Reovirus outer-capsid disassembly and the mechanism of membrane penetration

During cell entry, reovirus particles with a diameter of 70-80 nm must penetrate the cellular membrane to access the cytoplasm. The mechanism of penetration, without the benefit of membrane fusion, is not well characterized for any such nonenveloped animal virus. The 76-kDa μ1 protein 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 (ISVPs), 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 set of studies, work characterizing both the ISVP[arrow right]ISVP* conversion and the subsequent membrane interaction are presented. Thermostable mutants were selected from ISVPs. All of the mutants were found to have determinative mutations in μ1, and the heat-resistance phenotype was mapped to μ1 by both recoating and reassortant genetics. Rate constants of heat inactivation were determined, and the dependence of inactivation rate on temperature was consistent with the Arrhenius relationship. In addition, thermolabilizing intragenic pseudoreversions of one thermostabilizing mutation were isolated and characterized. ISVP[arrow right]ISVP* conversion was found to approximate a second-order reaction at high particle concentrations, and a positive feedback mechanism of promoting conversion was characterized. Released peptide μ1N was identified as a virus-derived promoting factor. Lysis of erythrocytes is an in vitro assay for the membrane perforation activity of reovirus; however, the mechanism of lysis has been unknown. Here, osmotic-protection experiments revealed that reovirus-induced lysis of erythrocytes occurs osmotically, after formation of small size-selective pores. Consistent results were obtained by monitoring leakage of fluorophore-tagged dextrans from the interior of resealed erythrocyte ghosts. Gradient fractionations showed that whole virus particles, as well as the myristoylated fragment μ1N that is released from particles, are recruited to membranes in association with pore formation. We propose that formation of small pores is a discrete, intermediate step in the reovirus membrane-penetration pathway, which may be shared by other nonenveloped animal viruses.