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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 viral polymerase and viral RNAs interact with DDX19.(a,b) A549 cells were infected with WSN (5 pfu/cell). At 3.5 hpi, DDX19 proteins were purified in the absence or presence of RNAse, using anti-DDX19 antibodies (α-DDX19) or control immunoglobulins (Ctrl IgG). (a) Inputs and α-DDX19/Ctrl IgG eluates were analyzed by immunoblot to detect DDX19, PB2-Strep and the IgG antibodies (upper, middle and lower panel, respectively). Results representative of two independent experiments are shown. The star indicates a non-specific band. (b) The levels of NP mRNAs and vRNAs co-purified with DDX19 are expressed as the mean ± SEM of four independent experiments. The background level of detection in control IgG eluates was 2.9 × 106 and 3.9 × 105 copies for NP vRNAs and NP mRNAs, respectively. Co-immunoprecipitated to input copy ratios between NP mRNAs and vRNAs differed significantly (multivariate linear model with an interaction term between sample type and RNA type, p = 0.0465). (c,d) HEK-293T cells expressing Strep-tagged wild-type DDX19B, mutant DDX19B or NXF1 were infected with WSN (5 pfu/cell). At 6 hpi, Strep-tagged proteins were purified, and the levels of co-purified mRNAs were determined (c). For wild-type DDX19B and NXF1, co-purified/total mRNAs ratios were normalized to the one obtained with the DDX19 mutant. The data represent the mean ± SEM of three independent experiments. Statistical analysis (multivariate linear model) did not demonstrate significance. (d) Lysates and eluates were analyzed by immunoblot using Strep-Tactin. The dashed line indicates the juxtaposition of non-adjacent lanes from the same immunoblot. (e,f) HEK-293T cells expressing the Gluc2-DDX19B variants were infected with the WSN-PB2-Gluc1 virus at a m.o.i. >1 pfu/cell. At 6 hpi, cells were lysed and normalized luminescence ratios (NLRs) were determined (e). Four independent experiments in triplicate were performed. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the DDX19B wild-type condition. The significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (****p < 0.0001). (f) Lysates were analyzed by immunoblot using anti-Gluc or anti-GAPDH antibodies. Cropped blots are shown in (a,d,f). Corresponding full-length blots are shown in Figures S7, S8 and S9, respectively.
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f5: The viral polymerase and viral RNAs interact with DDX19.(a,b) A549 cells were infected with WSN (5 pfu/cell). At 3.5 hpi, DDX19 proteins were purified in the absence or presence of RNAse, using anti-DDX19 antibodies (α-DDX19) or control immunoglobulins (Ctrl IgG). (a) Inputs and α-DDX19/Ctrl IgG eluates were analyzed by immunoblot to detect DDX19, PB2-Strep and the IgG antibodies (upper, middle and lower panel, respectively). Results representative of two independent experiments are shown. The star indicates a non-specific band. (b) The levels of NP mRNAs and vRNAs co-purified with DDX19 are expressed as the mean ± SEM of four independent experiments. The background level of detection in control IgG eluates was 2.9 × 106 and 3.9 × 105 copies for NP vRNAs and NP mRNAs, respectively. Co-immunoprecipitated to input copy ratios between NP mRNAs and vRNAs differed significantly (multivariate linear model with an interaction term between sample type and RNA type, p = 0.0465). (c,d) HEK-293T cells expressing Strep-tagged wild-type DDX19B, mutant DDX19B or NXF1 were infected with WSN (5 pfu/cell). At 6 hpi, Strep-tagged proteins were purified, and the levels of co-purified mRNAs were determined (c). For wild-type DDX19B and NXF1, co-purified/total mRNAs ratios were normalized to the one obtained with the DDX19 mutant. The data represent the mean ± SEM of three independent experiments. Statistical analysis (multivariate linear model) did not demonstrate significance. (d) Lysates and eluates were analyzed by immunoblot using Strep-Tactin. The dashed line indicates the juxtaposition of non-adjacent lanes from the same immunoblot. (e,f) HEK-293T cells expressing the Gluc2-DDX19B variants were infected with the WSN-PB2-Gluc1 virus at a m.o.i. >1 pfu/cell. At 6 hpi, cells were lysed and normalized luminescence ratios (NLRs) were determined (e). Four independent experiments in triplicate were performed. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the DDX19B wild-type condition. The significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (****p < 0.0001). (f) Lysates were analyzed by immunoblot using anti-Gluc or anti-GAPDH antibodies. Cropped blots are shown in (a,d,f). Corresponding full-length blots are shown in Figures S7, S8 and S9, respectively.

Mentions: To confirm the interaction of the endogenous DDX19 protein with the viral polymerase during infection and to assess whether this interaction requires the presence of RNAs, DDX19 was immunoprecipitated from lysates of HEK-293T cells infected with a recombinant WSN virus expressing a Strep-tagged PB2 protein (WSN-PB2-Strep) in the presence or absence of RNAse (Fig. 5a). Upon DDX19 immunoprecipitation (Fig. 5a, upper panel), the PB2-Strep protein was specifically co-immunoprecipitated in conditions where RNA integrity was preserved (Fig. 5a, middle panel, -RNase) or not (Fig. 5a, middle panel, +RNase). RNAs from cell lysates and eluates were extracted and the viral NP mRNAs and vRNAs were quantified by strand-specific RT-qPCR. They could not be detected in RNase-treated samples (data not shown), thus demonstrating the efficiency of RNase treatment. In RNase-free samples, a higher proportion of NP mRNAs was co-immunoprecipitated with DDX19 (8.6 × 106 copies −0.79% of the input NP mRNAs) compared to NP vRNAs (4.8 × 106 copies −0.10% of the input NP vRNAs) (p = 0.0465) (Fig. 5b).


Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs
The viral polymerase and viral RNAs interact with DDX19.(a,b) A549 cells were infected with WSN (5 pfu/cell). At 3.5 hpi, DDX19 proteins were purified in the absence or presence of RNAse, using anti-DDX19 antibodies (α-DDX19) or control immunoglobulins (Ctrl IgG). (a) Inputs and α-DDX19/Ctrl IgG eluates were analyzed by immunoblot to detect DDX19, PB2-Strep and the IgG antibodies (upper, middle and lower panel, respectively). Results representative of two independent experiments are shown. The star indicates a non-specific band. (b) The levels of NP mRNAs and vRNAs co-purified with DDX19 are expressed as the mean ± SEM of four independent experiments. The background level of detection in control IgG eluates was 2.9 × 106 and 3.9 × 105 copies for NP vRNAs and NP mRNAs, respectively. Co-immunoprecipitated to input copy ratios between NP mRNAs and vRNAs differed significantly (multivariate linear model with an interaction term between sample type and RNA type, p = 0.0465). (c,d) HEK-293T cells expressing Strep-tagged wild-type DDX19B, mutant DDX19B or NXF1 were infected with WSN (5 pfu/cell). At 6 hpi, Strep-tagged proteins were purified, and the levels of co-purified mRNAs were determined (c). For wild-type DDX19B and NXF1, co-purified/total mRNAs ratios were normalized to the one obtained with the DDX19 mutant. The data represent the mean ± SEM of three independent experiments. Statistical analysis (multivariate linear model) did not demonstrate significance. (d) Lysates and eluates were analyzed by immunoblot using Strep-Tactin. The dashed line indicates the juxtaposition of non-adjacent lanes from the same immunoblot. (e,f) HEK-293T cells expressing the Gluc2-DDX19B variants were infected with the WSN-PB2-Gluc1 virus at a m.o.i. >1 pfu/cell. At 6 hpi, cells were lysed and normalized luminescence ratios (NLRs) were determined (e). Four independent experiments in triplicate were performed. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the DDX19B wild-type condition. The significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (****p < 0.0001). (f) Lysates were analyzed by immunoblot using anti-Gluc or anti-GAPDH antibodies. Cropped blots are shown in (a,d,f). Corresponding full-length blots are shown in Figures S7, S8 and S9, respectively.
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f5: The viral polymerase and viral RNAs interact with DDX19.(a,b) A549 cells were infected with WSN (5 pfu/cell). At 3.5 hpi, DDX19 proteins were purified in the absence or presence of RNAse, using anti-DDX19 antibodies (α-DDX19) or control immunoglobulins (Ctrl IgG). (a) Inputs and α-DDX19/Ctrl IgG eluates were analyzed by immunoblot to detect DDX19, PB2-Strep and the IgG antibodies (upper, middle and lower panel, respectively). Results representative of two independent experiments are shown. The star indicates a non-specific band. (b) The levels of NP mRNAs and vRNAs co-purified with DDX19 are expressed as the mean ± SEM of four independent experiments. The background level of detection in control IgG eluates was 2.9 × 106 and 3.9 × 105 copies for NP vRNAs and NP mRNAs, respectively. Co-immunoprecipitated to input copy ratios between NP mRNAs and vRNAs differed significantly (multivariate linear model with an interaction term between sample type and RNA type, p = 0.0465). (c,d) HEK-293T cells expressing Strep-tagged wild-type DDX19B, mutant DDX19B or NXF1 were infected with WSN (5 pfu/cell). At 6 hpi, Strep-tagged proteins were purified, and the levels of co-purified mRNAs were determined (c). For wild-type DDX19B and NXF1, co-purified/total mRNAs ratios were normalized to the one obtained with the DDX19 mutant. The data represent the mean ± SEM of three independent experiments. Statistical analysis (multivariate linear model) did not demonstrate significance. (d) Lysates and eluates were analyzed by immunoblot using Strep-Tactin. The dashed line indicates the juxtaposition of non-adjacent lanes from the same immunoblot. (e,f) HEK-293T cells expressing the Gluc2-DDX19B variants were infected with the WSN-PB2-Gluc1 virus at a m.o.i. >1 pfu/cell. At 6 hpi, cells were lysed and normalized luminescence ratios (NLRs) were determined (e). Four independent experiments in triplicate were performed. The results are expressed as the mean percentages ± SEM of luciferase activity relative to the DDX19B wild-type condition. The significance was tested with a Holm-Sidak’s multiple comparisons test using GraphPad Prism software (****p < 0.0001). (f) Lysates were analyzed by immunoblot using anti-Gluc or anti-GAPDH antibodies. Cropped blots are shown in (a,d,f). Corresponding full-length blots are shown in Figures S7, S8 and S9, respectively.
Mentions: To confirm the interaction of the endogenous DDX19 protein with the viral polymerase during infection and to assess whether this interaction requires the presence of RNAs, DDX19 was immunoprecipitated from lysates of HEK-293T cells infected with a recombinant WSN virus expressing a Strep-tagged PB2 protein (WSN-PB2-Strep) in the presence or absence of RNAse (Fig. 5a). Upon DDX19 immunoprecipitation (Fig. 5a, upper panel), the PB2-Strep protein was specifically co-immunoprecipitated in conditions where RNA integrity was preserved (Fig. 5a, middle panel, -RNase) or not (Fig. 5a, middle panel, +RNase). RNAs from cell lysates and eluates were extracted and the viral NP mRNAs and vRNAs were quantified by strand-specific RT-qPCR. They could not be detected in RNase-treated samples (data not shown), thus demonstrating the efficiency of RNase treatment. In RNase-free samples, a higher proportion of NP mRNAs was co-immunoprecipitated with DDX19 (8.6 × 106 copies −0.79% of the input NP mRNAs) compared to NP vRNAs (4.8 × 106 copies −0.10% of the input NP vRNAs) (p = 0.0465) (Fig. 5b).

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