Enterovirus-induced cleavage of Mitofusin 2 generates mitophagosomes for enveloped virion release

Fig. 1.. Enterovirus D68 depolarizes mitochondria and induces mitophagosome formation. (A) H1HeLa cells were either treated with 10 μM CCCP (4 hours) or infected with EV-D68 at an MOI of 25 for 4 hours. Mitochondrial membrane potential was determined using the JC-10 mitochondrial membrane potential kit. (B) Cells were treated with 10 μM CCCP for 12 hours or infected with EVD-68 for 4 hours, and the intracellular calcium levels were measured by the Fluo-8 No Wash Calcium Assay Kit. (C) H1HeLa cells were treated with CCCP or infected with EV-D68 (MOI = 25) for the indicated time points. The cells were then fractionated using a mitochondrial fractionation kit, and a Western blot was performed against the indicated antibodies. Numbers indicate the ratio of Parkin to TOMM20 signal. (D) H1HeLa cells were infected with EV-D68 for 5 hours at an MOI of 25. The cells were fixed, and IFA was performed against TOMM20 and VP3. Scale bars, 3 μm. (E to G) H1HeLa cells were mock-infected or infected with EV-D68 (MOI = 30) for 4 hours for EM imaging. (E) Normal cell morphology and intact mitochondrial network in mock-infected cells (left) versus membrane rearrangement and replication organelle formation…

Fig. 2.. EV-D68 cleaves the mitochondrial fusion protein, MFN-2. (A) H1HeLa cells were infected for the indicated time points with EV-D68 (MOI = 25). Western blot was then performed against the mitochondria-associated proteins. Lysates from cells treated with 10 μM CCCP or 10 μM oligomycin-A for 12 hours were included as positive controls. (B) H1HeLa cells were infected with the indicated enteroviruses at MOI = 25 for 5 hours, after which lysates were prepared for Western blot. (C) A549 cell lysates were incubated with and without the 3C protease for an in vitro cleavage assay for Western blot against MFN-2. (D and E) H1HeLa cells were transfected with pcDNA3.1 MFN-2–HA for 24 hours. The cells were either mock-infected or infected with EV-D68 (MOI = 25) for 5 hours for Western blot using anti–MFN-2 (D) or anti-HA (E) antibodies. (F) H1HeLa cells were transfected with the indicated constructs for 24 hours. The cells were then mock-infected or infected with EV-D68 for 5 hours, followed by a Western blot using anti-HA antibodies. n = 3 independent repeats for all experiments. Ab, antibody; CL-MFN-2, cleavage product of MFN-2.

Fig. 3.. Knockdown or overexpression of MFN-2 attenuated EV-D68 release. (A) H1HeLa cells were transfected with control (Scramble) or MFN-2 siRNAs for 48 hours. The cells were then infected with EV-D68 for 5 hours, followed by viral titer measurement by plaque assay. (B) A549 cells were transfected and infected as in (A) for viral titer determination by a plaque assay. (C) H1HeLa and A549 cells were transfected with the indicated small interfering RNAs (siRNAs) for 48 hours, after which the whole cell lysates were collected and prepared for a Western blot against the indicated antibodies. (D) H1HeLa and A549 cells were infected with EV-D68 (MOI = 30) for 5 hours for Western blot against MFN-2 and VP3. (E and F) H1HeLa cells were transfected with the indicated constructs for 24 hours. (G) The cells were then infected with EV-D68 (MOI = 0.1) for 5 hours, and viral intracellular (E) and extracellular (F) titers were determined by a plaque assay. n = 3 independent repeats for all Western blot experiments. Error bars indicate mean ± SEM of at least two independent repeats. Unpaired Student’s t test was used for the statistical analyses (**P < 0.01 and *P < 0.05). ns, not significant; PFU,…

Fig. 4.. Mitochondria colocalize with viral dsRNA late during EV-D68 infection. H1HeLa cells were infected with EV-D68 (MOI = 25) for the indicated time points. The cells were fixed and subjected to IFA against dsRNA and TOMM20. Scale bars, 50 μm.

Fig. 5.. Active mitophagy signaling is essential for EV-D68 release. (A) H1HeLa cells were transfected with control (Scramble) and Parkin siRNAs for 48 hours. The cells were then infected with EV-D68 (MOI = 0.1) for 5 hours, and viral titers were measured by a plaque assay. (B) A549 cells were transfected with scramble, Parkin, and PINK1 siRNAs for 48 hours. The cells were infected with EV-D68 for 5 hours, and viral titers were determined by a plaque assay. (C) H1HeLa cells were transfected with siRNAs against the mitophagy receptor, BCL2L13, for 48 hours. The cells were then infected as in (A) for a plaque assay–based viral titer measurement. (D) A549 cells were transfected with the indicated mitophagy-associated siRNAs for 48 hours, followed by viral infection and titer determination as in (A). (E) H1HeLa cells were transfected with control (Scramble) or siParkin siRNAs for 48 hours. The cells were then infected with EV-D68 (MOI = 25) for 5 hours. IFA was then conducted against endogenous LC3 and TOMM20. Scale bars, 2.3 μm. (F) A549 cells were transfected with the indicated siRNAs for 48 hours. (G) H1HeLa cells were transfected with the indicated siRNAs for 48 hours. Cell lysates were…

Fig. 6.. Overexpression of mCh-Parkin induces mitophagy and attenuates EV-D68 release. (A) H1HeLa cells transfected with mCh-Parkin expressing plasmids for 24 hours were left untreated (mock), treated with 10 μM CCCP for 5 hours, or infected with EV-D68 (MOI = 25) for 5 hours. The cells were then fixed, and IFA was performed against TOMM20. Scale bars, 3.3 μm. (B and C) HeLa cells were transfected with pcDNA3.1– and mCh-Parkin–expressing plasmids for 48 hours. Cells were collected and prepared for Western blot against the indicated proteins (C) or infected with EV-D68 (MOI = 0.1) for 5 hours (B). A plaque assay was then used to determine the viral titers. (D) A549 cells were infected for 30 min (adsorption), then washed, and replenished with complete media (infection) or media containing 10 μM CCCP for 5 hours. A plaque assay was used to determine the viral titer. (E) A549 cells were left untreated or treated with 10 μM CCCP for 5 hours. Lysates were collected and processed for Western blot. (F) H1HeLa and ATG5 KO cells were transfected with the indicated constructs for 24 hours, after which lysates were prepared for Western blot. (G) HeLa and ATG5 KO–HeLa cells were transfected as in (F)…