A bluetongue serogroup-reactive epitope in the amino terminal half of the major core protein VP7 is accessible on the surface of bluetongue virus particles.

Immunoelectron microscopy has been used to confirm that the core protein VP7 is accessible on the surface of bluetongue virus (BTV) particles. Monospecific antibodies generated to vaccinia virus-expressed VP7 and an anti-VP7 monoclonal antibody (MAb 20E9) bound to native virus particles and were localized by protein A-gold. In contrast, MAb 20E9 labeled directly with gold failed to gain access and bind, suggesting that VP7 is neither adventitiously adsorbed to the virion surface nor exposed in a manner such as protrusion through the outer capsid. Thus the surface layer of BTV may be considered as a net which only partially obscures the underlying core particle. Sequencing of VP7 revealed it to be an extremely hydrophobic protein, 350 amino acids in length with cysteine residues at positions 15, 65, and 154. Examination of VP7 in the cytosol of cells infected with either BTV or a vaccinia virus recombinant expressing VP7 indicated that the protein may exist as an oligomer, whose constituent monomers are not linked by intermolecular disulfide bonds. The cysteine residues in sodium dodecyl sulfate (SDS)-denatured, dithiothreitol (DTT)-treated VP7 were labeled with the fluorescent iodoacetamide AEDANS and the protein was cleaved by V8 protease. The size of the labeled peptides and knowledge of the location of potential V8 cleavage sites suggested that the enzyme cleaved VP7 at three locations (glutamic acid residues at positions 61, 104 (or 108), and 132 (or 134 or 135). Analysis of the fluorescent peptides generated by V8 protease cleavage of VP7 labeled with AEDANS in the absence of DTT (i.e., with any putative intramolecular disulfide bonds intact) suggested that the cysteine at position 154 was the only one accessible to AEDANS. The cysteines at positions 15 and 65 may therefore be linked via a disulfide bond. Denaturation of VP7 with SDS did not eliminate the capacity of the protein to bind MAb 20E9. However, the sensitivity of the epitope to reduction and acetylation and its resistance to either of these processes alone suggest that it may be located near a disulfide bond linking cysteines at positions 15 and 65. Confirmation that the epitope lay in the amino-terminal half of the VP7 came from immunoelectron microscopy experiments in which thin sections of bacteria expressing the complete VP7 and the amino-terminal half were probed with MAb 20E9 and protein A-gold.