Putative autocleavage of outer capsid protein 1, allowing release of myristoylated peptide 1N during particle uncoating, is critical for cell entry by reovirus

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein 1 of mammalian or-thoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated frag-ments 1N (4 kDa; myristoylated) and 1C (72 kDa). In this study, we found that 1 cleavage to yield 1N and 1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective 1 (asparagine 3 alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including 1 conformational change and 1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin -sarcin). In addition, after these particles were allowed to enter cells, the region of 1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective 1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absenc