Journal: mBio

Article Title: Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling

doi: 10.1128/mbio.01915-23

Figure Lengend Snippet: Long-term time course of EV infection in A375- and myeloid antigen-presenting cells. ( A and B ) A375 cells were infected with CAV21 or PVSRIPO (MOI, 10 [ A ]) and with HRV16 or PVSRIPO (MOI, 10 [ B ]). HRV16 infections were carried out at 33°C. Lysates were analyzed by immunoblot for viral translation (eIF4G cleavage, 2C/2BC); TBK1 activation [p-IRF3(S386)/IRF3]; and type-I IFN signaling [p-STAT1(Y701)/STAT1, p-IRF7(S477)/IRF7, MDA5] over time (*eIF4G1 cleavage products). ( C ) IFN-α/IFN-β immunoblots. ( D and E ) Expression levels of viral 2C and the indicated (phospho)-proteins in PVSRIPO-infected cells ( D ) or in CAV21-infected cells ( E ) were quantified. The values represent the average abundance of proteins in three independent series (% max. detected, normalized to GAPDH; mean ± standard error of the mean). The assays were performed in at least three independent series; representative results are shown. ( F and G ) Long-term time course of CAV21 (MOI, 10 [ F ]) and PVSRIPO (MOI, 10 [ G ]) infection in human monocyte-derived antigen-presenting cells. Lysates collected at the indicated intervals were analyzed by immunoblot for the parameters shown. Cells from two distinct donors were tested with CAV21 infection; #, loss of signal with the tubulin loading control (and all other proteins) at 24–48 hpi are due to rampant cytolysis ( F ). PVSRIPO infection of MDMs ( G ) was analyzed by immunoblot of samples collected at the indicated intervals. Cells from three distinct donors were tested in five independent series; representative results are shown with samples from one donor (the arrow indicates weak signal for viral 2C; # indicates prominent non-specific background bands).

Article Snippet: Primary antibodies used were against eIF4G1 (#2469), GAPDH (#2118), IRF3 (#119040), p-IRF3(S386) (#37829), STAT1 (#9172), p-STAT1(Y701) (#9167), IRF7 (#49200), p-IRF7(S477) (#12390), CD54 (#67836), CD155 (#13544), IFN-β (#736710), dsRNA (K1, #28764), MDA5 (#5321), IFIT1 (#14769), and NUP98 (#2598) (all Cell Signaling Tech); and puromycin (#MABE343) (Millipore); IFN-α (#18013-1-AP), and YTHDF3 (#25537–1-AP) (both Proteintech).

Techniques: Infection, Western Blot, Activation Assay, Expressing, Derivative Assay

Journal: mBio

Article Title: Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling

doi: 10.1128/mbio.01915-23

Figure Lengend Snippet: Effect of PI4KIIIβ inhibition on the innate antiviral type-I IFN response to CAV21 and PVSRIPO (see Fig. S3 for extended data). A375 cells were infected with CAV21 (A) or PVSRIPO (B) as described for in the presence or absence of 10 nM PI4KIIIβ-IN-10. Cell lysates collected at the indicated hpi were analyzed by dot blot for accumulation of viral dsRNA (upper panels) or by immunoblot for viral protein synthesis (2C), eIF4G cleavage, and the innate antiviral response [p-STAT1(Y701), p-IRF7(S477), IFN-α/IFN-β, and MDA5] (bottom panels; *eIF4G1 cleavage products). All assays were performed in at least three independent series; representative results are shown. Levels of dsRNA accumulation (C, CAV21; D, PVSRIPO), levels of p-STAT1(Y701)/2C in the presence of absence of inhibitor (E, CAV21), and levels of pIRF7(S477)/p-STAT1/2C in mock-treated cells (F, PVSRIPO) were quantified and plotted in the graphs (% max. normalized to GAPDH, means ± standard error of the mean).

Article Snippet: Primary antibodies used were against eIF4G1 (#2469), GAPDH (#2118), IRF3 (#119040), p-IRF3(S386) (#37829), STAT1 (#9172), p-STAT1(Y701) (#9167), IRF7 (#49200), p-IRF7(S477) (#12390), CD54 (#67836), CD155 (#13544), IFN-β (#736710), dsRNA (K1, #28764), MDA5 (#5321), IFIT1 (#14769), and NUP98 (#2598) (all Cell Signaling Tech); and puromycin (#MABE343) (Millipore); IFN-α (#18013-1-AP), and YTHDF3 (#25537–1-AP) (both Proteintech).

Techniques: Inhibition, Infection, Dot Blot, Western Blot

Journal: mBio

Article Title: Early enterovirus translation deficits extend viral RNA replication and elicit sustained MDA5-directed innate signaling

doi: 10.1128/mbio.01915-23

Figure Lengend Snippet: The effect of inhibiting vRNA replication with dipyrimadole (DIP) on innate type-I IFN responses to CAV21 and PVSRIPO. Time course of A375 infection with CAV21 (A) or PVSRIPO (B) performed in a manner similar to that in in the absence (dimethyl sulfoxide[DMSO]) or presence of 100 μM DIP. Cell lysates were analyzed by dot blot for vRNA replication (dsRNA, top panels) and by immunoblot for eIF4G cleavage, viral translation (2C), and innate type-I IFN response signatures (middle panels; *eIF4G1 cleavage products, **p-IRF7 with decreased electrophoretic mobility detected with IRF7 antibody). Assays were performed in at least three independent series; representative results are shown. Accumulation of dsRNA and 2C protein in CAV21-infected cells was quantified from three independent experiments and plotted in the bottom graph (% max. normalized to GAPDH, means ± standard error of the mean). (C) A repeat series of the assay shown in panel A with high detection sensitivity was conducted to rigorously document eIF4G1 status in CAV21-infected cells in the presence of DIP.

Article Snippet: Primary antibodies used were against eIF4G1 (#2469), GAPDH (#2118), IRF3 (#119040), p-IRF3(S386) (#37829), STAT1 (#9172), p-STAT1(Y701) (#9167), IRF7 (#49200), p-IRF7(S477) (#12390), CD54 (#67836), CD155 (#13544), IFN-β (#736710), dsRNA (K1, #28764), MDA5 (#5321), IFIT1 (#14769), and NUP98 (#2598) (all Cell Signaling Tech); and puromycin (#MABE343) (Millipore); IFN-α (#18013-1-AP), and YTHDF3 (#25537–1-AP) (both Proteintech).

Techniques: Infection, Dot Blot, Western Blot