Adaptation of reovirus to growth in the presence of protease inhibitor E64segregates with a mutation in the carboxy terminus of viral outer-capsid protein sigma 3

Reovirus virions are internalized into cells by receptor-mediated endocytos is. Within the endocytic compartment, the viral outer capsid undergoes acid -dependent proteolysis leading to degradation of sigma3 protein and proteol ytic cleavage of mu1/mu 1C protein. E64 is a specific inhibitor of cysteine -containing proteases that blocks disassembly of reovirus virions. To ident ify domains in reovirus proteins that influence susceptibility to E64-media ted inhibition of disassembly, we selected variant viruses by serial passag e of strain type 3 Dearing (T3D) in murine L929 cells treated with E64, E64 -adapted variant viruses (D-EA viruses) produced 7- to 17-fold-greater yiel ds than T3D did after infection of cells treated with 100 muM E64, Viral ge nes that segregate with growth of D-EA viruses in the presence of E64 were identified by using reassortant viruses isolated from independent crosses o f E64-sensitive strain type I Lang and two prototype D-EA viruses. Growth o f reassortant viruses in the presence of E64 segregated with the S4 gene, w hich encodes outer-capsid protein sigma3, Sequence analysis of S4 genes of three D-EA viruses isolated from independent passage series revealed a comm on tyrosine-to-histidine mutation at amino acid 354 in the deduced amino ac id sequence of sigma3, Proteolysis of D-EA virions by endocytic protease ca thepsin L occurred with faster kinetics than proteolysis of wild-type T3D v irions. Treatment of D-EA virions, but not T3D virions, with cathepsin D re sulted in proteolysis of sigma3, a property that also was found to segregat e with the D-EA S4 gene. These results indicate that a region in sigma3 pro tein containing amino acid 354 influences susceptibility of sigma3 to prote olysis during reovirus disassembly.