Reovirus Outer Capsid Protein Μ1 Research Articles

Cell Entry-Independent Role for the Reovirus μ1 Protein in Regulating Necroptosis and the Accumulation of Viral Gene Products

The reovirus outer capsid protein μ1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized μ1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized μ1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of μ1 also affects aspects of viral replication. We found that, while μ1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus-strand RNA, plus-strand RNA, and proteins that are not targeted by the μ1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when μ1 is knocked down. Following μ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of μ1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to μ1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.IMPORTANCE We used mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for the reovirus outer capsid protein μ1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection; μ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

Multi-organ lesions in suckling mice infected with SARS-associated mammalian reovirus linked with apoptosis induced by viral proteins μ1 and σ1.

We reported the isolation and characterization of a novel mammalian reassortant reovirus BYD1 that may have played an accomplice role with SARS-coronavirus during the 2003 SARS pandemic. The pathogenic mechanism of this novel reovirus is unknown. Reovirus pathogenicity has been associated with virus-induced apoptosis in cultured cells and in vivo. The reovirus outer capsid protein μ1 is recognized as the primary determinant of reovirus-induced apoptosis. Here, we investigated the apoptosis induced by BYD1, its outer capsid protein μ1, and its cell-attachment protein σ1 to understand the pathogenesis of BYD1. We also investigated BYD1 caused systemic complications in suckling mice. Under electron microscopy, BYD1-infected cells showed characteristics typical of apoptosis. Notably, ectopically expressed μ1 and σ1 induced similar pathological apoptosis, independent of BYD1 infection, in host cells in which they were expressed, which suggests that μ1 and σ1 are both apoptotic virulence factors. Consistent with previous reports of reovirus pathogenicity, suckling mice intracranially inoculated with BYD1 developed central nerve damage, myocarditis, and pneumonia. Collectively, our data suggest that BYD1 μ1- and σ1-induced apoptosis is involved in the multi-organ lesions in a suckling mouse BYD1 infection model.

The μ1 72-96 Loop Controls Conformational Transitions during Reovirus Cell Entry

The reovirus outer capsid protein μ1 forms a lattice surrounding the viral core. In the native state, μ1 determines the environmental stability of the viral capsid. Additionally, during cell entry, μ1 undergoes structural rearrangements that facilitate delivery of the viral cores across the membrane. To determine how the capsid-stabilizing functions of μ1 impinge on the capacity of μ1 to undergo conformational changes required for cell entry, we characterized viruses with mutations engineered at charged residues within the μ1 loop formed by residues 72 to 96 (72-96 loop). This loop is proposed to stabilize the capsid by mediating interactions between neighboring μ1 trimers and between trimers and the core. We found that mutations at Glu89 (E89) within this loop produced viruses with compromised efficiency for completing their replication cycle. ISVPs of E89 mutants converted to ISVP*s more readily than those of wild-type viruses. The E89 mutants yielded revertants with second-site substitutions within regions that mediate interaction between μ1 trimers at a site distinct from the 72-96 loop. These viruses also contained changes in regions that control interactions within μ1 trimers. Viruses containing these second-site changes displayed restored plaque phenotypes and were capable of undergoing ISVP-to-ISVP* conversion in a regulated manner. These findings highlight regions of μ1 that stabilize the reovirus capsid and demonstrate that an enhanced propensity to form ISVP*s in an unregulated manner compromises viral fitness.

A Proapoptotic Peptide Derived from Reovirus Outer Capsid Protein μ1 Has Membrane-Destabilizing Activity

The reovirus outer capsid protein μ1 is responsible for cell membrane penetration during virus entry and contains determinants necessary for virus-induced apoptosis. Residues 582 to 611 of μ1 are necessary and sufficient for reovirus-induced apoptosis, and residues 594 and 595 independently regulate the efficiency of viral entry and reovirus-induced cell apoptosis, respectively. Two of three α-helices within this region, helix 1 (residues 582 to 611) and helix 3 (residues 644 to 675), play a role in reovirus-induced apoptosis. Here, we chemically synthesized peptides representing helix 1 (H1), H1:K594D, H1:I595K, and helix 3 (H3) and examined their biological properties. We found that H1, but not H3, was able to cause concentration- and size-dependent leakage of molecules from small unilamellar liposomes. We further found that direct application of H1, but not H1:K594D, H1:I595K, or H3, to cells resulted in cytotoxicity. Application of the H1 peptide to L929 cells caused rapid elevations in intracellular calcium concentration that were independent of phospholipase C activation. Cytotoxicity of H1 was not restricted to eukaryotic cells, as the H1 peptide also had bactericidal activity. Based on these findings, we propose that the proapoptotic function of the H1 region of μ1 is dependent on its capacity to destabilize cellular membranes and cause release of molecules from intracellular organelles that ultimately induces cell necrosis or apoptosis, depending on the dose.

Reovirus Infection or Ectopic Expression of Outer Capsid Protein μ1 Induces Apoptosis Independently of the Cellular Proapoptotic Proteins Bax and Bak

Mammalian orthoreoviruses induce apoptosis in vivo and in vitro; however, the specific mechanism by which apoptosis is induced is not fully understood. Recent studies have indicated that the reovirus outer capsid protein μ1 is the primary determinant of reovirus-induced apoptosis. Ectopically expressed μ1 induces apoptosis and localizes to intracellular membranes. Here we report that ectopic expression of μ1 activated both the extrinsic and intrinsic apoptotic pathways with activation of initiator caspases-8 and -9 and downstream effector caspase-3. Activation of both pathways was required for μ1-induced apoptosis, as specific inhibition of either caspase-8 or caspase-9 abolished downstream effector caspase-3 activation. Similar to reovirus infection, ectopic expression of μ1 caused release into the cytosol of cytochrome c and smac/DIABLO from the mitochondrial intermembrane space. Pancaspase inhibitors did not prevent cytochrome c release from cells expressing μ1, indicating that caspases were not required. Additionally, μ1- or reovirus-induced release of cytochrome c occurred efficiently in Bax(-/-)Bak(-/-) mouse embryonic fibroblasts (MEFs). Finally, we found that reovirus-induced apoptosis occurred in Bax(-/-)Bak(-/-) MEFs, indicating that reovirus-induced apoptosis occurs independently of the proapoptotic Bcl-2 family members Bax and Bak.

Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein μ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation

Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein micro 1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to micro 1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of micro 1-based thermostabilization can occur by a general mechanism of micro 1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between micro 1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in micro 1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.

Thermostabilizing mutations in reovirus outer-capsid protein μ1 selected by heat inactivation of infectious subvirion particles

The 76-kDa μ1 protein of nonfusogenic mammalian reovirus is a major component of the virion outer capsid, which contains 200 μ1 trimers arranged in an incomplete T = 13 lattice. In virions, μ1 is largely covered by a second major outer-capsid protein, σ3, which limits μ1 conformational mobility. In infectious subvirion particles, from which σ3 has been removed, μ1 is broadly exposed on the surface and can be promoted to rearrange into a protease-sensitive and hydrophobic conformer, leading to membrane perforation or penetration. In this study, mutants that resisted loss of infectivity upon heat inactivation (heat-resistant mutants) were selected from infectious subvirion particles of reovirus strains Type 1 Lang and Type 3 Dearing. All of the mutants were found to have mutations in μ1, and the heat-resistance phenotype was mapped to μ1 by both recoating and reassortant genetics. Heat-resistant mutants were also resistant to rearrangement to the protease-sensitive conformer of μ1, suggesting that heat inactivation is associated with μ1 rearrangement, consistent with published results. Rate constants of heat inactivation were determined, and the dependence of inactivation rate on temperature was consistent with the Arrhenius relationship. The Gibbs free energy of activation was calculated with reference to transition-state theory and was found to be correlated with the degree of heat resistance in each of the analyzed mutants. The mutations are located in upper portions of the μ1 trimer, near intersubunit contacts either within or between trimers in the viral outer capsid. We propose that the mutants stabilize the outer capsid by interfering with unwinding of the μ1 trimer.

Reovirus Outer Capsid Protein μ1 Induces Apoptosis and Associates with Lipid Droplets, Endoplasmic Reticulum, and Mitochondria

The mechanisms by which reoviruses induce apoptosis have not been fully elucidated. Earlier studies identified the mammalian reovirus S1 and M2 genes as determinants of apoptosis induction. However, no published results have demonstrated the capacities of the proteins encoded by these genes to induce apoptosis, either independently or in combination, in the absence of reovirus infection. Here we report that the mammalian reovirus micro1 protein, encoded by the M2 gene, was sufficient to induce apoptosis in transfected cells. We also found that micro1 localized to lipid droplets, endoplasmic reticulum, and mitochondria in both transfected cells and infected cells. Two small regions encompassing amphipathic alpha-helices within a carboxyl-terminal portion of micro1 were necessary for efficient induction of apoptosis and association with lipid droplets, endoplasmic reticulum, and mitochondria in transfected cells. Induction of apoptosis by micro1 and its association with lipid droplets and intracellular membranes in transfected cells were abrogated when micro1 was coexpressed with sigma3, with which it is known to coassemble. We propose that micro1 plays a direct role in the induction of apoptosis in infected cells and that this property may relate to the capacity of micro1 to associate with intracellular membranes. Moreover, during reovirus infection, association with sigma3 may regulate apoptosis induction by micro1.