Thennostabilizing mutations in reovirus outer-capsid protein mu 1 selected by heat inactivation of infectious subvirion particles

摘要

The 76-kDa mu 1 protein of nonfusogenic mammalian reovirus is a major component of the virion outer capsid, which contains 200 mu 1 trimers arranged in an incomplete T=13 lattice. In virions, mu 1 is largely covered by a second major outer-capsid protein, sigma 3, which limits mu 1 conformational mobility. In infectious subvirion particles, from which sigma 3 has been removed, mu 1 is broadly exposed on the surface and can be promoted to rearrange into a protease-sensitive and hydrophobic conformer, leading to membrane perforation or penetration. In this study, mutants that resisted loss of infectivity upon heat inactivation (heat-resistant mutants) were selected from infectious subvirion particles of reovirus strains Type 1 Lang and Type 3 Dearing. All of the mutants were found to have mutations in mu 1, and the heat-resistance phenotype was mapped to mu 1 by both recoating and reassortant genetics. Heat-resistant mutants were also resistant to rearrangement to the protease-sensitive conformer of mu 1, suggesting that heat inactivation is associated with mu 1 rearrangement, consistent with published results. Rate constants of heat inactivation were determined, and the dependence of inactivation rate on temperature was consistent with the Arrhenius relationship. The Gibbs free energy of activation was calculated with reference to transition-state theory and was found to be correlated with the degree of heat resistance in each of the analyzed mutants. The mutations are located in upper portions of the mu 1 trimer, near intersubunit contacts either within or between trimers in the viral outer capsid. We propose that the mutants stabilize the outer capsid by interfering with unwinding of the It I trimer. (c) 2006 Elsevier Inc. All rights reserved.