Enterovirus D68 proteases cleave several nucleoporins.

(A) HEK cells were transfected with Enterovirus D68 2Apro, Poliovirus 1 2Apro, or empty vector control, and the indicated nucleoporins were quantified by western blot, normalized to control=100%. EIF4G was included as a non-nucleoporin positive control. Mean ± S.D. of 3 independent replicates. Statistical comparison was by multiple t-tests of the EV-D68 2Apro vs control, with multiple comparisons correction by the false discovery rate method. Red asterisks represent significance with FDR<10%, blue asterisks represent samples for which a cleavage product was visible on the Western blot irrespective of significance. (B) As in A, except transfecting with 3Cpro. The positive control was CREB. (C) Nuclear lysates from HEK cells were incubated at 37°C with recombinant EV-D68 2Apro at a ratio of 1:50 (protease:lysate by mass) for the indicated time. Grey bars represent lysate incubated for 4 hours at 37°C in the absence of protease as a negative control. Mean ± S.D. of 3 independent replicates. Statistical comparison was by t-test between the with- and without-protease samples at 4 hours. (D) As in C, except with EV-D68 3Cpro at a ratio of 1:200. *p<0.05, **p<0.01, ***p<0.001.

Enterovirus D68 2A protease impairs nucleocytoplasmic transport of protein cargoes and disrupts the nuclear pore complex permeability barrier.

(A) tdTomato reporter assay showing nuclear-cytoplasmic ratios at steady state 20 hours after transfection of IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Mean values of three independent replicates n=29,404 GFP+ cells in total, error bars are S.D. Statistical comparison was by two-way ANOVA with Šídák’s multiple comparisons test. (B) Example output of image analysis protocol. In the overlay image, yellow represents the nuclei of transfected cells, defined by the area of expression of H2A-iRFP670 in GFP+ cells only. The surrounding dark blue ring is the perinuclear cytoplasm. The intensity of reporter construct was measured in the red channel and reported as a ratio of nuclear to perinuclear cytoplasmic signal. (C) Example images from A. Dotted outlines mark the nuclei. (D) Kinetics of nuclear import (left) and export (right) measured by photoinducible LINus and LEXY reporter assays, respectively, in HeLa cells 20 hours after transfection with IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Mean values of 3 biologically independent replicates, n=999 GFP+ cells for LINus and 1059 for LEXY in total. Error bars are S.E.M. Statistical comparison was by two-way ANOVA. (E) Example images from D. Dotted outlines mark GFP+ cells. (F) Dextran exclusion assay was performed in HeLa cells 15 hours after transfection with IRES-iRFP670-H2A, IRES-iRFP670-H2A-(2A)-2Apro, or IRES- iRFP670-H2A-(2A)-3Cpro. n=45-46 cells per group pooled from 3 biologically independent replicates. Statistical comparison was by two-way ANOVA. (H) Propidium iodide viability assay. Mean values of 3 independent replicates, n=8042 cells in total. Error bars are S.D. Statistical comparison was by one-way ANOVA with Tukey’s multiple comparison test.

Enterovirus D68 proteases do not significantly alter RNA export.

(A) Steady state mRNA levels were measured by Poly-A FISH in HeLa cells 20 hours after transfection with IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Data represent the mean ± S.D. of four independent replicates, n=55,177 GFP+ cells in total. Statistical comparison was by one-way ANOVA with Šídák’s multiple comparisons test. (B) Nuclear export of EU-labeled RNAs in HEK cells by 1hr pulse followed by 0-8hr chase. EU labeling began 20hr after transfection with IRES-GFP, IRES-GFP-2Apro, or IRES-GFP-3Cpro. Pulse labeling of total RNA or primarily the products of the listed RNA polymerases was performed in the presence of pairs of combinations of actinomycin D, α-amanitin, and CAS 577784-91-9 as described in Materials and Methods. Data represent the mean ± S.E.M. of three independent replicates. n=253,015 GFP+ cells analyzed in total. Statistical comparison was by two-way ANOVA. (C) Representative images from the experiment described in B. (D) Nuclear export of EU-labeled RNAs in RD cells by 1hr pulse followed by 0-4hr chase. EU labeling began 24 hours after infections with EV-D68 strains at MOI 5 or mock infection as indicated. Data represent the mean ± S.E.M. of three independent replicates. n=125,401 cells analyzed in total. Statistical comparison was by two-way ANOVA. (E) Representative images from the experiment described in D.

Effects of 2A protease during Enterovirus D68 infection on NPC function and motor neuron toxicity.

(A) Western blots of diMN lysates collected 24 hours after infection with the indicated strains of EV-D68 at MOI 5 followed by treatment with telaprevir at 0.3, 1, 3, and 10 µM. Total protein loading control was Biorad stainfree imaging. One representative example out of three biologically independent replicates. (B) tdTomato reporter assays in RD cells showing nuclear-cytoplasmic ratios 24 hours after mock infection or infection with EV-D68 US/MO/2014-18947. Telaprevir was added following infection at the indicated concentrations. Data are mean of three independent replicates, n=11,847 cells in total for NLS-tdTomato and n=8941 for tdTomato-NES. (C) Representative images of tdTomato signal from B. (D) tdTomato reporter assays in diMN showing nuclear-cytoplasmic ratios 24 hours after mock infection or infection with EV-D68 US/MO/2014-18947, treated with DMSO or 3µM telaprevir. Means of three replicates from independent iPSC lines. n=3388 cells in total for NLS-tdTomato and. n=3371 for tdTomato-NES. (E) Representative images from panel D. (F) Survival of diMNs following infection with EV-D68 at MOI 5 with the indicated EV-D68 strains in the presence of varying concentrations of telaprevir or DMSO control. diMNs were generated from four independent iPSC lines. n=8512 and 7046 cells counted for US/MO/2014-18947 and US/MD/2018-23209 respectively. Statistical comparisons were by Cox proportional hazard regression. (G) Representative images of diMNs from the experiment presented in panel F. Note fragmentation of neurites and loss of cell body shape signifying cell death following EV-D68 infection, and relative preservation following treatment with 3µM telaprevir. (H) Growth curve of EV-D68 in the presence of varying concentrations of telaprevir or DMSO control. Infections were performed with the indicated strains at MOI 0.5, using diMNs generated from four independent iPSC lines for each virus. Error bars are SEM. Statistical comparisons were by two-way ANOVA with Dunnet’s multiple comparisons test between DMSO and telaprevir-treated groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Western blots demonstrating evidence of 2Apro and 3Cpro protease activity on well-established substrates following transfection with each of the expression vectors used in this study.

Source data for Figure 2A, showing individual cell level data for three independent replicates.

Bars are mean ± S.D.

Source data for Figure 2D, showing mean ± S.D. values from three independent replicates for LINus (A) and LEXY (C) nucleocytoplasmic transport reporters.

Individual cell level data is also presented at t=10 min for LINus (B) and LEXY (D). Bars are mean ± S.D.

Source data for Figure 3A, demonstrating individual cell-level data across four independent replicates.

Bars are mean ± S.D.

Source data for figures 4C (A) and 4D (B), showing individual cell-level data for three independent replicates in diMNs derived from iPSC lines CS0002, CS0003, and CS88.

Bars are mean ± S.D.