Putative autocleavage of reovirus μ1 protein in concert with outer-capsid disassembly and activation for membrane permeabilization

Max L. Nibert, Amy L. Odegard, Melina A. Agosto, Kartik Chandran, Leslie A. Schiff

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Abstract

Capsid proteins of several different families of non-enveloped animal viruses with single-stranded RNA genomes undergo autocatalytic cleavage (autocleavage) as a maturation step in assembly. Similarly, the 76 kDa major outer-capsid protein μ1 of mammalian orthoreoviruses (reoviruses), which are non-enveloped and have double-stranded RNA genomes, undergoes putative autocleavage between residues 42 and 43, yielding N-terminal N-myristoylated fragment μ1N and C-terminal fragment μ1C. Cleavage at this site allows release of μ1N, which is thought to be critical for penetration of the host-cell membrane during cell entry. Most previous studies have suggested that cleavage at the μ1N/μ1C junction precedes addition to the outer capsid during virion assembly, such that only a small number of the μ1 subunits in mature virions remain uncleaved at that site (∼5%). In this study, we varied the conditions for disruption of virions before running the proteins on denaturing gels and in several circumstances recovered much higher levels of uncleaved μ1 (up to ∼60%). Elements of the disruption conditions that allowed greater recovery of uncleaved protein were increased pH, absence of reducing agent, and decreased temperature. These same elements allowed comparably higher levels of the μ1δ protein, in which cleavage at the μ1N/δ junction has not occurred, to be recovered from particle uncoating intermediates in which μ1 had been previously cleaved by chymotrypsin in a distinct protease-sensitive region near residue 580. The capacity to recover higher levels of μ1δ following disruption of these particles for electrophoresis was lost, however, in concert with a series of structural changes that activate the particles for membrane permeabilization, suggesting that the putative autocleavage is itself one of these changes.

Original languageEnglish (US)
Pages (from-to)461-474
Number of pages14
JournalJournal of Molecular Biology
Volume345
Issue number3
DOIs
StatePublished - Jan 21 2005

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Keywords

  • autolytic
  • cell entry
  • reovirus
  • virus assembly
  • virus disassembly

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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