Virus-Cell Interactions

Cleavage of the C-Terminal Fragment of Reovirus μ1 Is Required for Optimal Infectivity

Anthony J. Snyder, Pranav Danthi
Susana López, Editor
DOI: 10.1128/JVI.01848-17

ABSTRACT

The mammalian orthoreovirus (reovirus) outer capsid, which is composed of 200 μ1/σ3 heterohexamers and a maximum of 12 σ1 trimers, contains all of the proteins that are necessary for attaching to and entering host cells. Following attachment, reovirus is internalized by receptor-mediated endocytosis and acid-dependent cathepsin proteases degrade the σ3 protein. This process generates a metastable intermediate, called infectious subviral particle (ISVP), in which the μ1 membrane penetration protein is exposed. ISVPs undergo a second structural rearrangement to deposit the genome-containing core into the host cytoplasm. The conformationally altered particle is called ISVP*. ISVP-to-ISVP* conversion culminates in the release of μ1 N- and C-terminal fragments, μ1N and Φ, respectively. Released μ1N is thought to facilitate core delivery by generating size-selective pores within the endosomal membrane, whereas the precise role of Φ, particularly in the context of viral entry, is undefined. In this report, we characterize a recombinant reovirus that fails to cleave Φ from μ1 in vitro. Φ cleavage, which is not required for ISVP-to-ISVP* conversion, enhances the disruption of liposomal membranes and facilitates the recruitment of ISVP*s to the site of pore formation. Moreover, the Φ cleavage-deficient strain initiates infection of host cells less efficiently than the parental strain. These results indicate that μ1N and Φ contribute to reovirus pore forming activity.

IMPORTANCE Host membranes represent a physical barrier that prevents infection. To overcome this barrier, viruses utilize diverse strategies, such as membrane fusion or membrane disruption, to access internal components of the cell. These strategies are characterized by discrete protein-protein and protein-lipid interactions. The mammalian orthoreovirus (reovirus) outer capsid undergoes a series of well-defined conformational changes, which conclude with pore formation and delivery of the viral genetic material. In this report, we characterize the role of the small, reovirus-derived Φ peptide in pore formation. Φ cleavage from the outer capsid enhances membrane disruption and facilitates the recruitment of virions to membrane-associated pores. Moreover, Φ cleavage promotes the initiation of infection. Together, these results reveal an additional component of the reovirus pore forming apparatus and highlight a strategy for penetrating host membranes.

INTRODUCTION

To launch infection, viruses must gain access to internal components of a cell; however, host membranes represent a physical barrier to invasion. Enveloped viruses, which contain a host-derived lipid bilayer, overcome this barrier by membrane fusion (1, 2). In contrast, nonenveloped viruses deliver their genomic material by compromising membrane integrity (3–6), fully transiting lipid bilayers (7), or forming small, proteinaceous pores (8–13). Mammalian orthoreovirus (reovirus) is composed of two concentric, protein shells: the inner core, which encapsidates 10 segments of genomic, double-stranded RNA, and the outer capsid, which contains all of the proteins that are necessary for attaching to and entering host cells (14–16). During entry, reovirus generates pores within host membranes to deliver its intact core particle (16–24).

The reovirus outer capsid is composed of 200 μ1/σ3 heterohexamers and a maximum of 12 σ1 trimers (14–16). To initiate infection of a cell, the σ1 attachment protein engages proteinaceous receptors (e.g., junctional adhesion molecule A) (25, 26) or serotype-specific glycans (e.g., sialic acid or GM2) (27–30). The μ1 protein also influences virus-receptor interactions (31). Following attachment, virions are delivered to clathrin-coated vesicles via receptor-mediated endocytosis (32–36). The internalized particles traffic to Rab7-positive late endosomes in a β1 integrin-, Src kinase-, and microtubule-dependent manner (34, 35, 37–41). Within the endosome, reovirus undergoes a series of stepwise disassembly events. First, acid-dependent cathepsin proteases degrade σ3 and cleave μ1 into μ1δ (i.e., uncleaved μ1N-δ) and Φ (Fig. 1A) (36, 42–48). This step generates a metastable intermediate called infectious subviral particle (ISVP) (14, 15). Within the gastrointestinal or respiratory tract, ISVPs are generated extracellularly by luminal proteases (49–51). Virion-to-ISVP conversion is recapitulated in vitro by treating purified virus with chymotrypsin (52, 53). Second, ISVPs undergo a conformational change that deposits the genome-containing core into the host cytoplasm (termed ISVP-to-ISVP* conversion) (19). Neighboring μ1 trimers unwind and separate (54, 55), and μ1N is cleaved from μ1δ via autocatalytic activity (Fig. 1A) (16, 21, 22). Cleaved μ1N and Φ are released from the particle and facilitate core delivery by interacting with host membranes (16–24). In vitro, ISVP-to-ISVP* conversion is triggered using nonspecific factors, such as heat and large, monovalent cations (19, 56–58) or factors that ISVPs are more likely to encounter during infection, such as released μ1N and lipids (59–61).

FIG 1
FIG 1

Sequence, protein composition, and size distribution of the Φ cleavage mutant. (A) Schematic of μ1 cleavage fragments. (B) μ1 amino acid sequence alignments. The residue corresponding to the chymotrypsin cleavage site at the δ-Φ junction is in bold. T1L, reovirus type 1 Lang; T2J, reovirus type 2 Jones; T3D, reovirus type 3 Dearing. (C) Protein composition. Virions and chymotrypsin-generated ISVPs of T1L/T3D M2 and T1L/T3D M2 Y581A were analyzed by SDS-PAGE. The gel was Coomassie brilliant blue stained. Migration of reovirus capsid proteins is indicated on the left. μ1 resolves as μ1C, and μ1δ resolves as δ (21). μ1N and Φ are too small to resolve on the gel (n = 3 independent replicates; results from 1 representative experiment are shown). (D) Particle size distribution profile. Virions and chymotrypsin-generated ISVPs were analyzed by dynamic light scattering. T1L/T3D M2 (gray) and T1L/T3D M2 Y581A (black) size distribution profiles are overlaid (n = 3 independent replicates; results from 1 representative experiment are shown).

Reovirus pore forming activity is attributed to the μ1 N- and C-terminal fragments, μ1N and Φ, respectively. Released μ1N is necessary and sufficient for generating 4- to 6-nm pores within target membranes (17, 20, 24). Lipid-associated μ1N also recruits virions to the site of pore formation (20, 61). Recoated particles that contain cleavage-deficient μ1N are noninfectious and fail to disrupt membranes (23). Nonetheless, how μ1N and Φ cooperate during viral entry is not well established. Released Φ interacts with membranes following pore formation (20); this interaction is likely mediated by a predicted, amphipathic α-helix (48). ISVP-like particles, which fail to generate cleaved Φ, are competent to induce hemolysis (23, 62); however, pore formation is most efficient when μ1N and Φ are present (20). Together, these data suggest that Φ functions as a μ1N chaperone to facilitate pore formation.

In this work, we utilized genetic, biochemical, and cell-based approaches to investigate the components of the reovirus pore forming apparatus. The μ1 protein is composed of three fragments: N-terminal μ1N (residues 2 to 42), central δ (residues 43 to 581), and C-terminal Φ (residues 582 to 708) (14). μ1N generates size-selective pores within target membranes (17, 20, 24), whereas the precise role of Φ, particularly in the context of viral entry, is undefined. Thus, we generated a single residue mutation (μ1 Y581A) within the reovirus type 1 Lang (T1L)/reovirus type 3 Dearing (T3D) M2 genetic background. T1L and T3D represent the prototype strains for their respective mammalian orthoreovirus serotypes; T1L × T3D reassortant viruses are used extensively to investigate many aspects of the reovirus replication cycle (14). T1L/T3D M2, which contains a T3D-derived M2 gene (encoding μ1) and nine genes from T1L, served as the parental strain (i.e., wild type). We show the following: (i) the Y581A change blocked chymotrypsin-mediated cleavage at the δ-Φ junction, (ii) Φ cleavage was not required for particle attachment or internalization or for ISVP-to-ISVP* conversion, (iii) cleaved Φ enhanced pore formation and mediated the recruitment of ISVP*s to model membranes, and (iv) cleaved Φ enhanced reovirus the infection of L929 cells (L cells). Together, these results associate pore forming activity with the μ1-derived Φ peptide.

RESULTS

Recombinant reovirus with mutation μ1 Y581A.Reovirus entry is characterized by a series of stepwise disassembly events. During virion-to-ISVP conversion, the σ3 protein is degraded and the membrane penetration protein, μ1, is cleaved into μ1δ and Φ (Fig. 1A) (36, 42–48). During ISVP-to-ISVP* conversion, neighboring μ1 trimers unwind and separate (54, 55), and the μ1 N-terminal fragment, μ1N, is cleaved from μ1δ (Fig. 1A) (16, 21, 22). Cleaved μ1N is necessary and sufficient for generating size-selective pores within target membranes (17, 20, 24); however, a precise role for cleaved Φ, which is predicted to have an amphipathic α-helix (48), in pore formation is unclear. To evaluate the role of cleaved Φ, we mutated μ1 residue 581 within recombinant reovirus. Changes to the viral capsid that define reovirus entry can be recapitulated in vitro; ISVPs are produced by digesting purified virions with chymotrypsin (52, 53). Therefore, the mutation at residue 581 (Y581A) was designed to prevent chymotrypsin cleavage at the δ-Φ junction (Fig. 1B) (48, 63). The mutant virus was made in the T1L/T3D M2 background, which contains a wild-type or mutated T3D M2 gene (encoding μ1) and nine genes from T1L. Compared to the wild type, T1L/T3D M2 Y581A virions displayed no unexpected defects in protein composition or stoichiometry. μ1C (i.e., uncleaved δ-Φ) was present in T1L/T3D M2 Y581A ISVPs, whereas the δ fragment was present in T1L/T3D M2 ISVPs (Fig. 1C). These results demonstrate that mutating residue 581 blocked chymotrypsin cleavage at the δ-Φ junction. To rule out the possibility of gross structural changes, we analyzed virions and ISVPs by dynamic light scattering (DLS) (Fig. 1D). For each strain, we detected a single peak at the expected hydrodynamic diameter (data not shown).

The Y581A change enhances reovirus attachment.To initiate infection of a host cell, the reovirus σ1 protein engages proteinaceous or carbohydrate receptors, such as junctional adhesion molecule A and glycans (25–30). Despite not interacting directly with σ1, the μ1 protein can also influence receptor binding (31). Surprisingly, T1L/T3D M2 Y581A contained higher levels of σ1 than T1L/T3D M2 (Fig. 2A). Thus, to test the cell binding properties of each strain, we utilized a fluorescence-based, quantitative binding assay (31). Adherent L929 cells were adsorbed with an equivalent number of T1L/T3D M2 or T1L/T3D M2 Y581A particles. Particle internalization was blocked by incubating the cells at 4°C. Bound virus was then detected using an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Binding index was quantified by the ratio of bound virus to total cells. We performed all cell culture-based experiments (see Fig. 2 to 4 and 9) in the absence or presence of ammonium chloride (AC). AC, which prevents acidification of the endosomal-lysosomal system, blocks particle disassembly (47, 64, 65). At each multiplicity of infection tested, T1L/T3D M2 Y581A virions attached more efficiently than T1L/T3D M2 virions (Fig. 2B). We observed similar trends when cells were adsorbed with ISVPs (Fig. 2C). To ensure that the anti-reovirus serum detected each strain equivalently, we tested its reactivity to T1L/T3D M2 or T1L/T3D M2 Y581A, which were coated onto plates (Fig. 2D). We observed no differences in antibody recognition. Thus, differences in cell binding efficiency were not an artifact of the detection method. Together, these results indicate that T1L/T3D M2 Y581A contained more σ1 and attached more efficiently than T1L/T3D M2. Although the Y581A change was correlated with increased levels of particle-associated σ1, the basis for this observation was not the subject of the present study.

FIG 2
FIG 2

The Φ cleavage mutant attaches to cells more efficiently than wild-type virus. (A) Protein composition. T1L/T3D M2 and T1L/T3D M2 Y581A virions and ISVPs were analyzed by SDS-PAGE. The levels of μ1C/δ and σ1 were determined by Western blotting. Migration of reovirus proteins is indicated on the left of each blot. μ1 resolves as μ1C, and μ1δ resolves as δ (21) (n = 3 independent replicates; results from 1 representative experiment are shown). (B and C) Cell attachment. Adherent L929 cells were adsorbed with the indicated concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions (B) or ISVPs (C). All experiments were performed in the absence (top graphs) or presence (bottom graphs) of ammonium chloride (AC). Attached virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Total cells were labeled with a fluorescent DNA stain. Attached virus was detected using an infrared scanner, and binding index was quantified by the ratio of bound virus to total cells. Data are presented as means ± SDs. *, P ≤ 0.05 (n = 3 independent replicates). (D) Antibody reactivity. The indicated concentrations of virions (top) or ISVPs (bottom) of T1L/T3D M2 or T1L/T3D M2 Y581A were coated onto high-affinity polystyrene plates. Plate-bound virus was labeled with an anti-reovirus primary antibody followed by a fluorophore-conjugated secondary antibody. Fluorescent intensity of staining was detected using an infrared scanner. Data are presented as means ± SDs (n = 3 independent replicates).

To evaluate the role of Φ cleavage during reovirus entry, we determined the concentrations of T1L/T3D M2 (1.0 × 103 particles/cell) and T1L/T3D M2 Y581A (0.4 × 103 particles/cell) that conferred equivalent relative attachment (i.e., wild-type and Y581A reovirus attached equal numbers of particles/cell) (Fig. 3A). Thus, equivalent attachment units, which control for differences in binding efficiency, were used for the remaining cell-based experiments (see Fig. 3, 4, and 9).

FIG 3
FIG 3

The Φ cleavage mutant displays wild type-like internalization kinetics. (A) Normalization of particle attachment. Adherent L929 cells were adsorbed with T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) virions (left side) or ISVPs (right side). All experiments were performed in the absence or presence of ammonium chloride (AC). Following attachment, the cells were lysed and total RNA was extracted. Relative attachment was quantified via qRT-PCR using primers against the T1L S2 gene segment and murine GAPDH mRNA. Data are presented as means ± SDs (n = 3 independent replicates). (B and C) Particle internalization. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) virions (B) or ISVPs (C). All experiments were performed in the absence or presence of AC. At the indicated times postinfection, the cells were labeled with a membrane-impermeative biotinylation reagent. The labeled cells were lysed and biotinylated protein was affinity purified (AP) with streptavidin agarose resin (SAR). Lysates (input) and AP:SAR samples were analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus μ1C/δ and host protein tubulin by Western blotting. μ1 resolves as μ1C, and μ1δ resolves as δ (21) (n = 3 independent replicates; results from 1 representative experiment are shown).

Φ cleavage is not required for reovirus internalization.Reovirus is internalized by receptor-mediated endocytosis (32–36). Once inside clathrin-coated vesicles, particles are delivered to Rab7-positive late endosomes in a β1 integrin-, Src kinase-, and microtubule-dependent manner (34, 35, 37–41). To test uptake kinetics, L929 cells were adsorbed with equivalent attachment units of TIL/T3D M2 or T1L/T3D M2 Y581A. At various times postinfection, cell surface-exposed particles (i.e., particles that have not undergone endocytosis) were modified with a membrane-impermeative biotinylation reagent and affinity purified with streptavidin agarose resin (66). In the absence (Fig. 3B, left side) or presence (Fig. 3B, right side) of AC, endocytosis of T1L/T3D M2 and T1L/T3D M2 Y581A virions was complete by 1 h postinfection. Internalized virus was present in cell lysates (see lysate blots) but exhibited resistance to biotinylation (see AP:SAR blots). We next analyzed T1L/T3D M2 and T1L/T3D M2 Y581A ISVPs (Fig. 3C); like for virions, endocytosis of each strain was complete by 1 h postinfection. Similar uptake kinetics were observed for reovirus infection of NIH 3T3 cells (66).

The reovirus core particle, which contains 10 segments of genomic, double-stranded RNA, is delivered intact during viral entry (14–24). Thus, the intracellular levels of any gene segment (e.g., S2) represent the extent of core internalization. To confirm that each strain delivered equal genome copies, cells were infected with equivalent attachment units of T1L/T3D M2 or T1L/T3D M2 Y581A in the absence or presence of AC. At 1 h postinfection, the cells were lysed and the S2 gene segment was detected by quantitative reverse transcription-PCR (qRT-PCR). These experiments were performed in the presence of 200 μM ribavirin, which blocks positive-strand RNA synthesis (67). In T1L/T3D M2- and T1L/T3D M2 Y581A-infected cells, the intracellular levels of S2 were not significantly different (data not shown). These results suggest that each strain delivered an equal number of core particles to the host cell.

Y581A reovirus fails to generate cleaved Φ within AC-treated cells.As discussed earlier, the Y581A change blocked chymotrypsin-mediated cleavage at the δ-Φ junction (Fig. 1C). To test if μ1 cleavage (i.e., processing of μ1C into δ) was affected by this mutation during a bona fide infection, L929 cells were infected with equivalent attachment units of T1L/T3D M2 or T1L/T3D M2 Y581A. At various times postinfection, the cells were lysed and the status of the μ1 protein was analyzed by Western blotting. In cells infected with virions (Fig. 4A, top two blots), both strains generated the δ fragment by 2 h postinfection. In cells infected with T1L/T3D M2 Y581A ISVPs (Fig. 4A, bottom two blots), the δ fragment was generated by 1 h postinfection. These results indicate that Y581A does not block in-cell processing of μ1. Within the endosomes of infected cells, cathepsin proteases degrade the σ3 protein and cleave Φ from μ1; endosomal acidification is required for optimal protease activity (36, 42–48). Thus, to test if disassembly of the Y581A virus required active cathepsin proteases, L929 cells were pretreated with AC prior to infection. As a consequence of protease inhibition, virions failed to disassemble (Fig. 4B, top two blots). To bypass the block in σ3 degradation, AC treated cells were infected with ISVPs (Fig. 4B, bottom two blots). Processing of μ1C into δ was not observed in T1L/T3D M2 Y581A ISVP-infected cells. These results establish conditions under which the role of Φ cleavage can be evaluated during reovirus entry.

FIG 4
FIG 4

The Φ cleavage mutant fails to generate the δ fragment within ammonium chloride-treated cells. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) virions or ISVPs. All experiments were performed in the absence (A) or presence (B) of AC. At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus μ1C/δ and host protein tubulin by Western blotting. μ1 resolves as μ1C, and μ1δ resolves as δ (21) (n = 3 independent replicates; results from 1 representative experiment are shown).

Φ cleavage is not required for reovirus to undergo entry related conformational changes.During reovirus entry, ISVP-to-ISVP* conversion culminates in the release of the genome-containing core particle into the host cytoplasm (16–24). To test if Φ cleavage enhances this conformational change, we conducted 20-min heat inactivation experiments over a range of temperatures (Fig. 5A). Inducing ISVP-to-ISVP* conversion in vitro with heat renders the particles noninfectious. Thus, thermal inactivation (i.e., loss of infectivity) can be used as an indirect readout for ISVP* formation (58). Following incubation at 39°C, T1L/T3D M2 ISVPs and T1L/T3D M2 Y581A ISVPs were reduced in titer by ∼2.0 log10 units relative to control virus that was incubated at 4°C. Concurrent with ISVP-to-ISVP* conversion, the reovirus μ1 protein adopts a protease-sensitive conformation (19). This structural rearrangement is assayed in vitro by heating ISVPs and determining the susceptibility of the δ fragment (a product of μ1 cleavage) to trypsin digestion (19, 58). Consistent with the thermal-inactivation results, the δ fragment in T1L/T3D M2 ISVPs and T1L/T3D M2 Y581A ISVPs became trypsin sensitive at equivalent temperatures (Fig. 5B, see 39°C lanes). Of note, the μ1 protein in T1L/T3D M2 Y581A ISVPs migrated as cleaved δ rather than uncleaved μ1C (compare Fig. 1C and 5B). Trypsin, which was used to probe for protease sensitivity following thermal inactivation, can cleave at the δ-Φ junction; cleavage occurs at residue 584 (Fig. 1B) (48).

FIG 5
FIG 5

The Φ cleavage mutant displays wild type-like thermostability. (A and C) Thermal inactivation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer in the absence (A) or presence (C) of EE liposomes for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 (n = 3 independent replicates). (B and D) Heat-induced ISVP-to-ISVP* conversion. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer in the absence (B) or presence (D) of EE liposomes for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained (n = 3 independent replicates; results from 1 representative experiment are shown).

The data presented above indicate that Φ cleavage does not enhance ISVP-to-ISVP* conversion. Lipids lower the temperature that is needed to induce this conformational change for two prototype reovirus strains (e.g., T1L and T3D) (60, 61). To evaluate the effects of lipids on the Y581A strain, we conducted heat inactivation experiments in the presence of early endosome (EE) liposomes (Fig. 5C); EE liposomes resemble the lipid composition of early endosomal membranes (60, 68). Following incubation at 36°C, T1L/T3D M2 ISVPs and T1L/T3D M2 Y581A ISVPs were reduced in titer by ∼4.0 log10 units and ∼3.0 log10 units, respectively; however, the difference was not statistically significant. Both strains were reduced in titer by ∼5.0 log10 units at 39°C. The thermal-inactivation results were confirmed using the trypsin sensitivity assay. The δ fragment in T1L/T3D M2 ISVPs and T1L/T3D M2 Y581A ISVPs became trypsin sensitive at equivalent temperatures (Fig. 5D, see 36°C lanes).

ISVP-to-ISVP* conversion culminates in the release of two μ1-derived fragments, μ1N and Φ, and the cell attachment protein σ1 (16–23). In addition to generating pores within target membranes (17, 20, 24), μ1N can facilitate ISVP-to-ISVP* conversion in trans, whereas Φ is mostly dispensable for promoting activity (59). To determine whether the released fragments from T1L/T3D M2 Y581A promote thermal inactivation as efficiently as the released fragments from the wild type, T1L/T3D M2 ISVPs were incubated with the supernatant of preconverted ISVP*s. Wild-type ISVP* supernatant contains μ1N, Φ, and σ1 (20), whereas T1L/T3D M2 Y581A ISVP* supernatant is expected to contain μ1N and σ1. Both were generated by incubating ISVPs for 5 min at 52°C (20, 59). The incubation temperature was selected to ensure that each strain underwent ISVP* formation. The reactions were centrifuged to pellet particles, and the supernatant (spin) was transferred to tubes containing target T1L/T3D M2 ISVPs. The supernatant was analyzed by plaque assay (Fig. 6A) and by Western blotting (Fig. 6B) to confirm the clearance of input virus. As expected, virus mixed with ISVP* supernatant was inactivated at a lower temperature than virus alone. The released fragments from T1L/T3D M2 and T1L/T3D M2 Y581A reduced the titer of input virus by ∼2.5 log10 units at 36°C, whereas virus alone was reduced in titer by ∼1.5 log10 units at 39°C (Fig. 6C). These results were confirmed using the trypsin sensitivity assay (Fig. 6D). When T1L/T3D M2 or T1L/T3D M2 Y581A ISVP* supernatant was included in the reaction, the δ fragment in input virus became trypsin sensitive at 33°C. Because each supernatant induced ISVP-to-ISVP* conversion equivalently, these results suggest that Φ does not contribute to the ISVP*-promoting activity of released μ1N.

FIG 6
FIG 6

The Φ cleavage mutant retains ISVP* promoting activity. (A and B) Generation of ISVP* supernatant. Input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 1012 particles/ml were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was centrifuged to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal inactivation reactions. Aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay (A) and for the presence of μ1C/δ by Western blotting (B). In panel A, data are presented as means ± SDs. *, P ≤ 0.05 (n = 3 independent replicates). (C) ISVP* supernatant-mediated thermal inactivation. T1L/T3D M2 ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as means ± SDs. *, P ≤ 0.05 (n = 3 independent replicates). (D) ISVP* supernatant-mediated ISVP-to-ISVP* conversion. T1L/T3D M2 ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer supplemented with the indicated ISVP* supernatants for 20 min at the indicated temperatures. Each reaction was then treated with trypsin for 30 min on ice. Following digestion, equal particle numbers from each reaction were analyzed by SDS-PAGE. The gels were Coomassie brilliant blue stained (n = 3 independent replicates; results from 1 representative experiment are shown).

Φ cleavage enhances reovirus-induced pore formation.To establish a productive infection, reovirus must deliver its genome-containing core to the host cytoplasm; this step is accomplished by perforating the plasma or endosomal membrane during viral entry. As a consequence of ISVP-to-ISVP* conversion, two μ1-derived fragments, μ1N and Φ, and the cell attachment protein σ1 are released from the particle (16–23). Released μ1N is necessary and sufficient for generating size-selective pores within model membranes (e.g., liposomes and red blood cells [RBCs]), whereas Φ is not sufficient and σ1 is dispensable for pore formation (17, 20, 24). Nonetheless, μ1N and Φ are required for optimal hemolytic activity (20). To further assess the role of Φ in pore formation, we compared the capacities of T1L/T3D M2 ISVPs and T1L/T3D M2 Y581A ISVPs to release carboxyfluorescein (CF) from EE liposomes. The reaction mixtures were incubated for 20 min at 42°C; thus, each strain was expected to undergo efficient ISVP-to-ISVP* conversion (Fig. 5). T1L/T3D M2 Y581A ISVPs induced ∼50% CF leakage, whereas T1L/T3D M2 ISVPs induced ∼80% CF leakage (Fig. 7A). These results confirm that Φ cleavage enhances pore formation efficiency.

FIG 7
FIG 7

The Φ cleavage mutant disrupts membranes less efficiently than wild-type virus. (A) ISVP-induced pore formation. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer supplemented with CF-loaded EE liposomes for 20 min at the indicated temperatures. After 20 min, the reactions were diluted 1:50 into virus storage buffer. The samples were equilibrated to room temperature for 15 min prior to measurement of fluorescence. Levels of 0 and 100% CF leakage were determined by incubating an equivalent number of CF-loaded liposomes in virus storage buffer alone or virus storage buffer supplemented with 0.5% Triton X-100, respectively. Data are presented as means ± SDs. *, P ≤ 0.05 (n = 3 independent replicates). (B and C) Osmotic protection of ISVP-induced hemolysis. T1L/T3D M2 (B) or T1L/T3D M2 Y581A (C) ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer supplemented with RBCs and the indicated PEG molecules for 1 h at 37°C. After 1 h, hemolysis was quantified by measuring the absorbance of the supernatant at 405 nm. Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. For each virus, relative hemolysis was normalized to the no-PEG control. Data are presented as means ± SDs (n = 3 independent replicates).

Reovirus ISVPs can induce hemolysis of RBCs (19, 56). Lysis occurs when water enters the RBC through lesions, causing swelling and subsequent rupture. The influx of water is driven by an osmotic gradient (17). Thus, large polyethylene glycol (PEG) molecules (molecular weight [MW], ≥8,000) can serve as osmotic protectants against reovirus-induced hemolysis. In contrast, small PEG molecules (MW, ≤4,000) can enter through membrane pores and rapidly equilibrate, conferring no osmotic protection (17, 69–71). To test if Φ regulates pore size (i.e., PEG size selectivity), we performed hemolysis experiments in the presence or absence of different PEG molecules (MWs, 1,000, 3,500, 6,000, 8,000, and 10,000). PEG does not inhibit ISVP-to-ISVP* conversion (17). T1L/T3D M2 ISVPs were incubated with RBCs for 1 h at 37°C (Fig. 7B). Compared to the no-PEG control, we observed no differences in relative hemolysis in the presence of PEG at MWs of 1,000 and 3,500, whereas PEG at a MW of 8,000 and PEG at a MW of 10,000 were strong osmotic protectants. In contrast, PEG at a MW of 6,000 conferred an intermediate phenotype. We obtained similar results with T1L/T3D M2 Y581A ISVPs (Fig. 7C), suggesting that both strains generate 4- to 6-nm pores within RBC membranes (17, 72, 73).

Φ cleavage facilitates the recruitment of reovirus to lipid membranes.Released μ1N associates with target membranes and forms size-selective pores (17, 20, 24). Moreover, μ1N-generated pores interact with ISVPs (or ISVP*s) (20, 61); however, it is unclear if Φ modulates this recruitment activity. When ISVP-to-ISVP* conversion is triggered in the presence of resealed ghosts (i.e., the membrane shell formed by the lysis of RBCs), Φ cosediments with lipid-associated μ1N (20). To test the hypothesis that cleaved Φ facilitates the recruitment of ISVP*s to membranes, we performed virus-liposome coflotation experiments. T1L/T3D M2 ISVPs or T1L/T3D M2 Y581A ISVPs were incubated with EE liposomes for 20 min at 4°C or 36°C. The reaction mixtures were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. When the reaction mixtures were incubated at 4°C prior to sedimentation, the μ1C/δ fragments for both strains were observed predominantly in fractions 7 and 8 (Fig. 8B, top two blots), similar to ISVPs alone (Fig. 8A). Thus, ISVP-liposome interactions were not detected. We next analyzed ISVPs that were incubated at 36°C (Fig. 8B, bottom two blots). Under these conditions, ISVP-to-ISVP* conversion is expected to occur (Fig. 5C and D). T1L/T3D M2 ISVP*s were observed predominantly in fractions 1 to 6 (i.e., top half of the gradient), whereas T1L/T3D M2 Y581A ISVP*s were observed in all fractions. Given the greater propensity for the wild type to cosediment with liposomes, these results indicate that Φ cleavage enhances the recruitment of ISVP*s to membranes.

FIG 8
FIG 8

The Φ cleavage mutant interacts with liposomes less efficiently than wild-type virus. (A) Virus incubated alone. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer for 20 min at 4°C. The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting (n = 3 independent replicates; results from one representative experiment are shown). (B) Virus incubated with liposomes. T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs at 2 × 1012 particles/ml were incubated in virus storage buffer supplemented with EE liposomes for 20 min at 4°C (top two blots) or 36°C (bottom two blots). The samples were then applied to the tops of sucrose gradients and sedimented by ultracentrifugation. Fractions were collected from the tops of the gradients. Equal volumes of each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting (n = 3 independent replicates; results from 1 representative experiment are shown).

Φ cleavage enhances reovirus infection of L929 cells.Our results demonstrate that Φ cleavage is not required for an early step in the reovirus entry pathway (i.e., attachment, internalization, or ISVP-to-ISVP* conversion) (Fig. 2, 3, 5, and 6). Nonetheless, Φ cleavage contributes to pore formation (Fig. 7) and mediates the recruitment of ISVP*s to lipid membranes (Fig. 8). To test if cleaved Φ facilitates the establishment of a bona fide infection, L929 cells were infected with equivalent attachment units of T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs. At early times postinfection (i.e., 0 to 12 h), cells were lysed and analyzed by Western blotting for expression of the reovirus nonstructural protein σNS. When cells were infected in the presence of AC (Fig. 9A, right side), T1L/T3D M2 expressed detectable levels of σNS 2 h earlier than T1L/T3D M2 Y581A. As discussed earlier, AC blocks in-cell processing of μ1 (i.e., cleavage at the δ-Φ junction) (Fig. 4). We observed similar trends when cells were infected in the absence of AC (Fig. 9A, left side); however, the difference between the wild type and Y581A mutant was less evident. We next determined the percentage of reovirus-positive cells at 18 h postinfection (Fig. 9B). In the absence or presence of AC, T1L/T3D M2 ISVPs infected a higher percentage of cells than T1L/T3D M2 Y581A ISVPs. Thus, inhibition of particle disassembly (Fig. 4) was correlated with a reduced efficiency to establish infection (Fig. 9).

FIG 9
FIG 9

The Φ cleavage mutant infects cells less efficiently wild-type virus. (A) Initiation of protein synthesis. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) ISVPs. All experiments were performed in the absence (left side) or presence (right side) of ammonium chloride (AC). At the indicated times postinfection, the cells were lysed and analyzed by SDS-PAGE. The gels were analyzed for the presence of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 by Western blotting (n = 3 independent replicates; results from 1 representative experiment are shown). (B) Establishment of infection. Adherent L929 cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) ISVPs. All experiments were performed in the absence or presence of AC. At 18 h postinfection, the percentage of reovirus-positive cells was quantified by indirect immunofluorescence. Data are presented as means ± SDs. *, P ≤ 0.05 (n = 3 independent replicates).

DISCUSSION

Reovirus entry culminates in the delivery of the genome-containing core particle to the host cytoplasm. The outer capsid releases μ1 N- and C-terminal fragments, μ1N and Φ, respectively (16–24). μ1N is necessary and sufficient for generating size-selective pores within target membranes (17, 20, 24). In this work, we explored a role for Φ in pore formation. Recombinant reovirus, which encoded a change at the μ1 δ-Φ junction (i.e., Y581A), failed to generate cleaved Φ in vitro (Fig. 1). The mutant strain attached to cells more efficiently than the wild type (Fig. 2); however, Φ cleavage did not affect the rate of particle internalization (Fig. 3). Moreover, processing at the μ1 δ-Φ junction was not required for ISVP-to-ISVP* conversion (Fig. 5) or for mediating the ISVP* promoting activity of released μ1N (Fig. 6). Nonetheless, the Φ cleavage-deficient strain induced pore formation (Fig. 7) and recruited ISVP*s to model membranes (Fig. 8) less efficiently than the wild type. Membrane penetration is an essential step for reovirus to gain access to internal components of a cell. Thus, cleavage at the δ-Φ junction (Fig. 4) was correlated with enhanced infection of L929 cells (Fig. 9). Together, these results indicate that μ1N and Φ contribute to reovirus pore forming activity.

Reovirus invades host cells by forming small, proteinaceous pores. μ1N (residues 2 to 42 of μ1), which dissociates from the outer capsid following ISVP-to-ISVP* conversion, is primarily responsible for membrane penetration (16–24). Released μ1N is sufficient to induce RBC hemolysis and to recruit particles to the site of pore formation (17, 20, 24, 61). When μ1N autocleavage is blocked by substitution μ1 N42A, particle infectivity and hemolytic capacity are severely attenuated; however, the transition to ISVP*s is not affected (23). Membrane disruption does not require additional reovirus components; synthetic μ1N peptides are sufficient to recapitulate pore forming activity (20, 24). μ1N contains an N-terminal myristoyl group that presumably anchors the peptide to the lipid bilayer (22). Secondary-structure and sequence analyses suggest that μ1N oligomerizes to form a β-barrel pore within target membranes. As measured by circular dichroism, lipid-associated μ1N contains significant β-strand content. Moreover, the μ1N sequence reveals two putative transmembrane regions (i.e., alternating nonpolar residues). Substitutions within these regions (e.g., μ1 V13N or μ1 L36D) impair membrane disruption (24). Similar structural and sequence features are observed for β-barrel toxins (74–77). Φ (residues 582 to 708 of μ1), which also dissociates from the outer capsid following ISVP-to-ISVP* conversion, is not required for pore formation (16–24). Inconsistent with the results presented here (Fig. 7), recoated particles that contain a Φ cleavage-deficient variant of μ1 are fully competent to induce hemolysis (63). Nonetheless, wild-type ISVP* supernatant, which contains μ1N and Φ, disrupts membranes more efficiently than ISVP* supernatant that contains μ1N but lacks Φ (20). Our work, which utilized infectious, recombinantly generated reovirus, also supports a model in which released Φ enhances pore formation (Fig. 7). The following possibilities may explain the discrepancy between our results and the results obtained using recoated particles: (i) how the virus is constructed (i.e., genetic background), (ii) how cleavage at the δ-Φ junction is abolished, (iii) how ISVP-to-ISVP* conversion is triggered, and (iv) the composition of the lipid bilayer.

Sequence analysis reveals a predicted, amphipathic α-helix at the N terminus of Φ. This feature is proposed to facilitate Φ-lipid interactions (48). Cleaved Φ is expected to enhance membrane penetration by two possible mechanisms. First, reovirus ISVPs (∼70 nm in diameter) are too large to pass through 4- to 6-nm pores (15, 17, 20, 24); however, internalized virions dissociate from endosomal and lysosomal markers (38, 78). To exit the endosome, pore expansion likely occurs, allowing for particle translocation and access to cytosolic components (17, 20, 24). Nonetheless, PEG protection experiments indicate that Φ cleavage does not impact pore size (Fig. 7). Thus, the addition of μ1N subunits to preexisting pores and/or osmotic lysis may be required for endosomal escape (17, 20, 24). Host factors, such as Hsc70, also contribute to core delivery (79). Second, peptides released from the outer capsid recruit reovirus to membrane pores (17, 20). Lipid-associated μ1N is sufficient to recapitulate recruitment activity (20, 61). Our results demonstrate that cleaved Φ also enhances ISVP*-membrane interactions (Fig. 8). Thus, cleaved Φ indirectly regulates pore formation by driving μ1N membrane insertion or by facilitating μ1N oligomerization (20). Alternatively, lipid-associated Φ could interact directly with the virus particle. It is unclear how recruiting an ISVP* to preexisting pores would enhance membrane disruption. One possibility is that the interaction between partially converted virions and lipid-associated μ1N/Φ induces complete ISVP-to-ISVP* conversion (60, 61). If so, newly released μ1N would be concentrated at the site of pore formation (i.e., in position to facilitate pore expansion).

One unexpected result from this study was the excess levels of σ1 on T1L/T3D M2 Y581A particles (Fig. 2). σ1 is required for attachment to cell surface receptors (25–30), whereas its function postinternalization (if any) has not been defined. Recoated particles that lack σ1 undergo virion-to-ISVP conversion and induce hemolysis as efficiently as native reovirus (63). Future studies are needed to investigate the incorporation of σ1 into newly assembled virions and to determine if σ1 affects late entry events.

Reovirus penetrates host membranes by forming small, proteinaceous pores. Studies thus far attribute pore forming activity to the μ1 N-terminal fragment, μ1N (17, 20, 23, 24). In this work, we provide evidence that the μ1 C-terminal fragment, Φ, also facilitates membrane disruption. In vitro, cleavage at the δ-Φ junction modulates pore forming activity, particle recruitment, and infectivity. Together, these results provide further evidence that reovirus utilizes two distinct, membrane-penetrating peptides to invade host cells. The requirement for two peptides to bypass membranes bears a striking resemblance to the entry mechanism utilized by picornaviruses (e.g., poliovirus). Poliovirus undergoes a conformational change that culminates in the release of capsid protein VP4 (9, 11, 13). Reovirus μ1N and poliovirus VP4 display common features: limited sequence similarity, an N-terminal myristoyl moiety, and cleavage at the C-terminal side of an asparagine residue (22). Moreover, poliovirus capsid protein VP1, like reovirus Φ, is predicted to interact with host membranes through an N-terminal, amphipathic α-helix (10). The combined activities of VP4 and VP1 are thought to form a channel that facilitates RNA delivery (8, 12). Despite similar entry mechanisms, reovirus and poliovirus deliver distinctly different cargos (i.e., core particle and genomic RNA, respectively). For each system, the events that follow pore formation and the events the promote payload delivery are poorly understood and warrant further investigation.

MATERIALS AND METHODS

Cells and viruses.Murine L929 (L) cells were grown at 37°C in Joklik's minimal essential medium (Lonza, Walkersville, MD) supplemented with 5% fetal bovine serum (FBS; Life Technologies, Carlsbad, CA), 2 mM l-glutamine (Invitrogen, Carlsbad, CA), 100 U/ml of penicillin (Invitrogen), 100 μg/ml of streptomycin (Invitrogen), and 25 ng/ml of amphotericin B (Sigma-Aldrich, St. Louis, MO). All virus strains used in this study were derived from reovirus type 3 Dearing (T3D) and reovirus type 1 Lang (T1L) and were generated by plasmid-based reverse genetics (80, 81). Mutations within the T3D M2 gene were generated by QuikChange site-directed mutagenesis (Agilent Technologies, Santa Clara, CA) with the following primer pair: forward primer 5′-GCAACTCGAAACTGGAGCTGGTGTGCGAATATTC-3′ and reverse primer 5′-GAATATTCGCACACCAGCTCCAGTTTCGAGTTGC-3′.

Virus purification.Recombinant reovirus strains T1L/T3D M2 and T1L/T3D M2 Y581A, which all contain a wild-type or mutated T3D M2 gene in an otherwise T1L background, were propagated and purified as previously described (80, 81). Briefly, L cells infected with second-passage reovirus stocks were lysed by sonication. Virus particles were extracted from lysates using Vertrel-XF specialty fluid (Dupont, Wilmington, DE) (82). The extracted particles were layered onto 1.2- to 1.4-g/cm3 CsCl step gradients. The gradients were then centrifuged at 187,000 × g for 4 h at 4°C. Bands corresponding to purified virus particles (∼1.36 g/cm3) (83) were isolated and dialyzed into virus storage buffer (10 mM Tris [pH 7.4], 15 mM MgCl2, and 150 mM NaCl). Following dialysis, the particle concentration was determined by measuring the optical density of the purified virus stocks at 260 nm (OD260; 1 unit at OD260 = 2.1 × 1012 particles/ml) (83).

Generation of ISVPs.T1L/T3D M2 or T1L/T3D M2 Y581A virions (2 × 1012 particles/ml or 4 × 1012 particles/ml) were digested with 200 μg/ml of TLCK (Nα-p-tosyl-l-lysine chloromethyl ketone)-treated chymotrypsin (Worthington Biochemical, Lakewood, NJ) in a total volume of 100 μl for 20 min at 32°C (52, 53). After 20 min, the reaction mixtures were incubated on ice for 20 min and quenched by the addition of 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich, St. Louis, MO). The generation of infectious subviral particles (ISVPs) was confirmed by SDS-PAGE and Coomassie brilliant blue (Sigma-Aldrich) staining.

DLS.T1L/T3D M2 or T1L/T3D M2 Y581A virions or ISVPs (2 × 1012 particles/ml) were analyzed using a Zetasizer Nano S dynamic light scattering (DLS) system (Malvern Instruments, Malvern, United Kingdom). All measurements were made at room temperature in a quartz Suprasil cuvette with a 3.00-mm path length (Hellma Analytics, Mullheim, Germany). For each sample, the hydrodynamic diameter was determined by averaging readings across 15 iterations.

Assessment of σ1 levels within purified particles.T1L/T3D M2 or T1L/T3D M2 Y581A virions or ISVPs (2 × 1012 particles/ml) were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE. The levels of reovirus μ1C/δ and σ1 were determined by Western blotting using an anti-reovirus primary antibody (84) and an anti-T1L σ1 head primary antibody (85), respectively.

Plate-based cell attachment assay.Quantification of reovirus attachment was performed as previously described, with some exceptions (31). Briefly, L929 cells grown in 96-well plates (Greiner Bio-One, Kremsmunster, Austria) were chilled for 15 min at 4°C. The chilled cells were adsorbed with the concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions or ISVPs indicated in the figure legend for 1 h at 4°C. When indicated, the cells were treated with 20 mM ammonium chloride prior to (15 min at 4°C) and during virus adsorption. After 1 h, the cells were washed three times with chilled phosphate-buffered saline (PBS) and blocked with PBS supplemented with 5% bovine serum albumin (PBS-BSA) for 10 min at 4°C. The cells were then incubated with an anti-reovirus primary antibody (84) diluted 1:2,500 into PBS-BSA for 30 min at 4°C. The cells were washed three times with PBS-BSA, followed by incubation with an Alexa Fluor 750-labeled secondary antibody (Invitrogen, Carlsbad, CA) diluted 1:1,000 into PBS-BSA for 30 min at 4°C. After two washes with PBS-BSA, total cells were labeled with a 1:1,000 dilution of DRAQ5 (Cell Signaling Technology, Danvers, MA) for 5 min at 4°C. The cells were washed three times with PBS-BSA and then fixed with 4% formaldehyde for 20 min at room temperature. Fixed plates were scanned using an Odyssey imaging system (LI-COR, Lincoln, NE). The binding index was quantified by the ratio of green (attached virus) and red (total cells) fluorescence using Image Studio Lite software (LI-COR).

Assessment of antibody reactivity.High-affinity-binding polystyrene plates (Thermo Fisher Scientific, Waltham, MA) were coated with the concentrations of T1L/T3D M2 or T1L/T3D M2 Y581A virions or ISVPs indicated in the figure legend for 18 h at 4°C. Prior to binding, each virus was diluted into 0.1 M carbonate-bicarbonate buffer (pH 9.5). Plates with bound virus were blocked with virus storage buffer (see above) supplemented with 2.5% BSA (VB-BSA) for 1 h at 4°C and then washed three times with wash buffer (10 mM Tris [pH 7.4], 15 mM MgCl2, 150 mM NaCl, 0.1% BSA, and 0.05% Tween). The plates were incubated with an anti-reovirus primary antibody (84) diluted 1:5,000 into VB-BSA for 1 h at room temperature. The plates were washed three times with wash buffer, followed by incubation with an Alexa Fluor 750-labeled secondary antibody (Invitrogen) diluted 1:1,000 into VB-BSA for 1 h at room temperature. Plates were washed three times with wash buffer and scanned using an Odyssey imaging system (LI-COR).

Normalization of attachment units.L929 cells grown in 24-well plates (Greiner Bio-One) were chilled for 15 min at 4°C. The chilled cells were adsorbed with T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) virions or ISVPs for 1 h at 4°C. When indicated, the cells were treated with 20 mM ammonium chloride prior to (15 min at 4°C) and during virus adsorption. After 1 h, the cells were washed three times with chilled PBS and lysed with Tri Reagent (Molecular Research Center, Cincinnati, OH). Total RNA was extracted from cell lysates by following the manufacturer's instructions. For quantitative reverse transcription-PCR (qRT-PCR), 1 μg of RNA was reverse transcribed using the High Capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA) and gene-specific primers against the T1L S2 gene segment (5′-ATGGCTTCGTACGGAACATC-3′) and murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (5′-GGATGCAGGGATGATGTTCT-3′). The cDNA was diluted 1:10 into ultrapure H2O, mixed with forward and reverse detection primers (T1L S2 forward [5′-AAGCGTTGGCAGATCAAACT-3′] and T1L S2 reverse [5′-ATGGCTTCGTACGGAACATC-3′] or GAPDH forward [5′-ACCCAGAAGACTGTGGATGG-3′] and GAPDH reverse [5′-GGATGCAGGGATGATGTTCT-3′]) and SYBR Select master mix (Applied Biosystems), and then subjected to PCR using the StepOnePlus real-time PCR system (Applied Biosystems). Multiple qRT-PCR measurements were made for each sample. ΔCT values were calculated by subtracting the threshold cycle (CT) values of GAPDH from the CT values of the T1L S2 gene segment. Relative attachment with respect to the control sample (i.e., T1L/T3D M2) was quantified using the ΔΔCT method (86).

Assessment of particle internalization.Assessment of reovirus internalization was performed as previously described, with some exceptions (66). L929 cells grown in 24-well plates (Greiner Bio-One) were chilled for 15 min at 4°C. The chilled cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) virions or ISVPs for 1 h at 4°C. After 1 h, the cells were washed three times with chilled PBS and incubated in growth medium at 37°C. When indicated, the cells were treated with 20 mM ammonium chloride prior to (15 min at 4°C), during, and after virus adsorption. At the times postinfection indicated on the figure, the cells were washed three times with PBS and labeled with 1 mM sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-biotin) (Thermo Fisher Scientific). The biotinylation reagent was prepared in PBS immediately before use. The infected monolayers were incubated with the biotinylation reagent for 30 min at room temperature. The biotinylated cells were washed twice with PBS and once with virus storage buffer and then lysed with RIPA buffer (50 mM Tris [pH 7.5], 50 mM NaCl, 1% Triton X-100, 1% deoxycholate [DOC], 0.1% SDS, and 1 mM EDTA). The cell lysates were incubated with streptavidin agarose resin (SAR; Thermo Fisher Scientific) for 1 h at 4°C with rocking. The SAR was equilibrated in RIPA buffer immediately before use. After binding, the SAR was sedimented at 2,500 × g for 2 min at 4°C and then washed with RIPA buffer; this step was repeated for a total of 10 washes. After washing, the SAR was resuspended in reducing SDS sample buffer and boiled for 10 min at 95°C to elute bound proteins. The input lysates and eluted proteins were analyzed by SDS-PAGE. The levels of reovirus μ1C/δ and host protein tubulin were determined by Western blotting using an anti-reovirus primary antibody (84) and an anti-alpha-tubulin (B-5-1-2) primary antibody (Sigma-Aldrich), respectively.

Assessment of in cell particle disassembly.L929 cells grown in 24-well plates (Greiner Bio-One) were chilled for 15 min at 4°C. The chilled cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) virions or ISVPs for 1 h at 4°C. After 1 h, the cells were washed three times with PBS and incubated in growth medium at 37°C. When indicated, the cells were treated with 20 mM ammonium chloride prior to (15 min at 4°C), during, and after virus adsorption. At the times postinfection indicated on the figure, the infected monolayers were washed three times with PBS and lysed with RIPA buffer. The cell lysates were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE. The levels of reovirus μ1C/δ and host protein tubulin were determined by Western blotting using an anti-reovirus primary antibody (84) and anti-alpha-tubulin (B-5-1-2) primary antibody (Sigma-Aldrich), respectively.

Liposome preparation.The lipids used in this study (l-α-phosphatidylcholine [PC] from chicken egg, sphingomyelin [SM] from porcine brain, cholesterol [Chl] from ovine wool, l-α-phosphatidylethanolamine [PE] from chicken egg, l-α-phosphatidylserine [PS] from porcine brain, and lysobisphosphatidic acid [LBPA]) were purchased from Avanti Polar Lipids (Alabaster, AL). All lipids were dissolved in chloroform and stored at −20°C. Prior to liposome preparation, the lipids were dried under a stream of argon gas. Liposomes at 1 mM were prepared by resuspending the dried lipids in 250 μl of virus storage buffer and passing the resuspension (31 times) through an Avanti Mini Extruder with a 0.1-μm-pore size polycarbonate membrane (Avanti Polar Lipids). The composition of early endosome (EE) liposomes was Chl-PC-PE-SM-PS-LBPA (50:26:13:5:5:1 molar ratio) (60, 68).

Thermal-inactivation and trypsin sensitivity assays.T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs (2 × 1012 particles/ml) were incubated in the absence or presence of 1 mM EE liposomes for 20 min at the temperatures indicated on the figure in a Bio-Rad S1000 thermal cycler (Hercules, CA). When specified, ISVP* supernatant (15 μl of supernatant per 30 μl of total reaction volume) was included. The total volume of each reaction was 30 μl in virus storage buffer. For each reaction condition, an aliquot was also incubated for 20 min at 4°C. Following incubation, 10 μl of each reaction was diluted into 40 μl of ice-cold virus storage buffer, and infectivity was determined by plaque assay. The change in infectivity at a given temperature (T) was calculated using the following formula: log10(PFU/ml)T − log10(PFU/ml)4°C. Under each reaction condition, the titers of the 4°C control samples were between 5 × 109 and 5 × 1010 PFU/ml. The remaining 20 μl of each reaction was treated with 0.08 mg/ml of trypsin (Sigma-Aldrich) for 30 min on ice. Following digestion, equal particle numbers from each reaction were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE. The gels were Coomassie brilliant blue (Sigma-Aldrich) stained and imaged on an Odyssey imaging system (LI-COR).

Plaque assays.Plaque assays to determine infectivity were performed as previously described, with some exceptions (58, 87). Briefly, control or heat-treated virus samples were diluted into PBS supplemented with 2 mM MgCl2 (PBSMg). L cell monolayers in 6-well plates (Greiner Bio-One) were infected with 250 μl of diluted virus for 1 h at room temperature. Following the viral attachment incubation, the monolayers were overlaid with 4 ml of serum-free medium 199 (Sigma-Aldrich) supplemented with 1% Bacto agar (BD Biosciences), 10 μg/ml of TLCK-treated chymotrypsin (Worthington Biochemical), 2 mM l-glutamine (Invitrogen), 100 U/ml of penicillin (Invitrogen), 100 μg/ml of streptomycin (Invitrogen), and 25 ng/ml of amphotericin B (Sigma-Aldrich). The infected cells were incubated at 37°C, and plaques were counted 5 days postinfection.

Generation of ISVP* supernatant.The supernatant of preconverted ISVP*s was generated as previously described. Briefly, input T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs (2 × 1012 particles/ml) were incubated for 5 min at 52°C. The heat-inactivated virus (no spin) was then centrifuged at 16,000 × g for 10 min at 4°C to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T1L/T3D M2 ISVPs for thermal-inactivation reactions. To ensure that intact ISVPs did not contaminate the transferred ISVP* supernatant, aliquots of the no-spin and spin reactions were analyzed for residual infectivity by plaque assay and for the presence of μ1C/δ by Western blotting using an anti-reovirus primary antibody (84).

ISVP-induced hemolysis and PEG protection assay.Citrated bovine red blood cells (RBCs) (Colorado Serum Company, Denver, CO) were pelleted for 5 min by centrifugation at 500 × g and resuspended in ice-cold PBSMg. This step was repeated until the supernatant remained clear after pelleting. After being washed, the RBCs were resuspended in PBSMg at a 30% (vol/vol) concentration. Hemolysis efficiency was determined by incubating a suboptimal number of T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs (2 × 1012 particles/ml) in a 3% (vol/vol) solution of RBCs. When indicated, the reactions were supplemented with 30 mM PEG (Sigma-Aldrich). Levels of 0 and 100% hemolysis were determined by incubating an equivalent number of RBCs in virus storage buffer alone or virus storage buffer supplemented with 0.8% Triton X-100, respectively. The samples were incubated for 1 h at 37°C. Following incubation, the reaction mixtures were placed on ice for 20 min, followed by centrifugation for 5 min at 500 × g. To determine the amount of hemoglobin released, the supernatants were diluted 1:5 into virus storage buffer and the A405 was measured using a microplate reader (Molecular Devices, Sunnyvale, CA). Percent hemolysis was calculated using the following formula: [(Asample − Abuffer)/(ATX-100 − Abuffer)] × 100, where Abuffer contained buffer and RBCs and ATX-100 contained buffer, RBCs, and Triton X-100.

Generation of CF-loaded liposomes.Carboxyfluorescein (CF)-loaded liposomes were generated as previously described, with some exceptions (24). Briefly, CF was purchased from Acros Organics (Geel, Belgium). CF-loaded liposomes were prepared following the liposome preparation protocol described above, with the exception that dried lipids were resuspended in 250 μl of CF solution (22 mg/ml). CF solution was prepared by mixing CF in virus storage buffer and adding drops of 10 M NaOH until the mixture became clear. Following extrusion, CF-loaded liposomes were separated from unincorporated dye using 10DG desalting columns (Bio-Rad, Hercules, CA) that had been equilibrated with virus storage buffer following the manufacturer's protocol. Fractions of 0.5 ml were collected by elution with virus storage buffer. Fractions containing CF-loaded liposomes were identified by diluting 1 μl of each fraction into 99 μl of virus storage buffer alone or virion storage buffer supplemented with 0.5% Triton X-100 and measuring the fluorescence (excitation at 485 nm and emission at 528 nm) using a Synergy H1 hybrid plate reader (BioTek, Winooski, VT).

CF-loaded liposome pore formation assay.T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs (2 × 1012 particles/ml) were incubated with 10 μl of CF-loaded EE liposomes (final reaction volume, 20 μl) for 20 min at the temperatures indicated on the figure. Levels of 0 and 100% CF leakage were determined by incubating equivalent volumes of CF-loaded EE liposomes in virus storage buffer alone or virion storage buffer supplemented with 0.5% Triton X-100, respectively. After 20 min, the reaction mixtures were diluted 1:50 into virus storage buffer. The samples were allowed to equilibrate for 15 min at room temperature, and the fluorescence (excitation at 485 nm and emission at 528 nm) was measured using a Synergy H1 hybrid plate reader (BioTek). The percent CF leakage was calculated using the following formula: [(Asample − Abuffer)/(ATX-100 − Abuffer)] × 100, where Abuffer contained buffer and CF-loaded EE liposomes and ATX-100 contained buffer, CF-loaded EE liposomes, and Triton X-100.

Virus-liposome coflotation assay.T1L/T3D M2 or T1L/T3D M2 Y581A ISVPs (2 × 1012 particles/ml) were incubated in the absence or presence of EE liposomes for 20 min at the temperatures indicated on the figure. The samples were then applied to the tops of 15 to 60% linear sucrose gradients (mass/volume in virus storage buffer) and sedimented at 77,000 × g for 2 h at 4°C in an SW 41 Ti rotor (Beckman Coulter, Brea, CA). Following ultracentrifugation, 1-ml fractions were collected from the tops of the gradients and solubilized in reducing SDS sample buffer. Equal volumes from each fraction were analyzed by SDS-PAGE. The gels were analyzed for the presence of μ1C/δ by Western blotting using an anti-reovirus primary antibody (84).

Assessment of infectivity by initiation of protein synthesis.L929 cells grown in 24-well plates (Greiner Bio-One) were chilled for 15 min at 4°C. The chilled cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) ISVPs for 1 h at 4°C. After 1 h, the cells were washed three times with PBS and incubated in growth medium at 37°C. When indicated, the cells were treated with 20 mM ammonium chloride prior to (15 min at 4°C), during, and after virus adsorption. At the times postinfection indicated on the figure, the infected monolayers were washed three times with PBS and lysed with RIPA buffer. The cell lysates were solubilized in reducing SDS sample buffer and analyzed by SDS-PAGE. The levels of reovirus σNS and the PSTAIR epitope of the host protein Cdk1 were determined by Western blotting using an anti-σNS primary antibody (88) and an anti-PSTAIR primary antibody (Sigma-Aldrich), respectively.

Assessment of infectivity by indirect immunofluorescence.L929 cells grown in 96-well plates (Greiner Bio-One) were chilled for 15 min at 4°C. The chilled cells were adsorbed with equivalent attachment units of T1L/T3D M2 (1.0 × 103 particles/cell) or T1L/T3D M2 Y581A (0.4 × 103 particles/cell) ISVPs for 1 h at 4°C. After 1 h, the cells were washed three times with PBS and incubated in growth medium for 18 h at 37°C. When indicated, the cells were treated with 20 mM ammonium chloride prior to (15 min at 4°C), during, and after virus adsorption. The infected monolayers were fixed with 100 μl of methanol for 30 min at −20°C, washed three times with PBS, and blocked with PBS supplemented with 2.5% BSA and 0.25% Triton X-100 for 30 min at room temperature. The blocked cells were incubated with an anti-reovirus primary antibody (84) diluted 1:5,000 into PBS supplemented with 0.25% Triton X-100 for 30 min at 37°C. The cells were washed three times with PBS, followed by incubation with an Alexa Fluor 488-labeled secondary antibody (Invitrogen) diluted 1:5,000 into PBS supplemented with 0.25% Triton X-100 for 30 min at 37°C. The cells were washed three times with PBS and stained with 0.5 μg/ml of 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) diluted 1:10,000 into PBS. The monolayers were washed three times with PBS, and infected cells were visualized by indirect immunofluorescence using a fluorescein isothiocyanate (FITC)/DAPI filter set on an Olympus 1X71 microscope (Olympus, Center Valley, PA). Infected cells were identified by intense cytoplasmic fluorescence. No background staining of uninfected control monolayers was noted. Reovirus antigen-positive cells were quantified by counting fluorescent cells in random fields in triplicate wells at a magnification of ×20. Percentages of reovirus-positive cells were calculated by counting infected cells per total (DAPI-positive) cells.

Statistical analyses.The reported values represent the means from three independent biological replicates. Error bars indicate standard deviations. P values were calculated using Student's t test (two-tailed; unequal variance assumed).

ACKNOWLEDGMENTS

We thank members of our laboratory and the Indiana University Bloomington virology community for helpful suggestions. Dynamic light scattering was performed in the Indiana University Bloomington Physical Biochemistry Instrumentation Facility.

Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under award numbers 1R01AI110637 (to P.D.) and F32AI126643 (to A.J.S.) and by Indiana University Bloomington.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the funders.

FOOTNOTES

    • Received 23 October 2017.
    • Accepted 20 December 2017.
    • Accepted manuscript posted online 3 January 2018.

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KEYWORDS

reovirus
cell entry
membrane penetration
nonenveloped virus
pore-forming peptides

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