From virion to viral factory

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 μNS 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 μ1. We demonstrate that μ1 fragments, μ1N and φ, 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 μl fragment δ to yield the cytoplasmic core particle, which can then interact with μNS. An in vitro assay with reticulocyte lysate recapitulated the release of intact δ molecules and demonstrated the requirement for Hsc70 in this process. We present evidence consistent with the involvement of Hsc70 in δ release in cells as well. δ release either accompanies or occurs soon after particle translocation across the membrane. In the third part we show that μNS contains a conserved clathrin-box motif, by which it effectively recruits clathrin to both reovirus factories and factory-matrix structures formed by μNS alone. Mutations of this μNS motif disrupt its association with clathrin, but do not completely inhibit factory-matrix formation. The data implicate μNS 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 μNS uncoating from core particles in preparation for outer-capsid assembly.