Membrane association of African horsesickness virus nonstructural protein NS3 determines its cytotoxicity

The smallest RNA genome segment of African horsesickness virus (AHSV) encod es the nonstructural protein NS3 (24K). NS3 localizes in areas of plasma me mbrane disruption and is associated with events of viral release. Conserved features in all AHSV NS3 proteins include the synthesis of a truncated NS3 A protein from the same open reading frame as that of NS3, a proline-rich r egion, a region of strict sequence conservation and two hydrophobic domains . To investigate whether these features are associated with the cytotoxicit y of NS3 or altered membrane permeability, a series of mutants were constru cted and expressed in the BAC-TO-BAC baculovirus-expression system. Our res ults indicate that mutations in either of the two hydrophobic domains do no t prevent membrane targeting of the mutant proteins but abolish their membr ane anchoring. This prevents their localization to the cell surface and obv iates their cytotoxic effect. The cytotoxicity of NS3 is therefore dependen t on its membrane topography and thus involves both hydrophobic domains. NS 3 has many of the characteristics of lytic viral proteins that play a centr al role in viral pathogenesis through modifying membrane permeability. (C) 2001 Academic Press.