Figure 3.
Figure 3. ZMPSTE24 inhibits viral entry, but catalytic activity is dispensable for antiviral function. (A) HEK293 cells were transfected with vector, ZMPSTE24-FLAG, or the indicated ZMPSTE24 mutants. After 24 h, cells were infected with 1 MOI PR8-GLuc for 16 h. Luciferase activity assays were performed. Each group was tested in three experiments. Mean ± SD; *, P < 0.05. (B) Zmpste24+/+, zmpste24−/−, and zmpste24−/− MEFs transduced with retrovirus expressing FLAG-tagged GFP, ZMPSTE24, E336A mutant ZMPSTE24, or IFITM3-HA were infected with IAV-GLuc, VSV-Luc, or VACV-Luc for 16 h. Relative luciferase levels are shown. Representative data from one of three experiments are shown. Mean ± SD; *, P < 0.05. Western blot demonstrates expression levels. (C) A549 cells transfected with ZMPSTE24 were infected with 50 MOI PR8 (2 h) and then stained with LysoTracker red, DAPI, and anti-NP. The arrowhead indicates nuclear localization of influenza NP protein. Arrows indicate representative colocalized puncta. Pearson’s r coefficients are denoted. Bars, 10 µm. (D) Zmpste24+/+ and zmpste24−/− cells were infected with IAV or MLV VLPs and then loaded with CCF2. Cleavage of CCF2 was determined by fluorescence emission shift from 520 nm (uncleaved CCF2) to 447 nm (cleaved CCF2). Data are representative of two experiments. (E) Zmpste24+/+ and zmpste24−/− cells were infected with IAV (HA1, HA5, and HA7), VSV, and Ebola VLP (2 h) and then assayed for viral entry with CCF2. Data are representative of two or more experiments per group. Mean ± SD; *, P < 0.05. Percentages of positive zmpste24+/+ MEFs were 5.2%, 3.8%, 3.4%, 5.6%, and 4.2%, respectively. (F) HEK293 cells were transfected with ZMPSTE24 for 24 h and then infected with the indicated VLPs. Data are representative of two or more experiments per group. Mean ± SD; *, P < 0.05. Percentages of infected cells in the vector transfected control group were 23%, 17%, 18%, 39%, and 26%, respectively. (G) IfitmDel−/− and ifitmDel−/− MEFs expressing GFP-FLAG, ZMPSTE24-FLAG, or IFITM3-HA were infected with 1 MOI PR8-GLuc, VSV-Luc, or VACV-Luc (16 h). Mean ± SD; *, P < 0.05. Western blot (WB) shows expression levels. Data are representative of two independent experiments. (A, B, and G) Molecular mass (MM) is indicated in kilodaltons. (H) IfitmDel−/− and wild-type MEFs were transfected with nontargeting control or ZMPSTE24 siRNA#2. After 48 h, cultures were infected with VSV-Luc or VACV-Luc reporter virus. Luciferase levels were monitored 16 h after infection (three experiments). Mean ± SD; *, P < 0.05.

ZMPSTE24 inhibits viral entry, but catalytic activity is dispensable for antiviral function. (A) HEK293 cells were transfected with vector, ZMPSTE24-FLAG, or the indicated ZMPSTE24 mutants. After 24 h, cells were infected with 1 MOI PR8-GLuc for 16 h. Luciferase activity assays were performed. Each group was tested in three experiments. Mean ± SD; *, P < 0.05. (B) Zmpste24+/+, zmpste24−/−, and zmpste24−/− MEFs transduced with retrovirus expressing FLAG-tagged GFP, ZMPSTE24, E336A mutant ZMPSTE24, or IFITM3-HA were infected with IAV-GLuc, VSV-Luc, or VACV-Luc for 16 h. Relative luciferase levels are shown. Representative data from one of three experiments are shown. Mean ± SD; *, P < 0.05. Western blot demonstrates expression levels. (C) A549 cells transfected with ZMPSTE24 were infected with 50 MOI PR8 (2 h) and then stained with LysoTracker red, DAPI, and anti-NP. The arrowhead indicates nuclear localization of influenza NP protein. Arrows indicate representative colocalized puncta. Pearson’s r coefficients are denoted. Bars, 10 µm. (D) Zmpste24+/+ and zmpste24−/− cells were infected with IAV or MLV VLPs and then loaded with CCF2. Cleavage of CCF2 was determined by fluorescence emission shift from 520 nm (uncleaved CCF2) to 447 nm (cleaved CCF2). Data are representative of two experiments. (E) Zmpste24+/+ and zmpste24−/− cells were infected with IAV (HA1, HA5, and HA7), VSV, and Ebola VLP (2 h) and then assayed for viral entry with CCF2. Data are representative of two or more experiments per group. Mean ± SD; *, P < 0.05. Percentages of positive zmpste24+/+ MEFs were 5.2%, 3.8%, 3.4%, 5.6%, and 4.2%, respectively. (F) HEK293 cells were transfected with ZMPSTE24 for 24 h and then infected with the indicated VLPs. Data are representative of two or more experiments per group. Mean ± SD; *, P < 0.05. Percentages of infected cells in the vector transfected control group were 23%, 17%, 18%, 39%, and 26%, respectively. (G) IfitmDel−/− and ifitmDel−/− MEFs expressing GFP-FLAG, ZMPSTE24-FLAG, or IFITM3-HA were infected with 1 MOI PR8-GLuc, VSV-Luc, or VACV-Luc (16 h). Mean ± SD; *, P < 0.05. Western blot (WB) shows expression levels. Data are representative of two independent experiments. (A, B, and G) Molecular mass (MM) is indicated in kilodaltons. (H) IfitmDel−/− and wild-type MEFs were transfected with nontargeting control or ZMPSTE24 siRNA#2. After 48 h, cultures were infected with VSV-Luc or VACV-Luc reporter virus. Luciferase levels were monitored 16 h after infection (three experiments). Mean ± SD; *, P < 0.05.

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