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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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The nuclear export of IAV mRNAs is dependent on DDX19.(a,b) A549 cells were treated with control (dark grey bars) or DDX19 (light grey bars) siRNAs and infected with WSN (5 pfu/cell). Cytoplasmic and nuclear extracts were prepared at 2 hpi (a) or 4 hpi (b), and poly(A)+ mRNAs were purified. The levels of viral mRNAs were determined by RT-qPCR. The percentages of mRNAs detected in the cytoplasmic fraction (solid bars) and nuclear fraction (hatched bars) are shown. The results are expressed as the mean ± SEM of three independent experiments, and the significance was tested with paired t test using GraphPad Prism software (*p < 0.05; ***p < 0.001). (c,d) HEK-293T cells were transfected with DDX19 or control siRNAs and then with siRNA-resistant DDX19B plasmids or empty vector. After 24 h, cells were infected with the WSN-PB2-Nanoluc virus (0.01 pfu/cell) and luciferase activities were measured at 24 hpi (c). The results are expressed as the mean percentages ± SEM of luciferase activity relative to the control siRNA + empty vector condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (*p < 0.05). (d) Lysates from the RNAi rescue experiment shown in c were analyzed by immunoblot using Strep-Tactin or anti-GAPDH antibodies. The ⦸, symbol indicates cells transfected with the empty vector. Cropped blots are shown. The corresponding full-length blots are shown in Figure S6.
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f4: The nuclear export of IAV mRNAs is dependent on DDX19.(a,b) A549 cells were treated with control (dark grey bars) or DDX19 (light grey bars) siRNAs and infected with WSN (5 pfu/cell). Cytoplasmic and nuclear extracts were prepared at 2 hpi (a) or 4 hpi (b), and poly(A)+ mRNAs were purified. The levels of viral mRNAs were determined by RT-qPCR. The percentages of mRNAs detected in the cytoplasmic fraction (solid bars) and nuclear fraction (hatched bars) are shown. The results are expressed as the mean ± SEM of three independent experiments, and the significance was tested with paired t test using GraphPad Prism software (*p < 0.05; ***p < 0.001). (c,d) HEK-293T cells were transfected with DDX19 or control siRNAs and then with siRNA-resistant DDX19B plasmids or empty vector. After 24 h, cells were infected with the WSN-PB2-Nanoluc virus (0.01 pfu/cell) and luciferase activities were measured at 24 hpi (c). The results are expressed as the mean percentages ± SEM of luciferase activity relative to the control siRNA + empty vector condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (*p < 0.05). (d) Lysates from the RNAi rescue experiment shown in c were analyzed by immunoblot using Strep-Tactin or anti-GAPDH antibodies. The ⦸, symbol indicates cells transfected with the empty vector. Cropped blots are shown. The corresponding full-length blots are shown in Figure S6.

Mentions: We next examined the potential role of DDX19 in the export of viral mRNAs. To this end, siRNA-treated A549 cells were infected and subjected to subcellular fractionation at 2 and 4 hpi. The levels of cytoplasmic and nuclear PB2, NP, NA, NS1 and NS2 mRNAs were then determined by RT-qPCR (Fig. 4a,b). At 2 hpi in control cells, a substantial proportion of each mRNA species (29% to 57%) was found in the cytoplasm (Fig. 4a, dark grey solid bars). In DDX19-silenced cells, the proportions of cytoplasmic viral mRNAs were strikingly reduced (3% to 8%) (Fig. 4a, light grey solid bars). The sensitivity of the M1- and M2-specific RT-qPCR was insufficient to evaluate accurately the levels of cytoplasmic and nuclear M1 and M2 mRNAs at 2 hpi. At 4 hpi in control cells, the viral mRNAs appeared equally distributed in both fractions (PB2, NP, NA) or predominantly cytoplasmic for NS1 and NS2 mRNAs (Fig. 4b). In contrast, in DDX19-silenced cells, the viral mRNAs remained predominantly nuclear, with only 24% to 34% of the viral mRNAs being detected in the cytoplasm (Fig. 4b). Although these differences were repeatedly observed (n = 3), the effect of DDX19 silencing at 4 hpi was generally not significant upon statistical analysis.


Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs
The nuclear export of IAV mRNAs is dependent on DDX19.(a,b) A549 cells were treated with control (dark grey bars) or DDX19 (light grey bars) siRNAs and infected with WSN (5 pfu/cell). Cytoplasmic and nuclear extracts were prepared at 2 hpi (a) or 4 hpi (b), and poly(A)+ mRNAs were purified. The levels of viral mRNAs were determined by RT-qPCR. The percentages of mRNAs detected in the cytoplasmic fraction (solid bars) and nuclear fraction (hatched bars) are shown. The results are expressed as the mean ± SEM of three independent experiments, and the significance was tested with paired t test using GraphPad Prism software (*p < 0.05; ***p < 0.001). (c,d) HEK-293T cells were transfected with DDX19 or control siRNAs and then with siRNA-resistant DDX19B plasmids or empty vector. After 24 h, cells were infected with the WSN-PB2-Nanoluc virus (0.01 pfu/cell) and luciferase activities were measured at 24 hpi (c). The results are expressed as the mean percentages ± SEM of luciferase activity relative to the control siRNA + empty vector condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (*p < 0.05). (d) Lysates from the RNAi rescue experiment shown in c were analyzed by immunoblot using Strep-Tactin or anti-GAPDH antibodies. The ⦸, symbol indicates cells transfected with the empty vector. Cropped blots are shown. The corresponding full-length blots are shown in Figure S6.
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Related In: Results  -  Collection

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f4: The nuclear export of IAV mRNAs is dependent on DDX19.(a,b) A549 cells were treated with control (dark grey bars) or DDX19 (light grey bars) siRNAs and infected with WSN (5 pfu/cell). Cytoplasmic and nuclear extracts were prepared at 2 hpi (a) or 4 hpi (b), and poly(A)+ mRNAs were purified. The levels of viral mRNAs were determined by RT-qPCR. The percentages of mRNAs detected in the cytoplasmic fraction (solid bars) and nuclear fraction (hatched bars) are shown. The results are expressed as the mean ± SEM of three independent experiments, and the significance was tested with paired t test using GraphPad Prism software (*p < 0.05; ***p < 0.001). (c,d) HEK-293T cells were transfected with DDX19 or control siRNAs and then with siRNA-resistant DDX19B plasmids or empty vector. After 24 h, cells were infected with the WSN-PB2-Nanoluc virus (0.01 pfu/cell) and luciferase activities were measured at 24 hpi (c). The results are expressed as the mean percentages ± SEM of luciferase activity relative to the control siRNA + empty vector condition, and the significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (*p < 0.05). (d) Lysates from the RNAi rescue experiment shown in c were analyzed by immunoblot using Strep-Tactin or anti-GAPDH antibodies. The ⦸, symbol indicates cells transfected with the empty vector. Cropped blots are shown. The corresponding full-length blots are shown in Figure S6.
Mentions: We next examined the potential role of DDX19 in the export of viral mRNAs. To this end, siRNA-treated A549 cells were infected and subjected to subcellular fractionation at 2 and 4 hpi. The levels of cytoplasmic and nuclear PB2, NP, NA, NS1 and NS2 mRNAs were then determined by RT-qPCR (Fig. 4a,b). At 2 hpi in control cells, a substantial proportion of each mRNA species (29% to 57%) was found in the cytoplasm (Fig. 4a, dark grey solid bars). In DDX19-silenced cells, the proportions of cytoplasmic viral mRNAs were strikingly reduced (3% to 8%) (Fig. 4a, light grey solid bars). The sensitivity of the M1- and M2-specific RT-qPCR was insufficient to evaluate accurately the levels of cytoplasmic and nuclear M1 and M2 mRNAs at 2 hpi. At 4 hpi in control cells, the viral mRNAs appeared equally distributed in both fractions (PB2, NP, NA) or predominantly cytoplasmic for NS1 and NS2 mRNAs (Fig. 4b). In contrast, in DDX19-silenced cells, the viral mRNAs remained predominantly nuclear, with only 24% to 34% of the viral mRNAs being detected in the cytoplasm (Fig. 4b). Although these differences were repeatedly observed (n = 3), the effect of DDX19 silencing at 4 hpi was generally not significant upon statistical analysis.

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