Analysis of the structural and dsRNA binding activities of reovirus protein sigma3.

摘要

Mammalian reoviruses are non-enveloped viruses of the Reoviridae family, formed by two concentric protein capsids. The virions are highly stable, yet undergo a disassembly process to activate the putative membrane penetration protein, μ1. The σ3 protein, major component of the outer capsid, is believed to confer stability to the particle, and its proteolytically processing during entry activates μ1. σ3 also has dsRNA binding activity. The mode of σ3:dsRNA interaction and the role of this activity, remain unknown. This dissertation focuses on the proteolytic processing of σ3 during virion disassembly, and on the characterization of its dsRNA binding activity.; A first study developed a system to generate in vitro virion particles containing mutagenized forms of σ3, allowing to circumvent (partially) the lack of reovirus ‘reverse genetics’. Recombinant σ3 protein assembled onto purified infectious subvirion particles (ISVPs), and reconstituted the σ3 coat. The resulting particles, recoated ISVPs, were infectious and biochemically and functionally similar to virions. Thus, the ability to introduce mutations in the recombinant σ3 protein make recoated ISVPs a powerful system with which to study particle-bound σ3.; A second study, focused on the proteolytic processing of σ3 during particle disassembly, identified two regions within σ3 of high protease sensitivity (hypersensitivity regions (HSRs)), within which all the proteases tested in vitro, and the lysosomal proteases in vivo , cleaved σ3 as an early step in processing. We propose that processing of σ3 follows a defined proteolytic pathway, which initiates by cleavage within the HSRs. Mutagenic studies using rcISVPs identified σ3 residues 344, 347 and 353, as the main determinants of a strain difference in cleavage within the HSRs.; A third study proposes a new model for σ3's dsRNA binding activity. Alanine substitution analysis identified five residues required for dsRNA binding (Gln205, Arg208, Lys 287, Lys 293 and Gln329). The position of these residues on the σ3 structure defined a long and narrow strip that spans the two monomers of the crystallographic σ3 dimer. Substitution of residues within the σ3:σ3 interface also abrogated dsRNA binding. We propose that σ3 binds dsRNA as a dimer and that the long and narrow strip constitutes the putative dsRNA binding region.