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 orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments 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 genomedeficient 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 absence of 1N/1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal 1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

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The process by which nonenveloped viruses cross cell membranes during host cell entry remains poorly defined; however, common themes are emerging. Here, we use correlated in vivo and in vitro studies to understand the mechanism of Flock House virus (FHV) entry and membrane penetration. We demonstrate that low endocytic pH is required for FHV infection, that exposure to acidic pH promotes FHV-mediated disruption of model membranes (liposomes), and particles exposed to low pH in vitro exhibit increased hydrophobicity. In addition, FHV particles perturbed by heating displayed a marked increase in liposome disruption, indicating that membrane-active regions of the capsid are exposed or released under these conditions. We also provide evidence that autoproteolytic cleavage, to generate the lipophilic ␥ peptide (4.4 kDa), is required for membrane penetration. Mutant, cleavage-defective particles failed to mediate liposome lysis, regardless of pH or heat treatment, suggesting that these particles are not able to expose or release the requisite membrane-active regions of the capsid, namely, the ␥ peptides. Based on these results, we propose an updated model for FHV entry in which (i) the virus enters the host cell by endocytosis, (ii) low pH within the endocytic pathway triggers the irreversible exposure or release of ␥ peptides from the virus particle, and (iii) the exposed/released ␥ peptides disrupt the endosomal membrane, facilitating translocation of viral RNA into the cytoplasm.Flock House virus (FHV), a nonenveloped, positive-sense RNA virus, has been employed as a model system in several important studies to address a wide range of biological questions (reviewed in reference 55). FHV has been instrumental in understanding virus structure and assembly (17,19,45), RNA replication (2,3,37), and specific packaging of the genome (33,44,53,54). Studies of FHV infection in Drosophila melanogaster flies have provided valuable information about the antiviral innate immune response in invertebrate hosts (29,57). FHV is also used in nanotechnology applications as an epitope-presenting platform to develop novel vaccines and medical therapies (31,48). In this report, we use FHV as a model system to further elucidate the means by which nonenveloped viruses enter host cells and traverse cellular membranes.