CHARACTERIZATION OF 2 AFRICAN HORSE SICKNESS VIRUS NONSTRUCTURAL PROTEINS, NS1 AND NS3

Each of the ten segments of the African horse sickness virus (AHSV) ge nome encodes at least one viral polypeptide. This report focuses on th e nonstructural proteins NS1 and NS3, which are encoded by genome segm ents 5 and 10 respectively. The NS1 protein assembles into tubular str uctures, which are characteristically produced duping orbivirus replic ation in infected cells. NS1 expressed by a recombinant baculovirus in Sf9 cells also forms tubules, which were analysed electron microscopi cally. These tubules had an average diameter of 23 +/- 2 nm, which is less than half the width of the corresponding bluetongue virus (BTV) t ubules. They were also more fragile at high salt concentrations or pH. The cytotoxic effects produced by NS3 were examined by constructing o f mutated versions and expressing them in insect cells. Substitution o f amino acids 76-81 in a conserved region (highly conserved amongst al l AHSV NS3 proteins, as well as other orbiviruses) with similar amino acids, did not influence the cytotoxicity of the mutant protein. Howev er, mutation of four amino acids, from hydrophobic to charged amino re sidues, (aa 165-168) in a predicted transmembrane region of NS3, large ly abolished its cytotoxic effect. It is considered likely that the mu tant protein is unable to interact with cellular membrane components, thereby reducing its toxicity.