The proposed research uses mammalian reovirus (MRV) to define how a single capsid protein, μ1, influences distinct functions of the viral capsid. MRV particles are comprised of two concentric protein shells, the outer capsid and the inner core. The μ1 protein is a major component of the outer capsid. Published evidence and our preliminary data indicate that μ1 performs at least three functions. First, even though the two proteins are not in physical contact, properties of μ1 influence virus assembly in a way that alters the presentation of the sigma1 attachment protein on the virion. Second, two μ1 peptides, generated during virus disassembly, cooperate with host lipid membranes to facilitate further uncoating of the virus and permeabilize host membranes. Third, μ1 influences inter-particle interactions to form multivirion infectious units. With the aid of the following three Aims, the proposed research seeks to provide insight into the functions of μ1. In Aim 1, the role of μ1 in maintaining assembly fidelity will be determined. The position of sigma1 on the particle will be determined by biochemical studies and cryoelectron microscopy. The contribution of altering the strength of interaction between μ1 and adjacent capsid proteins on the presentation of sigma1 will be evaluated using genetic analysis. How changes to sigma1 encapsidation and sigma1 conformation influence virus replication in vivo will be determined using a mouse model of viral disease. In Aim 2, the function of μ1 in delivering core particles into the host cytoplasm will be defined. How μ1 peptides recruit entry intermediates to membranes will be determined by biochemical and genetic studies. The minimal number of μ1 peptides needed for successful recruitment of a virus entry intermediate, for pore formation and for successful infection will be quantified. The structure of the virus entry intermediate associated with the membrane will be determined by cryoelectron microscopy. Host proteins that associate with capsids following disassembly and influence the efficiency of infection will be identified by affinity purification and mass spectrometry. In Aim 3, the contribution of μ1 to MVIU formation will be identified. Regions important for inter-particle interactions will be determined by limited proteolysis and mass spectrometry. The relationship between MVIU formation, coinfection efficiency, and reassortment frequency will be determined. Whether MVIU formation also determines reassortment in vivo and if the determinants of MVIU formation and those that influence the recovery of reassortant progeny correlate, will be determined. Completion of this work will provide comprehensive insight into how μ1 completes each of these functions and define the properties of μ1 that influence the capacity of MRV to replicate in cell culture, produce reassortant progeny, and elicit disease.