX (p \u3c 0.001) and 1.71 times higher against denatured rCIX (p \u3c 0.001) than in the controls, and they remained high during the followup. No correlation was seen between antibody levels and clinical and laboratory findings. CONCLUSION: Our data show that patients with recent-onset RA have significantly elevated levels of autoantibodies to human rCIX. These autoantibodies were observed already at the early stages of the disease, which may reflect their diagnostic potential in RA