Enterovirus D68 capsid formation and stability requires acidic compartments

Fig 1. EV-D68 and PV display similarities in virus production kinetics and interaction with canonical autophagy players. (A) Titers of both cell-associated and extracellular PV and EV-D68 from a time course of infection. H1HeLa cells were infected at an MOI of 20. Intracellular and extracellular virus samples were collected at each hour post-infection. Viral titers were analyzed by plaque assay. (B) Impact of enterovirus D68 and poliovirus on host autophagy markers. H1HeLa cells were either untreated/mock (M) or infected with PV or EV-D68 at an MOI of 20 for 6 h, and samples were collected every hour during infection. Samples were subjected to western blot analysis for traditional autophagy markers: LC3B, p62(SQSTM1), and its Cp (cleavage product). Viral marker: VP3 (virus structural capsid protein 3). Beta-actin served as a loading control. (C–E) Intracellular EV-D68 and PV titers produced in parental H1HeLa versus KO cell lines. H1HeLa WT or KOs were infected with PV or EV-D68 at an MOI of 20 for 6 h. Viral titers were analyzed by plaque assay. KO cell lines represented: RB1CC1 KO (C), PI3K3 KO (D), and ATG7 KO(E), respectively. Unpaired Student’s t test was used for the statistical…

Fig 2. Poliovirus and EV-D68 respond differently to acidification inhibitors with major differences in virus production. (A) Left panel: titers of cell-associated PV and EV-D68 upon treatment with ammonium chloride (NH4Cl, 20 mM). H1HeLa were infected with PV or EV-D68 at an MOI of 20; after 30 min of virus adsorption at 37°C (indicated as 0 hpi time point), cells were washed twice to remove the uninternalized virus particles, then left either untreated/mock (M) or treated with ammonium 20 mM in a complete medium throughout the infection. Cells were collected at 6 hpi. Right panel: autophagy markers SQSTM1 and LC3B in mock samples of H1HeLas, untreated or treated with ammonium chloride (NH4Cl, 20 mM) for 2, 4, or 6 h, were analyzed by western blot. Beta-actin served as a loading control. The gradual accumulation of LC3B served as an indicator of blocked autophagy. (B) Titers of cell-associated PV and EV-D68 upon treatment with bafilomycin A1 (Baf A1, 0.1 µM). H1HeLa were infected with PV or EV-D68 at an MOI of 20; after 30 min of virus adsorption at 37°C (0 hpi), cells were washed two times to remove the initial virus input, left untreated, or treated with Baf1A (0.1 µM) in a complete…

Fig 3. Delay in EV-D68 production is associated with a delay in viral entry/fusion. (A) A schematic of the assays employed to investigate the effect of acidification inhibitors on the early steps of viral cycles. Except for the virucidal neutralization assay, H1HeLa cells were pre-chilled for 30 min on ice before each virus infection. All infections with PV or EV-D68 were performed at 0.1 MOI. Cells were washed two times with PBS (1×) between each incubation period to remove the residual virus after binding or before substituting drug-containing media with normal DMEM. End time points of infections were collected at 6 hpi. All intracellular titers were analyzed by plaque assay. Unpaired Student’s t test was used for the statistical analysis (***P < 0.001; **P < 0.01; *P ≤ 0.05; ns = not significant). (B) Effect of acidification inhibitors on viral entry/fusion. H1HeLa were pre-chilled before the infection of PV or EV-D68 at MOI 0.1. The virus binding/attachment step was allowed to run for 1 h at 4°C. Then the residual virus was washed at least two times and the media containing drugs at the defined concentrations (NH4Cl 20 mM, bafilomycin A 0.1 µM, CQ 100 µM) were added to cells and…

Fig 4. EV-D68 capsid production and virion maturation do not depend on cellular acidification; however, the production of several non-structural proteins does. (A) H1HeLa were infected at an MOI of 20, and the half of samples were treated with ammonium chloride added at 0 hpi and continued until the end of infection (5.5 hpi). Cells were labeled with 35S-methionine either from 2 hpi until collection at 4 hpi (A, upper panel), or from 3 hpi until collection at 5.5 hpi (A, lower panel). Cellular lysates were separated on 15–30% freshly prepared sucrose gradients and subjected to ultracentrifugation. Fractions were collected using the Fraction System and the counts per minute (CPM) were measured for each fraction. The experiments were independently repeated three times and the representative gradients are shown. (B) The collective three fractions of each determined peak (150S and 75S) were pooled and run on SDS-PAGE. The 35S-methionine labeled bands were visualized using autoradiography films. The bands are labeled according to the expected relative migration pattern, while VP2 is identified by its absence in the 75S peak. (C) Production of several non-structural proteins of EV-D68…

Fig 5. Vesicle acidification after the transition point of the EV-D68 cycle is essential for virion formation. (A) Titers of intracellular EV-D68 upon treatment with ammonium chloride (NH4Cl, 20 mM). H1HeLa were infected with EV-D68 at an MOI of 20; after 30 min of virus adsorption at 37°C (0 hpi), cells were washed two times to remove the initial virus input and the infection was allowed to run for 3 h. At 3 hpi ammonium chloride (20 mM in complete DMEM) was added to the cells until the end of the infection (6 hpi). Control samples (infection only) were left untreated until virus collection at 6 hpi. Intracellular titers were analyzed by plaque assay. Unpaired Student’s t test was used for the statistical analysis (***P < 0.001; **P < 0.01; *P ≤ 0.05; ns = not significant). (B) Effect of ammonium chloride treatment on viral RNA levels assessed by qRT-PCR. Cells were infected with EV-D68 at an MOI of 20 for 30 min at 37°C, then the residual virus was washed away, followed by either adding normal media (untreated infection, EV-D68; −NH4Cl) or media containing 20 mM of ammonium chloride (EV-D68; +NH4Cl) at different time points [at either 0, 3, 4, or 5 hpi until virus collection (6 hpi)].…

Fig 6. Disrupted EV-D68 capsid assembly through myristoylation inhibition results in decreased RNA levels. (A) Titers of intracellular EV-D68 upon treatment with ammonium chloride (NH4Cl, 20 mM), IMP-366 (5 µM), or both at indicated time points via time-of-drug assay. H1HeLa were infected with EV-D68 at an MOI of 20; after 30 min of virus adsorption at 37°C (0 hpi), cells were washed two times to remove the initial virus input and the infection was allowed to run for 3 h. At the indicated time points post-infection, ammonium chloride (20 mM in complete DMEM) or IMP-366 (5 µM) was added to the cells until the end of the infection (6 hpi). Control samples (infection only) were left untreated until virus collection at 6 hpi. (B) Effect of ammonium chloride and IMP-366 on EV-D68 RNA levels assessed by qRT-PCR. Cells were infected with EV-D68 at an MOI of 20 for 30 min at 37°C, then the residual virus was washed away, followed by either adding normal media (EV-D68 only) or media containing 20 mM of ammonium chloride (EV-D68; +NH4Cl), 5 µM of IMP-366 (EV-D68; +IMP-366), or both at indicated time points (at 0 or 3 hpi) until virus collection (6 hpi). Untreated infection (infection only, 6 hpi)…

Fig 7. De-acidification during the “transition point” of viral infection leads to structural changes in replication organelles (ROs) and multi-vesicular bodies (MVBs). Transmission electron micrographs of H1HeLa cells with mock infection or a 6 hr infection with EV-D68 (MOI = 20). H1HeLa were infected with EV-D68 at an MOI of 20; after 30 min of virus adsorption at 37°C (0 hpi), cells were washed two times to remove the initial virus input and the infection was allowed to run for 3 h. At 3 hpi ammonium chloride (20 mM) was added (EV-D68 + NH4Cl 3–6 hpi) or infection left untreated (EV-D68 only, 6 hpi). Mock cells were either left untreated (mock) or treated with ammonium chloride for 6 h (mock + NH4Cl for 6 h). At 6 h, all cells were fixed and subjected to TEM. Displayed images of the upper panel are followed by zoomed images of the same sample below, respectively. MT, mitochondria; MVB, multi-vesicular body; N, nucleus; RO, replication organelle. Scale bar: 2 µm or 500 nm (zoomed images) as indicated. TEM images are representative of one biological experiment.

Fig 8. Model. Time of infection progresses left to right. Early in infection, low pH is required for EV-D68 entry. During the log phase of virus growth, low pH compartments are required for assembly and maintenance of both empty and genome-containing capsids. Low pH is also required for normal localization and morphology of viral replication organelles (ROs). From 3 to 4 hpi, infections transition from the peak of vRNA replication, to EV-D68 capsid maturation and egress; after this transition point, low pH compartments are no longer required.

Fig 9. Summary of data. The effect of treatments (arrows) through different timepoints post-infection are shown. PV data are partially based on previous work (37).