Protease Cleavage of Reovirus Capsid Protein μ1/μ1C Is Blocked by Alkyl Sulfate Detergents, Yielding a New Type of Infectious Subvirion Particle

All viruses must cross a membrane barrier to gain access to the cytoplasm of their host cells. In enveloped animal viruses, entry is achieved by a viral surface protein(s) that mediates fusion between viral and cellular membranes (27). Viruses that lack a lipid envelope, however, cannot use membrane fusion as a generalized mechanism to penetrate host membranes. Instead, entry by nonenveloped viruses is conceived to occur via either the local disruption of a host membrane (3) or the formation of a membrane-spanning pore (48), as mediated by a viral surface protein analogous to the fusion proteins of enveloped viruses. A common theme that has emerged from studies of the fusion machinery of different enveloped viruses (e.g., togaviruses [36], orthomyxoviruses [32], and paramyxoviruses [49]) is the requirement for posttranslational proteolytic processing of fusion and/or accessory proteins prior to membrane penetration. In many instances, these cleavages prime the fusion proteins for membrane interaction by removing constraints on conformational changes needed for membrane insertion or by generating a new protein terminus that is hydrophobic and can insert into the membrane as a result of these conformational changes. Several groups of nonenveloped viruses, including the rotaviruses (2, 11, 31), adenoviruses (23), picornaviruses (35), and reoviruses (20, 30, 55, 59), also require proteolytic processing for efficient viral entry. To expand current knowledge of the requirement for proteolysis in membrane penetration by nonenveloped viruses, we performed studies to identify which proteolytic events make mammalian reoviruses competent for entry. The maximally stable infectious form of reovirus is the virion, composed of eight proteins ranging from 12 to 600 in copy number (see references 28 and 45 for reviews). These eight structural proteins are organized into two concentric icosahedral capsids (19). The inner capsid encloses the viral genome, which consists of 10 double-stranded RNA segments. The structural framework of the outer capsid is formed by proteins μ1 (600 copies primarily as cleaved fragments μ1N and μ1C, but also some uncleaved μ1) and λ2 (60 copies). Several lines of evidence indicate that μ1 and its fragments effect the membrane penetration step during infection (24–26, 37, 41, 43, 44, 57). λ2 forms pentameric spikes at the fivefold axes of the outer capsid (19, 61) and is involved in the capping of mRNAs extruded through these spikes in the transcribing particle (12, 50). The other two outer-capsid proteins, ς1 (36 to 48 copies) and ς3 (600 copies), decorate the primary lattice formed by μ1 and λ2. ς1 is found at the fivefold axes and mediates viral attachment to host cell receptors preceding endocytic uptake of the virus (34). ς3 engages in close interactions with the underlying μ1 molecules, suggesting that its primary role may be to stabilize virions (16, 17), probably by regulating the exposure and conformational status of μ1 (19, 37, 43). When virions are treated in vitro with exogenous proteases (such as chymotrypsin [CHT]), stepwise disassembly of the outer capsid is observed (5, 29, 51). ς3 is degraded, and μ1/μ1C is endoproteolytically cleaved to yield two particle-bound fragments (N-terminal μ1δ/δ and C-terminal φ [41]), producing infectious subvirion particles (ISVPs) (5, 29, 51). The cleavage of μ1/μ1C is initiated shortly after ς3 degradation commences (4, 7, 41). Although both virions and ISVPs are infectious, only ISVPs can lead to interactions with lipid bilayers in vitro (25, 26, 37, 43, 57). This fact, along with observations that ISVP-like particles are generated from infecting virions at early times postinfection (7, 10, 52, 53, 55), has been taken as evidence that the ISVP is a necessary intermediate in reovirus infection. Indeed, the processing of virion proteins by one or more lysosomal cysteine proteases appears to be required for the infectivity of virions, but not ISVPs, in murine L-cell fibroblasts (1, 30). These results suggest that ς3 plays a key regulatory role in infection by reovirus virions: it stabilizes the outer capsid until the particle enters a suitable hydrolytic compartment along the endosomal-lysosomal pathway, where ς3 is degraded, and μ1/μ1C is cleaved to μ1δ/δ and φ (43, 55). Membrane penetration, likely mediated by the cleavage products of μ1 (24–26, 37, 41, 43, 44, 57), can then ensue. While it seems nearly certain that ς3 removal from reovirus virions is an obligatory step in entry into cells, the importance of the μ1/μ1C cleavage is less clear because it has not been experimentally separable from ς3 degradation (4, 7, 41). μ1, like the fusion proteins of enveloped viruses, probably undergoes structural rearrangements in order to interact with its target membrane during penetration (9, 39). It thus seems reasonable to hypothesize that the cleavage of μ1/μ1C into fragments μ1δ/δ and φ is analogous to maturational cleavages that activate the fusion proteins of many enveloped viruses for membrane interaction, such as the cleavage of influenza virus hemagglutinin HA0 into fragments HA1 and HA2 (32). One possibility is that the cleavage of μ1/μ1C provides protein sequences on either side of the cleavage site with the conformational mobility needed to insert into the membrane bilayer during membrane disruption (41). In this report, we describe an investigation of the role played by the μ1/μ1C cleavage in reovirus infection.