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Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs

View Article: PubMed Central - PubMed

ABSTRACT

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19.

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Related in: MedlinePlus

IAV multiplication is reduced in DDX19-depleted cells.(a,b) A549 cells were treated with control non-target or NUP62 siRNAs (dark grey bars) or siRNAs targeting the indicated DDX (light grey bars) and infected with the WSN-PB2-Nanoluc virus (0.0001 pfu/cell). Luciferase activities were measured in cell lysates prepared at 24 hpi. Three independent experiments were performed in triplicate. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the non-target siRNA condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (**p < 0.01; ***p < 0.001). (c) At 48 hours post-transfection (hpt) with the control non-target siRNAs (C) or with individual siRNAs targeting DDX19A+B (#1, #2 and #3), the levels of DDX19 protein were evaluated by immunoblot using an antibody that recognizes both A and B forms of DDX19, and the levels of DDX19A and DDX19B mRNAs were determined by RT-qPCR and were expressed as percentages of the control. Cropped blots are shown. The corresponding full-length blots are shown in Figure S2. (d–g) A549 cells were treated with control (dark grey lines) or DDX19 siRNAs (light grey lines) and infected with the following viruses at the indicated m.o.i. in pfu/cell: A/WSN/33(H1N1 WSN, 0.0001); A/Udorn/307/72(H3N2) (Udorn, 0.01); A/Paris/908/97(H3N2) (P908, 0.01); A/Paris/650/2004(H1N1) (P650, 0.01); Vesicular Stomatitis Virus (VSV, 0.0001); Adenovirus 5 (AdV, 6). At the indicated times post-infection (d,f,g) or at 24 hpi (e), the viral titers were determined by plaque assay on MDCK cells (d–f) or immunostaining (g). The results are expressed as the mean ± SEM of triplicates and the significance was tested with an unpaired, 2-tailed Student t test using GraphPad Prism software (*p < 0.05; **p < 0.01; ***p < 0.001).
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f1: IAV multiplication is reduced in DDX19-depleted cells.(a,b) A549 cells were treated with control non-target or NUP62 siRNAs (dark grey bars) or siRNAs targeting the indicated DDX (light grey bars) and infected with the WSN-PB2-Nanoluc virus (0.0001 pfu/cell). Luciferase activities were measured in cell lysates prepared at 24 hpi. Three independent experiments were performed in triplicate. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the non-target siRNA condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (**p < 0.01; ***p < 0.001). (c) At 48 hours post-transfection (hpt) with the control non-target siRNAs (C) or with individual siRNAs targeting DDX19A+B (#1, #2 and #3), the levels of DDX19 protein were evaluated by immunoblot using an antibody that recognizes both A and B forms of DDX19, and the levels of DDX19A and DDX19B mRNAs were determined by RT-qPCR and were expressed as percentages of the control. Cropped blots are shown. The corresponding full-length blots are shown in Figure S2. (d–g) A549 cells were treated with control (dark grey lines) or DDX19 siRNAs (light grey lines) and infected with the following viruses at the indicated m.o.i. in pfu/cell: A/WSN/33(H1N1 WSN, 0.0001); A/Udorn/307/72(H3N2) (Udorn, 0.01); A/Paris/908/97(H3N2) (P908, 0.01); A/Paris/650/2004(H1N1) (P650, 0.01); Vesicular Stomatitis Virus (VSV, 0.0001); Adenovirus 5 (AdV, 6). At the indicated times post-infection (d,f,g) or at 24 hpi (e), the viral titers were determined by plaque assay on MDCK cells (d–f) or immunostaining (g). The results are expressed as the mean ± SEM of triplicates and the significance was tested with an unpaired, 2-tailed Student t test using GraphPad Prism software (*p < 0.05; **p < 0.01; ***p < 0.001).

Mentions: A549 cells were transfected with each of the non-toxic siRNAs and subsequently infected at a low multiplicity of infection (m.o.i.) with a recombinant A/WSN/33 virus carrying a luciferase reporter gene (WSN-PB2-Nanoluc). Luciferase activity was measured in cell lysates prepared at 24 hours post-infection (hpi) to monitor the efficiency of viral replication. As shown in Fig. 1a, IAV replication was significantly impaired upon silencing of 14 DDX proteins: DDX3X, DDX5, DDX13, DDX17, DDX19A, DDX19B, DDX24, DDX25, DDX28, DDX31, DDX39B, DDX41, DDX46 and DDX47. Among the DDX proteins that had been previously found to positively regulate IAV replication, DDX3X, DDX5 and DDX1719 and DDX39B15 were recovered in our screen, but not DDX2B, possibly due to low knock-down efficiency (Figure S1b). DDX21 silencing, shown by others to negatively regulate IAV replication14, had no significant effect in our screen. Upon co-silencing of DDX19 A and B forms (96% identity at the protein level), the reduction of luciferase signal was significantly greater than upon silencing of DDX19A or DDX19B alone (a 90% reduction compared to 48% and 59%, respectively, Fig. 1a), indicating a synergistic effect of DDX19A and DDX19B depletion. We repeated the experiment with three individual siRNAs targeting conserved regions between DDX19A and DDX19B (Fig. 1b,c). A strong 76 to 90% reduction of the luciferase signal was observed with all three individual siRNAs (Fig. 1b), thus ruling out any off-target effect. In the experiments described below, the combination of DDX19A and DDX19B siRNAs (thereafter named DDX19 siRNAs) was used.


Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs
IAV multiplication is reduced in DDX19-depleted cells.(a,b) A549 cells were treated with control non-target or NUP62 siRNAs (dark grey bars) or siRNAs targeting the indicated DDX (light grey bars) and infected with the WSN-PB2-Nanoluc virus (0.0001 pfu/cell). Luciferase activities were measured in cell lysates prepared at 24 hpi. Three independent experiments were performed in triplicate. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the non-target siRNA condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (**p < 0.01; ***p < 0.001). (c) At 48 hours post-transfection (hpt) with the control non-target siRNAs (C) or with individual siRNAs targeting DDX19A+B (#1, #2 and #3), the levels of DDX19 protein were evaluated by immunoblot using an antibody that recognizes both A and B forms of DDX19, and the levels of DDX19A and DDX19B mRNAs were determined by RT-qPCR and were expressed as percentages of the control. Cropped blots are shown. The corresponding full-length blots are shown in Figure S2. (d–g) A549 cells were treated with control (dark grey lines) or DDX19 siRNAs (light grey lines) and infected with the following viruses at the indicated m.o.i. in pfu/cell: A/WSN/33(H1N1 WSN, 0.0001); A/Udorn/307/72(H3N2) (Udorn, 0.01); A/Paris/908/97(H3N2) (P908, 0.01); A/Paris/650/2004(H1N1) (P650, 0.01); Vesicular Stomatitis Virus (VSV, 0.0001); Adenovirus 5 (AdV, 6). At the indicated times post-infection (d,f,g) or at 24 hpi (e), the viral titers were determined by plaque assay on MDCK cells (d–f) or immunostaining (g). The results are expressed as the mean ± SEM of triplicates and the significance was tested with an unpaired, 2-tailed Student t test using GraphPad Prism software (*p < 0.05; **p < 0.01; ***p < 0.001).
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f1: IAV multiplication is reduced in DDX19-depleted cells.(a,b) A549 cells were treated with control non-target or NUP62 siRNAs (dark grey bars) or siRNAs targeting the indicated DDX (light grey bars) and infected with the WSN-PB2-Nanoluc virus (0.0001 pfu/cell). Luciferase activities were measured in cell lysates prepared at 24 hpi. Three independent experiments were performed in triplicate. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the non-target siRNA condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (**p < 0.01; ***p < 0.001). (c) At 48 hours post-transfection (hpt) with the control non-target siRNAs (C) or with individual siRNAs targeting DDX19A+B (#1, #2 and #3), the levels of DDX19 protein were evaluated by immunoblot using an antibody that recognizes both A and B forms of DDX19, and the levels of DDX19A and DDX19B mRNAs were determined by RT-qPCR and were expressed as percentages of the control. Cropped blots are shown. The corresponding full-length blots are shown in Figure S2. (d–g) A549 cells were treated with control (dark grey lines) or DDX19 siRNAs (light grey lines) and infected with the following viruses at the indicated m.o.i. in pfu/cell: A/WSN/33(H1N1 WSN, 0.0001); A/Udorn/307/72(H3N2) (Udorn, 0.01); A/Paris/908/97(H3N2) (P908, 0.01); A/Paris/650/2004(H1N1) (P650, 0.01); Vesicular Stomatitis Virus (VSV, 0.0001); Adenovirus 5 (AdV, 6). At the indicated times post-infection (d,f,g) or at 24 hpi (e), the viral titers were determined by plaque assay on MDCK cells (d–f) or immunostaining (g). The results are expressed as the mean ± SEM of triplicates and the significance was tested with an unpaired, 2-tailed Student t test using GraphPad Prism software (*p < 0.05; **p < 0.01; ***p < 0.001).
Mentions: A549 cells were transfected with each of the non-toxic siRNAs and subsequently infected at a low multiplicity of infection (m.o.i.) with a recombinant A/WSN/33 virus carrying a luciferase reporter gene (WSN-PB2-Nanoluc). Luciferase activity was measured in cell lysates prepared at 24 hours post-infection (hpi) to monitor the efficiency of viral replication. As shown in Fig. 1a, IAV replication was significantly impaired upon silencing of 14 DDX proteins: DDX3X, DDX5, DDX13, DDX17, DDX19A, DDX19B, DDX24, DDX25, DDX28, DDX31, DDX39B, DDX41, DDX46 and DDX47. Among the DDX proteins that had been previously found to positively regulate IAV replication, DDX3X, DDX5 and DDX1719 and DDX39B15 were recovered in our screen, but not DDX2B, possibly due to low knock-down efficiency (Figure S1b). DDX21 silencing, shown by others to negatively regulate IAV replication14, had no significant effect in our screen. Upon co-silencing of DDX19 A and B forms (96% identity at the protein level), the reduction of luciferase signal was significantly greater than upon silencing of DDX19A or DDX19B alone (a 90% reduction compared to 48% and 59%, respectively, Fig. 1a), indicating a synergistic effect of DDX19A and DDX19B depletion. We repeated the experiment with three individual siRNAs targeting conserved regions between DDX19A and DDX19B (Fig. 1b,c). A strong 76 to 90% reduction of the luciferase signal was observed with all three individual siRNAs (Fig. 1b), thus ruling out any off-target effect. In the experiments described below, the combination of DDX19A and DDX19B siRNAs (thereafter named DDX19 siRNAs) was used.

View Article: PubMed Central - PubMed

ABSTRACT

Enhancing the knowledge of host factors that are required for efficient influenza A virus (IAV) replication is essential to address questions related to pathogenicity and to identify targets for antiviral drug development. Here we focused on the interplay between IAV and DExD-box RNA helicases (DDX), which play a key role in cellular RNA metabolism by remodeling RNA-RNA or RNA-protein complexes. We performed a targeted RNAi screen on 35 human DDX proteins to identify those involved in IAV life cycle. DDX19 was a major hit. In DDX19-depleted cells the accumulation of viral RNAs and proteins was delayed, and the production of infectious IAV particles was strongly reduced. We show that DDX19 associates with intronless, unspliced and spliced IAV mRNAs and promotes their nuclear export. In addition, we demonstrate an RNA-independent association between DDX19 and the viral polymerase, that is modulated by the ATPase activity of DDX19. Our results provide a model in which DDX19 is recruited to viral mRNAs in the nucleus of infected cells to enhance their nuclear export. Information gained from this virus-host interaction improves the understanding of both the IAV replication cycle and the cellular function of DDX19.

No MeSH data available.


Related in: MedlinePlus