MUTATIONS IN REOVIRUS OUTER-CAPSID PROTEIN SIGMA-3 SELECTED DURING PERSISTENT INFECTIONS OF L CELLS CONFER RESISTANCE TO PROTEASE INHIBITORE64

Mutations selected in reoviruses isolated from persistently infected c ultures (PI viruses) affect viral entry into cells. Unlike wild-type ( wt) viruses, PI viruses can grow in the presence of ammonium chloride, a weak base that blocks acid-dependent proteolysis of viral outer-cap sid proteins in cellular endosomes during viral entry. In this study, we show that E64, an inhibitor of cysteine proteases such as those pre sent in the endocytic compartment, blocks growth of wt reovirus by inh ibiting viral disassembly. To determine whether PI viruses can grow in the presence of an inhibitor of endocytic proteases, we compared yiel ds of wt and PI viruses in cells treated with E64, Prototype PI viruse s L/C, PI 2A1, and PI 3-1 produced substantially greater yields than w t viruses type 1 Lang (T1L) and type 3 Dearing (T3D) in E64-treated ce lls, To identify viral genes that segregate with growth of PI viruses in the presence of E64, we tested reassortant viruses isolated from in dependent crosses of T1L and each of the prototype PI viruses for grow th in cells treated with E64. Growth of reassortant viruses in the pre sence of E64 segregated exclusively with the S4 gene, which encodes vi ral outer-capsid protein sigma 3, These results suggest that mutations in sigma 3 protein selected during persistent infection alter its sus ceptibility to cleavage during viral disassembly. To determine the tem poral relationship of acid-dependent and protease-dependent steps in r eovirus disassembly, cells were infected with wt strain TIL or T3D, an d medium containing either ammonium chloride or E64d, a membrane-perme able form of E64, was added at various times after adsorption, Suscept ibility to inhibition by both ammonium chloride and E64 was abolished when either inhibitor was added at times greater than 60 min after ads orption, These findings indicate that acid-dependent and protease-depe ndent disassembly events occur with similar kinetics early in reovirus replication, which suggests that these events take place within the s ame compartment of the endocytic pathway.