Lipids Cooperate with the Reovirus Membrane Penetration Peptide to Facilitate Particle Uncoating

Virus-host interactions play a role in many stages of the viral lifecycle, including entry. Reovirus, a model system for studying the entry mechanisms of nonenveloped viruses, undergoes a series of regulated structural transitions that culminate in delivery of the viral genetic material. Lipids can trigger one of these conformational changes, infectious subviral particle (ISVP)-to-ISVP* conversion. ISVP* formation releases two virally encoded peptides, myristoylated μ1N (myr-μ1N) and Φ. Among these, myr-μ1N is sufficient to form pores within membranes. Released myr-μ1N can also promote ISVP* formation in trans. Using thermal inactivation as a readout for ISVP-to-ISVP* conversion, we demonstrate that lipids render ISVPs less thermostable in a virus concentration-dependent manner. Under conditions in which neither lipids alone nor myr-μ1N alone promotes ISVP-to-ISVP* conversion, myr-μ1N induces particle uncoating when lipids are present. These data suggest that the pore-forming activity and the ISVP*-promoting activity of myr-μ1N are linked. Lipid-associated myr-μ1N interacts with ISVPs and triggers efficient ISVP* formation. The cooperativity between a reovirus component and lipids reveals a distinct virus-host interaction in which membranes can facilitate nonenveloped virus entry.

This study investigates a mechanism for lipid-and myr-1Nmediated reovirus entry. Following cleavage and release from 1, myr-1N may serve two functions: (i) myr-1N generates pores within the host endosomal membrane (33,36,40) and (ii) myr-1N induces ISVP-to-ISVP* conversion in trans (46). We propose a model in which the pore-forming activity and the ISVP*-promoting activity are linked. First, myr-1N promotes ISVP* formation most efficiently when lipids are present. Second, the addition of exogenous myr-1N to a low concentration of ISVPs alters the reaction kinetics of ISVP-to-ISVP* conversion. Third, host proteins are not required for myr-1N to recruit ISVPs to a lipid bilayer. Thus, membrane-embedded myr-1N interacts with ISVPs and induces ISVP-to-ISVP* conversion. This work reveals a heretofore unknown mechanism in which membranes can actively participate in the entry pathway of a nonenveloped virus.

Results
Susceptibility of T3D ISVPs to Lipid-mediated Thermal Inactivation Is Particle Concentration-dependent-ISVPs undergo multiple structural transitions to generate ISVP*s. These conformational changes occur within an infected cell and are necessary for establishing a productive infection (35,44,45). Triggering ISVP-to-ISVP* conversion in vitro with heat renders the particles noninfectious (44). At high particle concentration (2 ϫ 10 12 particles/ml or 3 nM), liposomes facilitate ISVP* formation in a lipid composition-dependent manner (9). To test whether lipids also render reovirus particles noninfectious, we conducted 20-min heat inactivation experiments over a range of temperatures in the absence or presence of early endosome (EE), L-␣-phosphatidylcholine (PC), or PC/L-␣-phosphatidylethanolamine (PE) (2:1) liposomes. As described previously, liposomes composed of PC/PE induce ISVP-to-ISVP* conversion as efficiently as EE liposomes. Liposomes composed of PC alone can also trigger ISVP* formation, albeit to a lesser extent (9). At high particle concentration (Fig. 1A), reovirus type 3 Dearing (T3D) ISVPs were more susceptible to thermal inactivation in the presence of liposomes when compared with T3D ISVPs alone. At 43°C, T3D ISVPs incubated with EE or PC/PE (2:1) liposomes were reduced in titer by ϳ4.5 log 10 units relative to control virus incubated at 4°C. Consistent with a mechanism in which liposomes promote ISVP* formation in a lipid composition-dependent manner (9), T3D ISVPs incubated with PC liposomes were less susceptible to thermal inactivation when compared with virus incubated with EE or PC/PE (2:1) liposomes; viral titer was reduced by less than 0.5 log 10 unit at 43°C and by ϳ4 log 10 units at 46°C. T3D ISVPs alone were reduced in titer by only ϳ2 log 10 units following incubation at 49°C.
As a consequence of ISVP* formation, the reovirus outer capsid protein, 1, reorganizes into a protease-sensitive conformation (35). After incubating T3D ISVPs in the absence or presence of liposomes under conditions identical to Fig. 1A, this structural transition was assayed by determining the susceptibility of the ␦ fragment (a product of 1 cleavage during ISVP-to-ISVP* conversion) to trypsin digestion (Fig. 1B) (35,44). Consistent with the thermal inactivation curves shown in Fig.  1A, inclusion of EE, PC, or PC/PE (2:1) liposomes reduced the temperature that was required to render ␦ protease-sensitive.
At the high particle concentration used in Fig. 1, A and B (2 ϫ 10 12 particles/ml or 3 nM), ISVP* formation induced by heat does not follow first-order kinetics (34, 35, 46 -48). In contrast, ISVP-to-ISVP* conversion at low particle concentration (e.g. 2 ϫ 10 9 particles/ml or 0.003 nM) has been shown to approximate a first-order reaction (44,46). As such, by measuring the rate of infectivity loss at low particle concentration, three thermodynamic parameters can be calculated: enthalpy of activation (⌬H ‡ ), entropy of activation (⌬S ‡ ), and free energy of activation (⌬G ‡ ) (44,49). To test the hypothesis that lipids facilitate ISVP* formation by lowering the energy barrier of the reaction, T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) were incubated for 20 min over a range of temperatures in the absence or presence of EE, PC, or PC/PE (2:1) liposomes (Fig. 1C). At low particle concentration (i.e. conditions that are more likely to arise during infection), we expected ISVP-to-ISVP* conversion to occur most efficiently when lipids were present. Nonetheless, T3D ISVPs alone and T3D ISVPs incubated with liposomes were equally susceptible to thermal inactivation at 42, 45, and 55°C. At 49 and 52°C, virus incubated with liposomes was reduced in titer to a greater extent than virus alone; however, the difference in infectivity was less than 1 log 10 unit. Similar results were obtained using the trypsin sensitivity assay (Fig.  1D). At 49°C, the 1 ␦ fragment was only marginally more sensitive to trypsin digestion in virus incubated with liposomes when compared with virus alone.
To further quantify the effects of lipids on ISVP-to-ISVP* conversion (i.e. loss of infectivity), kinetic analyses were performed. T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) were incubated in the absence or presence of EE, PC, or PC/PE (2:1) liposomes at constant temperatures. Under each reaction condition, change in infectivity decreased linearly with respect to time (Fig. 2, plots on the left side of each panel); liposomes did not alter the first-order kinetics of ISVP* formation at low particle concentration. The slopes of the inactivation curves were used to generate Arrhenius plots (Fig. 2, plots on the right side of each panel) (44). ⌬H ‡ and ⌬S ‡ were then calculated from the slope and the y-intercept of the Arrhenius plots using the Eyring absolute equation (49). ⌬G ‡ was calculated for each reaction condition at 48°C using the following formula: ⌬G ‡ ϭ ⌬H ‡ Ϫ T ⌬S ‡ (Table 1) (44,49). The thermodynamic values derived from these reactions were not significantly different in the absence or presence of liposomes. These results demonstrate that lipids promote T3D ISVP thermal inactivation (i.e. ISVP-to-ISVP* conversion) only under conditions in which the particle concentration is high.
Lipids Enhance the ISVP-to-ISVP*-promoting Activity of Preconverted ISVP* Supernatant-The observation that liposomes promote thermal inactivation at high particle concentration, but not at low particle concentration, suggests that one or more of the reaction products interacts with one or more of the input components to drive the reaction forward (i.e. the reaction does not follow first-order kinetics). ISVP-to-ISVP* conversion culminates in the release of at least three reovirus-derived frag-ments: myr-1N, ⌽, and 1 (11,(33)(34)(35)(36)(37)(38)(39). Among these, myr-1N is sufficient to generate 4 -10-nm pores in target membranes (e.g. liposomes and bovine RBCs) (33,36,40) and to induce ISVP* formation in trans; however, its promoting activity is also enhanced by ⌽ (46). To determine whether the released fragments can facilitate lipid-mediated thermal inactivation, T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) supplemented with the supernatant of preconverted ISVP*s were incubated for 20 min over a range of temperatures in the absence or presence of EE, PC, or PC/PE (2:1) liposomes. ISVP* supernatant was generated by heating input T3D ISVPs for 5 min at 52°C (36,46). The reactions were then centrifuged to pellet particles, and the supernatant was transferred to tubes containing target ISVPs. The supernatant (spin) was analyzed by plaque assay (Fig. 3A) and by Western blotting (Fig. 3B) to confirm the clearance of input virus. As expected, virus incubated with ISVP* supernatant, which contains released myr-1N, ⌽, and 1 (36), was more susceptible to thermal inactivation than virus incubated without ISVP* supernatant (compare Figs. 1C and 3C). Furthermore, the released fragments functioned more efficiently in the presence of liposomes. At 46°C, virus incubated with ISVP* supernatant and EE liposomes was A and C, lipid-mediated thermal inactivation curves. T3D ISVPs at 2 ϫ 10 12 particles/ml (A) or 2 ϫ 10 9 particles/ml (C) were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) 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 mean Ϯ S.D., *, p Յ 0.01 (n ϭ 3 independent replicates for each reaction condition). B and D, liposome-mediated ISVP* formation. T3D ISVPs at 2 ϫ 10 12 particles/ml (B) or 2 ϫ 10 9 particles/ml (D) were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) 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. In panel B, the gels were Coomassie Brilliant Blue-stained. In panel D, the gels were analyzed for the presence of the 1 ␦ fragment by Western blotting (n ϭ 3 independent replicates for each reaction condition, one representative experiment is shown). reduced in titer by ϳ3 log 10 units, whereas virus incubated with ISVP* supernatant alone was reduced in titer by ϳ1 log 10 unit.
The thermal inactivation results were confirmed using the trypsin sensitivity assay (Fig. 3D). In the presence of liposomes, ISVP* supernatant induced ISVP-to-ISVP* conversion more efficiently (i.e. triggered reorganization of the 1 ␦ fragment at lower temperatures) than ISVP* supernatant that was incubated with virus alone. These results suggest that lipids potentiate the in trans promoting activity of one or more of the released fragments. T3D ISVPs at 2 ϫ 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes at the indicated temperatures. At the indicated time points, the change in infectivity relative to samples incubated at 4°C was determined by plaque assay (n ϭ 2 independent replicates for each reaction condition, one experiment is shown). A-D, plots on the right side of each panel, Arrhenius plots of lipid-mediated thermal inactivation. The rate constant, k, was determined for each thermal inactivation curve as described previously (44). The rate constants were plotted as ln(k/T) versus the reciprocal of the incubation temperature (T) in degrees Kelvin (n ϭ 2 independent replicates for each reaction condition, one experiment is shown).
The ISVP-to-ISVP*-promoting activity of myr-1N has been described previously (46). myr-1N cleavage and release were required for ISVP* supernatant to retain promoting activity and for ISVP* formation to occur in a virus concentration-dependent manner. To determine whether lipids enhance myr-1Nmediated ISVP* formation, T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) supplemented with a synthetic myr-1N peptide were incubated for 20 min at 42°C in the absence or presence of EE liposomes (Fig. 4B). As expected, myr-1N induced particle inactivation in a concentration-dependent manner. Promoting activity plateaued at 2.5 M peptide, which corresponds to the release of all 600 copies of myr-1N from each particle in ϳ3 ϫ 10 12 particles/ml (46). Moreover, T3D ISVPs incubated with 2.5 M myr-1N and EE liposomes were reduced in titer by ϳ2.5 log 10 units, whereas virus incubated with 2.5 M peptide alone was reduced in titer by ϳ1.5 log 10 units. We obtained similar results when the myr-1N peptide was incubated with a different prototype reovirus strain, T1L (data not shown).
Next, T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) supplemented with 2.5 M myr-1N were incubated for 20 min over a range of temperatures in the absence or presence of EE, PC, or PC/PE (2:1) liposomes. T3D ISVPs incubated with myr-1N and liposomes were reduced in titer to a greater extent than virus incubated with myr-1N alone (Fig. 4C). Moreover, T3D ISVPs incubated with myr-1N and PC liposomes inactivated less efficiently than virus incubated with myr-1N and EE or PC/PE (2:1) liposomes. The thermal inactivation results were confirmed using the trypsin sensitivity assay (Fig. 4D). In the presence of liposomes, myr-1N induced ISVP* formation at a lower temperature than myr-1N incubated with virus alone. Together, these data demonstrate that lipids promote ISVP-to-ISVP* conversion by maximizing the in trans promoting activity of released myr-1N.
Addition of Exogenous myr-1N Alters the Reaction Kinetics of T3D ISVP Thermal Inactivation-At high particle concentration, ISVP-to-ISVP* conversion does not follow first-order kinetics (34, 35, 46 -48). Given the observation that myr-1N promotes thermal inactivation ( Fig. 4) (46), we hypothesized that a low concentration of T3D ISVPs incubated with a high concentration of the trans-activating factor myr-1N would lose infectivity following non-first-order kinetics. T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) supplemented with 2.5 M myr-1N were incubated in the absence or presence of EE liposomes at constant temperatures. When liposomes were included in the reactions, the decrease in infectivity was biphasic: an initial, fast phase and a second, slower phase (Fig. 5B). The reactions did not obey first-order kinetics. Thus, the addition of myr-1N to the virus-liposome mixtures was sufficient to alter the kinetics of T3D ISVP thermal inactivation. In the absence of liposomes, the decrease in infectivity was biphasic at higher temperatures (Fig. 5A, see 50°C curve), but more linear at lower temperatures (Fig. 5A, see 44°C curve). Consistent with the results shown in Fig. 4, EE liposomes also enhanced the promoting activity of myr-1N (Fig. 5C). At 46°C, T3D ISVPs incubated with myr-1N and EE liposomes were reduced in titer by 1.5 log 10 units at ϳ15 min, whereas T3D ISVPs incubated with myr-1N alone were reduced in titer by 1.5 log 10 units at ϳ55 min. Together, these results provide direct, experimental evidence that released myr-1N is responsible for the particle concentration-dependent mechanism of ISVP-to-ISVP* conversion (i.e. the addition of exogenous myr-1N changes a first-order reaction into a non-first-order reaction) (46). ISVP* Supernatant and myr-1N Can Recruit T3D ISVPs to Liposomes-Our results demonstrate that lipids enhance the in trans promoting activity of myr-1N. Following ISVP-to-ISVP* conversion, released myr-1N and ⌽ are sufficient to form size-selective pores (4 -10 nm in diameter) within target membranes (e.g. liposomes and bovine RBCs) (33,36,40). Due to its membrane-targeting property (40), myr-1N may induce ISVP* formation most efficiently when lipid-associated. When formed within RBCs, myr-1N-generated pores can recruit reovirus ISVP*s (33,36); however, it is unclear whether RBC surface proteins are required for this interaction. Moreover, because these experiments induced ISVP-to-ISVP* conversion with heat, it is unknown whether pores can recruit ISVPs, in addition to ISVP*s, to membranes. When compared with ISVP*s, ISVPs are substantially less hydrophobic and present different portions of 1 on the surface of the particle (35).
To determine whether myr-1N can recruit reovirus to liposomes (i.e. membranes that lack host proteins), T3D ISVPs were incubated with EE, PC, or PC/PE (2:1) liposomes that were pretreated with the supernatant of preconverted ISVP*s or with 2.5 M myr-1N. The reactions were applied to sucrose gradients and sedimented by ultracentrifugation. When T3D ISVPs were mixed with liposomes alone (i.e. lacking ISVP* supernatant or myr-1N pretreatment), the 1 ␦ fragment was observed predominantly in fractions 7 and 8, similar to virus alone (Fig. 6A). Thus, virus-liposome interactions were not detected. In contrast, when T3D ISVPs were mixed with liposomes that were preincubated with ISVP* supernatant (Fig. 6B) or with myr-1N (Fig. 6C), the 1 ␦ fragment was observed predominantly in fractions 1-8; virus-liposome interactions prevented T3D ISVPs from fully sedimenting through the gradient. Furthermore, ISVP*s display a unique sedimentation profile. T3D ISVPs were converted to ISVP*s with heat in the absence or presence of liposomes. Following ultracentrifuga-  DECEMBER 23, 2016 • VOLUME 291 • NUMBER 52 tion, the majority of the 1 ␦ fragment was observed in fraction 12 (Fig. 6D). Together, these results suggest that myr-1N alone can recruit T3D ISVPs to membranes. Thus, we conclude that the pore-forming activity and the ISVP*-promoting activity of myr-1N are linked. Lipids facilitate ISVP-to-ISVP* conversion (Figs. 4 and 5) by increasing the efficiency of ISVP-myr-1N interactions.

Discussion
There is an increasing body of evidence that host lipids can play an active role in multiple stages of nonenveloped virus entry, including internalization and pore formation (7)(8)(9). For reovirus, membranes facilitate particle uncoating (i.e. ISVP-to-ISVP* conversion) in a lipid composition-dependent manner (9). In this work, we explore a potential mechanism by which lipids cooperate with reovirus to promote viral entry. Using thermal inactivation as a readout for ISVP-to-ISVP* conversion, we show that liposomes facilitate this conformational change at high particle concentration, but not at low particle concentration (Figs. 1 and 2). The concentration dependence is due to myr-1N (Figs. 4 and 5), a virally encoded peptide that is released from reovirus during entry (11, 33-39) and promotes particle inactivation through a positive-feedback loop (46).
Notably, myr-1N triggers ISVP-to-ISVP* conversion most efficiently when liposomes are present (Figs. 4 and 5). Moreover, host proteins are not required for myr-1N to recruit ISVPs to membranes (Fig. 6). Finally, under conditions in which thermal inactivation approximates first-order kinetics, we show that lipids alone do not affect the free energy of activation ( Fig. 2 and Table 1); however, the first-order behavior is altered by the inclusion of exogenous myr-1N (Fig. 5). This observation further supports the conclusion that myr-1N functions as an in trans promoting factor to induce ISVP-to-ISVP* conversion (46). Together, our results provide experimental evidence that lipids can facilitate reovirus entry by interacting with, and enhancing the ISVP*-promoting activity of, myr-1N.
At high particle concentration, reovirus ISVPs are susceptible to lipid-mediated ISVP-to-ISVP* conversion (Fig. 1). Even within the limited volume of an endosome, the virus concentration used in these experiments (2 ϫ 10 12 particles/ml or 3 nM) cannot be achieved biologically. There are at least two nonmutually exclusive strategies to overcome this limitation: (i) perform the reaction under conditions in which the concentration of input components can be reduced without affecting output and (ii) sequester the input components within a confined space. At low particle concentration (2 ϫ 10 9 particles/ml or Input T3D ISVPs at 2 ϫ 10 12 particles/ml were incubated at 52°C for 5 min. The heat-inactivated virus (No spin) was centrifuged to pellet particles. The supernatant (Spin) was immediately transferred to tubes containing target T3D 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 the 1 ␦ fragment by Western blotting (B). In panel A, data are presented as mean Ϯ S.D., *, p Յ 0.01 (n ϭ 3 independent preparations of ISVP* supernatant were analyzed). C, ISVP* supernatant-and liposome-mediated thermal inactivation curves. T3D ISVPs at 2 ϫ 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and ISVP* supernatant 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 mean Ϯ S.D., *, p Յ 0.01 (n ϭ 3 independent replicates for each reaction condition). D, ISVP* supernatant-and liposome-mediated ISVP* formation. T3D ISVPs at 2 ϫ 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and ISVP* supernatant 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 analyzed for the presence of the 1 ␦ fragment by Western blotting (n ϭ 3 independent replicates for each reaction condition, one representative experiment is shown). 0.003 nM), inclusion of liposomes alone or myr-1N alone only marginally enhanced ISVP-to-ISVP* conversion (Figs. 1, 2, and  4). In contrast, inclusion of lipids and myr-1N significantly reduced the thermal energy that was needed to inactivate ISVPs (Figs. 4 and 5). Thus, myr-1N, in cooperation with lipids, can promote ISVP-to-ISVP* conversion under conditions that are more relevant to a bona fide infection (i.e. low particle concentration). Following cleavage and release from 1, myr-1N, which alone can perforate liposomes and bovine RBCs, is expected to oligomerize within host membranes (33,36,40). Thus, it is presumed that a high local concentration of myr-1N is generated at the site of pore formation. Moreover, lipidassociated myr-1N recruits ISVPs to membranes (Fig. 6). Together, these observations suggest that membranes can act as a scaffold to bring two input components into close proxim-ity. High local concentrations of lipid-associated myr-1N and ISVPs increase the efficiency of ISVP-to-ISVP* conversion.
Membranes facilitate particle inactivation in a lipid composition-dependent manner; liposomes that contain phosphatidylcholine and phosphatidylethanolamine trigger ISVP* formation most efficiently (9). Given the membrane-targeting potential of myr-1N (33,36,40), we speculate that certain lipids, or certain lipid formulations, position myr-1N to interact with target ISVPs. The basis for lipid specificity is unclear; however, factors such as membrane fluidity or rigidity, composition of fatty acid tails, and/or composition of lipid head groups may influence pore formation, and thus, orientation of the inserted peptide. Furthermore, the properties that affect the conformation of embedded myr-1N and the properties that affect pore formation are likely not separable. The myr-1N  figure) in the absence (white bars) or presence (gray bars) of EE liposomes for 20 min at 42°C. The change in infectivity relative to samples incubated at 4°C was determined by plaque assay. Data are presented as mean Ϯ S.D., *, p Յ 0.01 (n ϭ 3 independent replicates for each reaction condition). C, myr-1N-and liposome-mediated thermal inactivation curves. T3D ISVPs at 2 ϫ 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and 2.5 M myr-1N 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 mean Ϯ S.D., *, p Յ 0.01 (n ϭ 3 independent replicates for each reaction condition). D, myr-1N-and liposome-mediated ISVP* formation. T3D ISVPs at 2 ϫ 10 9 particles/ml were incubated in virus storage buffer supplemented with EE, PC, or PC/PE (2:1) liposomes and 2.5 M myr-1N 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 analyzed for the presence of the 1 ␦ fragment by Western blotting (n ϭ 3 independent replicates for each reaction condition, one representative experiment is shown). DECEMBER 23, 2016 • VOLUME 291 • NUMBER 52 peptide contains two regions of alternating, hydrophobic residues. When mutations are made within these regions, the capacity of myr-1N to generate pores is reduced (40); however, whether these mutated peptides can interact with each other has not been demonstrated. As such, hydrophobic forces are likely important for oligomerization of myr-1N and for myr-1N-lipid interactions. Moreover, it is unknown whether ISVPs interact exclusively with pore-associated myr-1N. Thermal inactivation is induced by myr-1N in a dose-dependent manner (Fig. 4). Thus, it is possible that ISVPs interact with multiple, pore-associated peptides to promote particle conversion.

Lipids and 1N Induce Reovirus Uncoating
We propose a model in which lipids enhance the ISVP*-promoting activity of membrane-embedded myr-1N (Fig. 7). Reovirus ISVPs may exist in reversible equilibrium with a conformationally altered form, called ISVP ⌬ (54). The capsids of nonenveloped viruses can exhibit structural flexibility (55,56). Capsid "breathing" transiently and reversibly exposes normally internal peptides on the surface of the particle. ISVP ⌬ likely represents a flexible form of the ISVP. It was suggested that thermal energy induces spontaneous ISVP ⌬ -to-ISVP* conversion (46). ISVP* formation releases myr-1N, ⌽, and 1 (11,(33)(34)(35)(36)(37)(38)(39). myr-1N, which can perforate liposomes in the absence ⌽ or 1 (33,36,40), is predicted to interact with the endosomal membrane. When RBCs are incubated with ISVP* supernatant or with myr-1N, pores that are capable of recruiting ISVP*s are formed (33,36). In the absence of host proteins, myr-1N can also recruit ISVPs to membranes (Fig. 6). Finally, specific lipids (e.g. phosphatidylcholine and phosphatidylethanolamine) enhance the ISVP*-promoting activity of myr-1N (Figs. 4 and 5). Together, these results are consistent with myr-1N-lipid interactions serving two complementary functions: recruiting ISVPs to the endosomal membrane and augmenting myr-1N-mediated ISVP-to-ISVP* conversion. Thus, lipids function within a positive-feedback loop by artificially raising the concentration of myr-1N at the site of pore formation and by positioning myr-1N to interact with ISVPs more effectively. Nonetheless, questions regarding this model remain. The myr-1N binding site on target ISVPs, including the stoichiometry of the interaction (i.e. the minimum number of myr-1N peptides that is needed to convert a single ISVP), is currently unknown. The stability of the myr-1N-ISVP interaction at physiological conditions is also unknown. As discussed earlier, reovirus ISVPs can undergo "structural breathing" to form a conformationally altered intermediate (i.e. ISVP ⌬ ) (54). This structural transition may facilitate ISVP* formation by exposing internal epitopes that interact with lipidassociated myr-1N (46,54). Next, if membranes only position myr-1N to interact with ISVPs, the initial trigger of ISVP-to-ISVP* conversion is still unclear. Partial conversion, which releases a limited quantity of myr-1N, may occur at 37°C (35,46). Thus, the endosomal membrane would be in position to amplify the ISVP*-promoting activity. These questions are the subject of ongoing investigations.
A significant question remaining is whether lipids cooperate with myr-1N to facilitate reovirus entry into host cells. At high particle concentration, PC liposomes promote ISVP-to-ISVP* conversion (Fig. 1) (9). Thus, we attempted to enzymatically deplete PC from cells with phospholipases. PC constitutes a large proportion of the plasma and endosomal membranes (40 -50% of the phospholipid content) (57). Not surprisingly, reducing PC levels was severely cytotoxic, which precluded us from assessing its impact on viral entry (data not shown). Another question is whether lipid-associated myr-1N can FIGURE 5. myr-1N alters the reaction kinetics of T3D ISVP thermal inactivation. A and B, kinetics of myr-1N-and lipid-mediated thermal inactivation. T3D ISVPs at 2 ϫ 10 9 particles/ml were incubated in virus storage buffer supplemented with 2.5 M myr-1N in the absence (A) or presence (B) of EE liposomes at the indicated temperatures. At the indicated time points, the change in infectivity relative to samples incubated at 4°C was determined by plaque assay (n ϭ at least 2 independent replicates for each reaction condition, one experiment is shown). C, the results from panels A and B at 46°C are plotted on a bar graph. Data are presented as mean Ϯ S.D., *, p Յ 0.01 (n ϭ 3 independent replicates for each reaction condition).  DECEMBER 23, 2016 • VOLUME 291 • NUMBER 52

Lipids and 1N Induce Reovirus Uncoating
induce ISVP-to-ISVP* conversion under conditions that are likely to arise during a bona fide infection. As described earlier, high local concentrations of myr-1N and ISVPs may be generated on the endosomal membrane. At low particle concentration (e.g. a single ISVP within an endosome), the inclusion of lipids alone or myr-1N alone only marginally enhanced ISVP* formation (Figs. 1, 2, and 4). In contrast, this conformational change does occur when ISVPs are incubated with liposomes and myr-1N (Figs. 4 and 5). Moreover, lipids reduce the amount of myr-1N that is needed to inactivate ISVPs (Fig. 4). Together, these results suggest that the in vitro mechanism of myr-1N-mediated ISVP-to-ISVP* conversion might be biologically relevant. Lipids facilitate entry by enhancing the ISVP*-promoting activity of myr-1N.
Viruses utilize related pathways to penetrate the host and deliver their genetic material. In most cases, enveloped viruses mediate fusion between viral and host membranes, whereas the role of lipids during entry of nonenveloped viruses is an active area of research. Our work reveals that reovirus can usurp host membranes to amplify its own triggering activity. Thus, reovirus overcomes a limiting environment in which common promoting factors (e.g. virus-receptor interactions and low pH) do not facilitate the structural transitions required for entry.
Liposome Preparation-The lipids used in this study (PC from chicken egg, sphingomyelin (SM) from porcine brain, cholesterol (Chl) from ovine wool, PE from chicken egg, L-␣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 Ϫ20°C. Prior to liposome preparation, the lipids were dried under a stream of argon gas. 1 mM liposomes were prepared by resuspending the dried lipids in 250 l of virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl) and passing the resuspension (31 times) through an Avanti Mini Extruder with a 0.1-m-pore size polycarbonate membrane (Avanti Polar Lipids). The liposome compositions used in this study were PC alone and PC/PE (2:1 molar ratio). The composition of early endosome liposomes was Chl/PC/PE/SM/PS/ LBPA (50:26:13:5:5:1 molar ratio) (9,57).
Plaque Assays-Plaque assays to determine infectivity were performed as described previously (44,60). Briefly, control or heat-treated virus samples were diluted in PBS supplemented with 2 mM MgCl 2 . L cell monolayers in 6-well plates (Greiner Bio-One, Kremsmünster, Austria) were infected with 250 l 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 g/ml TLCK-treated chymotrypsin (Worthington Biochemical), 2 mM L-glutamine (Invitrogen), 100 units/ml penicillin (Invitrogen), 100 g/ml streptomycin (Invitrogen), and 25 ng/ml amphotericin B (Sigma-Aldrich) The infected cells were incubated at 37°C, and plaques were counted 5 days after infection.
Generation of ISVP* Supernatant-The supernatant of preconverted ISVP*s was generated as described previously (36,46). Briefly, input T3D ISVPs (2 ϫ 10 12 particles/ml or 3 nM) were incubated at 52°C for 5 min. The heat-inactivated virus (no spin) was then centrifuged at 16,000 ϫ g for 10 min at 4°C to pellet particles. The supernatant (spin) was immediately transferred to tubes containing target T3D ISVPs for thermal inactivation reactions (Fig. 3) or for ISVP-liposome coflotation experiments (Fig. 6B). To ensure that intact T3D 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 the 1 ␦ fragment by Western blotting using a rabbit, ␣-reovirus polyclonal antibody (63).
Synthetic myr-1N-The myr-1N peptide was synthesized by and purchased from ABI Scientific (Sterling, VA). The peptide comprised amino acids 2-42 of T3D 1 and was modified with an N-terminal N-myristoyl group (myr). To improve solubility, the peptide contained six additional amino acids (RGKGRC) at its C terminus (40). Purified peptide was dissolved in 100% dimethylformamide. When the myr-1N peptide was added to thermal inactivation (Figs. 4 and 5) or ISVP-liposome coflotation (Fig. 6C) reactions, the final dimethylformamide concentration, including the no peptide control, was adjusted to 0.25%. Figs. 1, 3, and 4, T3D ISVPs (2 ϫ 10 12 particles/ml (3 nM) or 2 ϫ 10 9 particles/ml (0.003 nM)) were incubated in the absence or presence of 1 mM EE, PC, or PC/PE (2:1) liposomes for 20 min at the indicated temperatures in a Bio-Rad S1000 thermal cycler. When specified, ISVP* supernatant (15 l of supernatant per 30 l of total reaction volume) (Fig. 3) or myr-1N peptide (concentrations shown in Fig. 4B or 2.5 M in Fig. 4, C and D) was included. Before adding virus, ISVP* supernatant or myr-1N was mixed with liposomes, vortexed briefly, and incubated on ice for 5 min. The total volume of each reaction was 30 l in virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl). For each reaction condition, an aliquot was also incubated for 20 min at 4°C. Following incubation, 10 l of each reaction was diluted into 40 l of ice-cold virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl), and infectivity was determined by plaque assay. The change in infectivity at a given temperature (T) was calculated using the following formula: log 10 (PFU/ml) T Ϫ log 10 (PFU/ml) 4°C . Under each reaction condition at high particle concentration (2 ϫ 10 12 particles/ml or 3 nM), the titers of the 4°C control samples were between 5 ϫ 10 9 and 5 ϫ 10 10 PFU/ml. Under each reaction condition at low particle concentration (2 ϫ 10 9 particles/ml or 0.003 nM), the titers of the 4°C control samples were between 5 ϫ 10 6 and 5 ϫ 10 7 PFU/ml. The remaining 20 l of each reaction was treated with 0.08 mg/ml 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. In Fig. 1B, the gels were Coomassie Brilliant Bluestained and imaged on an Odyssey imaging system (LI-COR, Lincoln, NE). In Figs. 1D, 3D, and 4D, the reaction mixtures were analyzed for the presence of the 1 ␦ fragment by Western blotting using a rabbit, ␣-reovirus polyclonal antibody (63).

Thermal Inactivation and Trypsin Sensitivity Assays-For experiments shown in
For the experiments shown in Figs. 2 and 5, T3D ISVPs (2 ϫ 10 9 particles/ml or 0.003 nM) were incubated in the absence or presence of 1 mM EE, PC, or PC/PE (2:1) liposomes for the indicated amounts of time at the indicated temperatures in a water bath. When specified, 2.5 M myr-1N peptide was included. Before adding virus, myr-1N was mixed with liposomes, vortexed briefly, and incubated for 5 min on ice. The total volume of each reaction was 2 ml in virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl). For each reaction condition, an aliquot was also incubated at 4°C. At the indicated time points, 10 l of each reaction was diluted into 40 l of ice-cold virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl), and infectivity was determined by plaque assay. The change in infectivity at a given temperature (T) was calculated using the following formula: log 10 (PFU/ml) T Ϫ log 10 (PFU/ml) 4°C . Under each reaction condition, the titers of the 4°C control samples were between 5 ϫ 10 6 and 5 ϫ 10 7 PFU/ml. The rate constant, k, was determined for each thermal inactivation curve as described previously (44). To generate Arrhenius plots, the rate constants were plotted as ln(k/T) versus the reciprocal of the incubation temperature (T) in degrees Kelvin. The thermodynamic values shown in Table 1 were calculated using the Eyring absolute equation as described previously (44,49). ⌬G ‡ was calculated at 48°C (321 K) using the following formula: ⌬G ‡ ϭ ⌬H ‡ Ϫ T ⌬S ‡ .
ISVP-Liposome Coflotation Assay-T3D ISVPs (2 ϫ 10 12 particles/ml or 3 nM) were incubated in the absence or presence of 1 mM EE, PC, or PC/PE (2:1) liposomes for 10 min on ice (Fig.  6, A-C) or for 20 min at 49°C (Fig. 6D). When indicated, liposomes were pretreated with ISVP* supernatant (10 l of supernatant per 100 l of total reaction volume) (Fig. 6B) or with 2.5 M myr-1N peptide (Fig. 6C) for 1 min at 37°C. All reactions were performed in virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl). The samples were then applied to the top of 15-60% linear sucrose gradients (mass/volume in virus storage buffer (10 mM Tris, pH 7.4, 15 mM MgCl 2 , 150 mM NaCl)) and sedimented at 77,000 ϫ g for 2 h at 4°C in a SW 41 Ti rotor (Beckman Coulter). Following ultracentrifugation, 1-ml fractions were collected from the top 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 the 1 ␦ fragment by Western blotting using a rabbit, ␣-reovirus polyclonal antibody (63).
Statistical Analyses-Unless stated otherwise, the reported values represent the mean of three independent, biological replicates. Error bars indicate the S.D. p values were calculated using Student's t test (two-tailed, unequal variance assumed).
Author Contributions-A. J. S. and P. D. designed the study; A. J. S. performed the experiments; A. J. S. and P. D. analyzed the data; and A. J. S. and P. D. wrote the manuscript.