Biochemical Evidence for the Oligomeric Arrangement of Bovine Rotavirus Nucleocapsid Protein and Its Possible Significance in the Immunogenicity of This Protein

Abstract

SUMMARYThe nucleocapsid protein of bovine rotavirus was shown to exist in trimeric units in both the virus particle and in infected cells, with the subunits linked by non-covalent interactions. These trimeric units complex further by disulphide bridges into larger units which may represent the hexameric structures observed by electron microscopy. Visualization of various nucleocapsid protein complexes was also achieved on polyacrylamide gels by treating virus preparations with urea at 37 °C or boiling in the presence and absence of 2-mercaptoethanol. Since virus particles devoid of nucleic acid were also broken down into trimeric subunits by such treatments, assembly of virus particles appears not to require an RNA-protein interaction. Four nucleocapsidspecific monoclonal antibodies with low neutralizing ability reacted with the monomeric (45 000 mol. wt., 45K), dimeric (90K), trimeric (135K) and trimeric pair (270K) subunits, indicating that a site responsible for neutralization is probably exposed after assembly of these subunits. Analysis of radiolabelled virus revealed that a high proportion (80~) of infectious particles could be immunoprecipitated by these monoclonal antibodies, suggesting that the virus particles are either partially doubleshelled or have the nucleocapsid exposed on the surface. The monoclonal antibodies also cross-reacted with the nucleocapsid proteins of simian (SA11), pig (OSU), bovine (NCDV and UK) and human (Wa and ST4) rotaviruses in an immunoblot ELISA

Assembly of double-layered virus-like particles in mammalian cells by coexpression of human rotavirus VP2 and VP6

“…When the immunoprecipitates of vv-Wa-6-infected cells were incubated at 37 °C, the monomeric form of VP6 observed in boiled samples (lane 1) disappeared and a product of high molecular mass was detected (lane 2). This band of about 140 kDa corresponds to the trimeric form of VP6 (Sabara et al, 1987). This result demonstrated that the expressed VP6 was in its native conformation.…”

Comparative Sequence Analysis of the Genomic Segment 6 of Four Rotaviruses Each with a Different Subgroup Specificity

“…140K (Fig. 3, 4) which corresponds to the trimeric form of VP6 (Gorziglia et al, 1985;Sabara et al, 1987). Fig.…”

“…The major inner capsid protein of rotaviruses, p45K (Novo & Esparza, 1981), also referred to as VP6 (Espejo et al, 1981;Ericson et al, 1982) is present in the virion in an oligomeric, possibly trimeric form (Gorziglia et al, 1985;Sabara et al, 1987). VP6 is an important component of the virion not only because of its abundance, accounting for approximately 80 ~ of viral protein, but also because of its constituent antigenic sites.…”

Monoclonal antibodies to the VP6 of porcine subgroup I rotaviruses reactive with subgroup I and non-subgroup I non-subgroup II strains

“…1 a). Omitting the heating step, an additional polypeptide of approximately 135K appears which replaces VP6 and corresponds to its trimeric form (Gorziglia et al, 1985;Sabara et al, 1987). In immunoblots nine of the 10 MAbs described and the subgroup I reference MAb recognized the trimeric but not the monomeric form of VP6 (Fig.…”