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.

Divalent metal ions are components of numerous icosahedral virus capsids. Flock House virus (FHV), a small RNA virus of the family Nodaviridae, was utilized as an accessible model system with which to address the effects of metal ions on capsid structure and on the biology of virus-host interactions. Mutations at the calcium-binding sites affected FHV capsid stability and drastically reduced virus infectivity, without altering the overall architecture of the capsid. The mutations also altered the conformation of gamma, a membranedisrupting, virus-encoded peptide usually sequestered inside the capsid, by increasing its exposure under neutral pH conditions. Our data demonstrate that calcium binding is essential for maintaining a pH-based control on gamma exposure and host membrane disruption, and they reveal a novel rationale for the metal ion requirement during virus entry and infectivity. In the light of the phenotypes displayed by a calcium site mutant of FHV, we suggest that this mutant corresponds to an early entry intermediate formed in the endosomal pathway.During cellular entry, the capsids of nonenveloped viruses undergo conformational changes triggered by various host factors. The transitions include the exposure of membrane-active, hydrophobic viral polypeptides and the destabilization and disassembly of the capsid, culminating in the delivery of the viral genome to the cytoplasm. A detailed, stepwise pathway of disassembly is unavailable for most viruses and requires molecular characterization of the intermediates formed during this process. Replication of disassembly intermediates in vitro necessitates careful treatment of native virions to simulate conditions likely to be encountered inside host cells. Receptor binding induces conformational changes in poliovirus, and the poliovirus entry intermediates, the 135S and 80S particles, can be generated in vitro by treating the native virion with the purified receptor, or by heating (6,10,19). Reovirus, which requires cathepsin-mediated proteolysis in the late endosomes in order to undergo entry-related changes, produces infectious subvirion particles (ISVP) and core particles as disassembly intermediates upon treatment with proteases in vitro (11,14).Apart from receptors and cellular proteases, other host determinants affecting viruses include low pH and Ca 2ϩ depletion in specialized cellular compartments. The removal of Ca 2ϩ from capsids could be particularly crucial for viruses that depend on metal ions for assembly and stability, such as rotavirus, mouse polyomavirus, and dragon grouper nervous necrosis virus (DGNNV) (1,39,43). A few studies have reported that mutations at the metal-binding sites decrease virus infectivity by impairing the early stages of virus-host interaction and genome release (23,24). When Ca 2ϩ is removed from the capsids of plant viruses such as cowpea chlorotic mottle virus (CCMV), tomato bushy stunt virus (TBSV), and turnip crinkle virus (TCV) by metal chelators (2, 33, 35), the virion swells by almost 10% and becomes suscept...