Putative Autocleavage of Reovirus ?1 Protein in Concert with Outer-capsid Disassembly and Activation for Membrane Permeabilization
Journal of Molecular Biology
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 ?1 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 ?1N and C-terminal fragment ?1C. Cleavage at this site allows release of ?1N, 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 ?1N/?1C junction precedes addition to the outer capsid during virion assembly, such that only a small number of the ?1 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 ?1 (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 ?1? protein, in which cleavage at the ?1N/? junction has not occurred, to be recovered from particle uncoating intermediates in which ?1 had been previously cleaved by chymotrypsin in a distinct protease-sensitive region near residue 580. The capacity to recover higher levels of ?1? 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.
Nibert, Max L, Amy L. Odegard, Melina A. Agosto, Kartik Chandran, and Leslie A. Schiff. "Putative Autocleavage of Reovirus ?1 Protein in Concert with Outer-Capsid Disassembly and Activation for Membrane Permeabilization." Journal of Molecular Biology. 345.3 (2005): 461-474. Print.