DDX21, a Host Restriction Factor of FMDV IRES-Dependent Translation and Replication

Abstract

In cells, the contributions of DEAD-box helicases (DDXs), without which cellular life is impossible, are of utmost importance. The extremely diverse roles of the nucleolar helicase DDX21, ranging from fundamental cellular processes such as cell growth, ribosome biogenesis, protein translation, protein-protein interaction, mediating and sensing transcription, and gene regulation to viral manipulation, drew our attention. We designed this project to study virus-host interactions and viral pathogenesis. A pulldown assay was used to investigate the association between foot-and-mouth disease virus (FMDV) and DDX21. Further insight into the DDX21-FMDV interaction was obtained through dual-luciferase, knockdown, overexpression, qPCR, and confocal microscopy assays. Our results highlight the antagonistic feature of DDX21 against FMDV, as it progressively inhibited FMDV internal ribosome entry site (IRES) -dependent translation through association with FMDV IRES domains 2, 3, and 4. To subvert this host helicase antagonism, FMDV degraded DDX21 through its non-structural proteins 2B, 2C, and 3C protease (3C^pro). Our results suggest that DDX21 is degraded during 2B and 2C overexpression and FMDV infection through the caspase pathway; however, DDX21 is degraded through the lysosomal pathway during 3C^pro overexpression. Further investigation showed that DDX21 enhanced interferon-beta and interleukin-8 production to restrict viral replication. Together, our results demonstrate that DDX21 is a novel FMDV IRES trans-acting factor, which negatively regulates FMDV IRES-dependent translation and replication.

Keywords: 2B; 2C; 3C protease; DDX21; IRES; foot-and-mouth disease virus; replication.

Figure 1

DDX21 co-precipitates with the FMDV IRES. (a) Schematic diagram of FMDV genome, which depict the various regions of FMDV genome. (b) PK-15 cells were harvested and lysed in RIPA buffer. Biotin-labeled FMDV 5′UTR, S-fragment, cre, IRES, and 3′UTR RNAs were added to the lysates and DDX21 was pulled down. Non-biotinylated RNA of each segment was used as a control. Anti-DDX21 antibodies were used for western blot analysis. (c, d) PK-15 cells were infected with FMDV at an MOI of 0.5 for 3 h. The cells were lysed in RIPA buffer, and the lysate was incubated with antibody against DDX21 for RNA immunoprecipitation. Negative controls included anti-IgG, no antibody, and ddH2O. RNA was isolated from immunoprecipitated samples, reverse transcribed, and amplified by PCR using primers directed against FMDV IRES, 3′UTR, RPL13, and GAPDH.

Figure 2

Regions of the FMDV IRES precipitate with the host protein DDX21. (a) Schematic diagram of FMDV IRES domains. (b) PK-15 cells in 100 mm cell culture dishes were transfected with 10 µg of Flag-DDX21. The lysate was collected in RIPA buffer and mixed with the in vitro synthesized biotinylated full-length FMDV IRES and domains D1–2, D3–5, D3, D4–5, D4, and D5. Following pulldown, beads were eluted with elution buffer, and 1× SDS loading buffer was added for Western blot analysis. (c) PK-15 cell in 100 mm cell culture dishes were transfected with 10 µg of Flag-nucleolin. The lysate was collected in RIPA buffer and mixed with the in vitro synthesized biotinylated full-length FMDV IRES and domains D1–2, D3–5, D3, D4–5, D4, and D5. Following pulldown, beads were eluted with elution buffer, and 1× SDS loading buffer was added for Western blot analysis.

Figure 3

DDX21 inhibits FMDV replication. (a) PK-15 cells in 12-well plates were transfected with Flag-DDX21 plasmid (1.5 µg) and incubated for 24 h at 37 °C. Infection with type O FMDV was performed at a MOI of 0.5 and cells were incubated for an additional 24 h at 37 °C. Samples were collected at 0, 1, 3, 5, 7, and 9 hpi. Cell lysates were analyzed by Western blotting. (b) PK-15 cells in 12-well plates were transfected with Flag-DDX21 plasmid (1.5 µg) and incubated for 24 h at 37 °C. Infection with type O FMDV was performed at a MOI of 0.5 and cells were incubated for 1 h at 37 °C. Samples were collected at 0, 1, 3, 5, 7, and 9 hpi using RNAiso Plus for RNA extraction and qRT-PCR analysis. (c) PK-15 cells in 12-well plates were transfected with Flag-DDX21 plasmid (1.5 µg) and incubated for 24 h at 37 °C. Infection with type O FMDV was performed at a MOI of 0.5 and cells were incubated for 1 h at 37 °C. Cells were washed with 1× PBS and incubated in DMEM supplemented with 1% FBS. Samples were collected at 0, 1, 3, 5, 7, and 9 hpi. Cellular supernatants were collected, centrifuged, and stored at −80 °C for TCID50 analysis. (d) DDX21 was knocked down using siRNA directed against DDX21. Cells were incubated for 36 h, followed by FMDV type O infection. Cell lysates were analyzed by Western blotting. (e) DDX21 was knocked down using siRNA directed against DDX21. Cells were incubated for 36 h, followed by FMDV type O infection. RNAiso Plus was added to collect samples for RNA extraction and qRT-PCR analysis. (f) DDX21 was knocked down using siRNA directed against DDX21. Cells were incubated for 36 h, followed by FMDV type O infection. Cellular supernatants were collected, centrifuged, and used for TCID₅₀ analysis. The data are presented as the mean and SD of three separate experiments (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 4

DDX21 inhibits FMDV, classical swine fever virus (CSFV), and Seneca Valley virus (SVV) IRES-dependent translation. (a) Schematic diagram of the bicistronic luciferase construct. (b) PK-15 cells in 24-well plates were co-transfected with psiCHECK-FMDV and Flag-DDX21, Flag-hnRNP K (positive control), or Flag-EV (negative control). Samples were harvested 24 h post-transfection using passive lysis buffer or 1× SDS loading buffer for Western blot analyses. The dual-luciferase assay was performed using the Dual-Luciferase Reporter Assay System. (c) PK-15 cells in 24-well plates were co-transfected with psiCHECK-CSFV or psiCHECK-SVV and Flag-DDX21 or Flag-EV. (d) PK-15 cells were transfected with Flag-DDX21 or Flag-EV and Flag-hnRNP K or Flag-EV. Samples were collected 24 h post-transfection for Western blot analyses. (e) PK-15 cells in 24-well plates were knocked down with siRNA-DDX21, siRNA-PTBP1 (positive control), or siNC (negative control). At 30 h post-knockdown, cells were transfected with psiCHECK-FMDV. After 24 h, samples were collected using passive lysis buffer for luciferase activity analysis. (f) PK-15 cells in 24-well plates were knocked down using siRNA-DDX21 or siNC. After 30 h, cells were transfected with psiCHECK-CSFV or psiCHECK-SVV. After an additional 24 h, samples were collected using passive lysis buffer for luciferase activity analysis. (g) PK-15 cells were knocked down with siRNA-DDX21, siRNA-PTBP1, or siNC. Samples were collected at 36 h post-transfection for Western blot analysis.

Figure 5

DDX21 is translocated into the cytoplasm of FMDV-infected cells. (a) PK-15 cells were cultured in glass-bottom cell culture dishes and transfected with Flag-DDX21, followed by mock or FMDV infection. Cells were fixed with 4% paraformaldehyde at 3 and 7 hpi. An indirect immunofluorescencent antibody test was performed using primary anti-Flag antibodies and secondary TRITC-conjugated antibodies (red); polyclonal pig antiserum was prepared in our laboratory [60], which was used to detect the viral proteins with secondary FITC-conjugated antibodies (green). Nuclei were stained blue with DAPI; the merged signal appeared yellow. (b) PK-15 cells on 100 mm dishes and transfected with 2 µg of Flag-DDX21. Cells were FMDV-infected, and samples were collected at 0, 1, 3, 5, 7, and 9 hpi. Samples were processed using the nuclear/cytosol fractionation assay. The nuclear and cytosol fractions were analyzed through Western blot assay.

Figure 6

FMDV degrades DDX21 during viral infection. (a) PK-15 cells in 12-well plates were mock-infected or infected with type O FMDV at a MOI of 0.5, and samples were harvested at 0, 1, 3, 5, 7, 9, and 12 hpi. Collected samples were analyzed by Western blotting. (b,c) PK-15 cells in 12-well plates were infected with type O FMDV, and cells were harvested using RNAiso Plus reagent for RNA collection. RNA was extracted, and qRT-PCR was performed. The data are presented as the mean and SD of three separate experiments (* p < 0.05, ** p < 0.01).

Figure 7

DDX21 is degraded by FMDV 2B, 2C, and 3C^pro. (a) PK-15 cells in six-well plates were co-transfected with Flag-DDX21 and VP0, VP1-2, VP3, L^pro, 2B, 2C, 3A, 3C^pro, 3Dpol, or Flag-EV. Cells were harvested after 24 h and 1× SDS loading buffer was added. The samples were analyzed by Western blot. (b) After the cells were grown to 80% confluence in 96 well plates, they were transfected with Flag-VP0, VP1-2, VP3, L^pro, 2B, 2C, 3A, 3C^pro, and 3D^pol or an empty vector for 24 h. For the MTS assay, 10 μL of CellTiter 96^® AQueous One Solution Cell Proliferation Assay reagent (Promega, WI, USA) was directly added to the cells, which were then incubated for 4 h. The absorbance at 490 nm was recorded. (c) PK-15 cells were co-transfected with HA-DDX21 (2 µg) and Flag-3C^pro (250, 500, 1000, or 2000 ng) or Flag-EV (2 µg). Cell lysates were collected in 1× SDS loading buffer and analyzed by Western blotting. (d) PK-15 cells on six-well plates were co-transfected with HA-DDX21 (2 µg) and Flag-3C^pro, H46Y, D84N, 163G, H205R, or Flag-EV (2 µg). Samples were collected at 24 h post-transfection and analyzed by Western blotting. (e) PK-15 cells on six-well plates were co-transfected with HA-DDX21 (2 µg) and Flag-2B (250, 500, 1000, or 2000 ng) or Flag-EV (2 µg). Cell lysates were collected 24 h post-transfection in 1× SDS loading buffer and analyzed by Western blotting. (f) PK-15 cells were co-transfected with HA-DDX21 (2 µg) and Flag-3C^pro (250, 500, 1000, or 2000 ng) or Flag-EV (2 µg). Cell lysates were collected 24 h post-transfection in 1× SDS loading buffer and analyzed by Western blotting. (g–i) PK-15 cells were cultured on a six-well plate. At 80% confluence, cells were transfected with an increasing concentration of Flag-2B, Flag-2C, and Flag-3C^pro (0, 250, 500, 1000, and 2000 ng). Twenty-four hours post transfection; RNA was extracted and the level of DDX21 mRNA was determined by qRT-PCR.

Figure 8

DDX21 does not interact with FMDV 2B, 2C, and 3C^pro. (a) PK-15 cells in 100 mm cell culture dishes were co-transfected with HA-DDX21 (8 µg) and Flag-2B (6 µg) and incubated for 24 h. Protein lysates were collected in RIPA buffer. Forward immunoprecipitation was performed with anti-HA and reverse immunoprecipitation was performed with anti-Flag. The immunocomplexes were analyzed by SDS-PAGE and Western blotting. (b) PK-15 cells in 100 mm cell culture dishes were co-transfected with HA-DDX21 (8 µg) and Flag-2C (6 µg) and incubated for 24 h. Protein lysates were collected in RIPA buffer. Forward immunoprecipitation was performed with anti-HA and reverse immunoprecipitation was performed with anti-Flag. The immunocomplexes were analyzed by SDS-PAGE and Western blotting. (c) PK-15 cells in 100 mm cell culture dishes were co-transfected with HA-DDX21 (8 µg) and Flag-3C^pro (6 µg) and incubated for 24 h. Protein lysates were collected in RIPA buffer. Forward immunoprecipitation was performed with anti-HA and reverse immunoprecipitation was performed with anti-Flag. The immunocomplexes were analyzed by SDS-PAGE and Western blotting.

Figure 9

DDX21 is degraded through the caspase pathway during FMDV infection. (a) PK-15 cells in six-well plates were infected with FMDV type O at a MOI of 0.5 and incubated for 1 h. Cells were washed with 1× PBS three times, and CQ was added at 50 to 100 µM to inhibit the lysosome pathway. After 11 h of incubation, samples were harvested in 1× SDS loading buffer, and Western blot analyses were performed. (b) PK-15 cells in six-well plates were infected with FMDV type O at a MOI of 0.5 and incubated for 1 h. Next, cells were washed with 1× PBS three times, and MG-132 was added at 10 to 20 µM to inhibit proteasome pathways. After 11 h of incubation, samples were harvested in 1× SDS loading buffer, and Western blot analyses were performed. (c) PK-15 cells in six-well plates were infected with FMDV type O at a MOI of 0.5 and incubated for 1 h. Next, cells were washed with 1× PBS three times, and Z-VAD-FMK was added at 10 to 50 µM for the inhibition of caspase pathways. After 11 h of incubation, samples were harvested in 1× SDS loading buffer, and Western blot analyses were performed. (d–l) PK-15 cells in six-well plates were co-transfected with HA-DDX21 (2 µg) and Flag-2B, 2C, 3C^pro, or Flag-EV (2 µg). At 6 h post-transfection, cells were washed with 1× PBS, CQ was added at 50 to 100 µM, MG-132 was added at 10 to 20 µM, and Z-VAD-FMK was added at 10 to 50 µM, and cells were incubated for an additional 18 h and collected in 1× SDS loading buffer. Samples were analyzed by SDS-PAGE and Western blot.

Figure 10

DDX21 induces IFN-β and IL-8 production during FMDV infection. (a,b) PK-15 cells in 12-well plates overexpressing Flag-DDX21 or Flag-EV were infected with FMDV type O at a MOI of 0.5. Samples were harvested with RNAiso Plus at 0, 1, 3, 5, 7, and 9 hpi. RNA was extracted and qRT-PCR analysis was performed. (c,d) PK-15 cells in 12-well plates were knocked down using siRNA-DDX21 or siNC, incubated for 36 h, and infected with FMDV type O. Samples were collected at 0, 1, 3, 5, 7, and 9 hpi using RNAiso Plus. RNA was extracted, and qRT-PCR was performed. The data reflect the means of three separate trials and error bars indicate standard deviations (SD) (* p < 0.05, ** p < 0.01, *** p < 0.001).