From virion to viral factory: Reovirus particle transformations leading to cell entry and productive infection

Nonfusogenic, mammalian orthoreovirus (reovirus) virion consists of two concentric protein capsids lacking a lipid envelope. The genome and the inner-capsid constitute the core particle. The outer-capsid proteins encapsidate the core and mediate its cytoplasmic delivery in a process involving stepwise outer-capsid disassembly and derepression of the core's transcriptional activity. Newly expressed nonstructural protein muNS then coats the transcriptionally-active, cytoplasmic core particle. This interaction seems to prevent outer-capsid assembly and to allow seeding of the viral factory, a cytoplasmic structure believed to represent the site of virus genome replication and virus particle assembly.; Data presented in the first part extends our understanding of the reovirus membrane-penetration mechanism. Recent in vitro work has demonstrated formation of small, size-selective membrane pores, in concert with structural rearrangements in the outer-capsid protein mu1. We demonstrate that mu1 fragments, mu1N and &phgr;, released from virus particles mediate membrane-pore formation. We further show that particle-associated sequences lack an independent membrane-association mechanism, but readily dock to preformed membrane pores. Particle docking to pores may represent a discrete step during membrane penetration.; In the second part we examine a final step in reovirus outer-capsid disassembly: release of the central mul fragment delta to yield the cytoplasmic core particle, which can then interact with muNS. An in vitro assay with reticulocyte lysate recapitulated the release of intact delta molecules and demonstrated the requirement for Hsc70 in this process. We present evidence consistent with the involvement of Hsc70 in delta release in cells as well. delta release either accompanies or occurs soon after particle translocation across the membrane.; In the third part we show that muNS contains a conserved clathrin-box motif, by which it effectively recruits clathrin to both reovirus factories and factory-matrix structures formed by muNS alone. Mutations of this muNS motif disrupt its association with clathrin, but do not completely inhibit factory-matrix formation. The data implicate muNS as a reovirus-encoded, adaptor-like protein, which recruits clathrin for roles different from allowing cell entry. We discuss several possible functions of clathrin recruitment, including one of providing a mechanistic basis for regulation of muNS uncoating from core particles in preparation for outer-capsid assembly.