Thermostabilizing mutations in reovirus outer-capsid protein μ1 selected by heat inactivation of infectious subvirion particles

The 76-kDa μ1 protein of nonfusogenic mammalian reovirus is a major component of the virion outer capsid, which contains 200 μ1 trimers arranged in an incomplete T = 13 lattice. In virions, μ1 is largely covered by a second major outer-capsid protein, σ3, which limits μ1 conformational mobility. In infectious subvirion particles, from which σ3 has been removed, μ1 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 μ1, and the heat-resistance phenotype was mapped to μ1 by both recoating and reassortant genetics. Heat-resistant mutants were also resistant to rearrangement to the protease-sensitive conformer of μ1, suggesting that heat inactivation is associated with μ1 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 μ1 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 μ1 trimer.