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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.

No MeSH data available.


The accumulation of viral proteins and RNAs is reduced in DDX19-depleted cells.A549 cells were treated with control (C) or DDX19 (19) siRNAs and infected with WSN (5 pfu/cell). (a) Total extracts were prepared at the indicated times post-infection and analyzed by immunoblots using antibodies directed against the indicated proteins. Results representative of 3 independent experiments are shown. Cropped blots are shown. The corresponding full-length blots are shown in Figure S3. (b–e) The levels of NP or NA mRNAs and vRNAs (b or c and d or e, respectively) were determined at the indicated times post-infection by strand specific RT-qPCR and were normalized to the level of the same RNA species at 3 hpi in cells treated with the control siRNAs. The results are expressed as the mean ± SEM of three independent experiments and the significance was tested with a one-sample t test using GraphPad Prism Software (*p < 0.05; **p < 0.01; ***p < 0.001). Dashed lines were used to indicate that the Y-axes have been segmented. Different scales were used for the mRNA and vRNA graphs.
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f2: The accumulation of viral proteins and RNAs is reduced in DDX19-depleted cells.A549 cells were treated with control (C) or DDX19 (19) siRNAs and infected with WSN (5 pfu/cell). (a) Total extracts were prepared at the indicated times post-infection and analyzed by immunoblots using antibodies directed against the indicated proteins. Results representative of 3 independent experiments are shown. Cropped blots are shown. The corresponding full-length blots are shown in Figure S3. (b–e) The levels of NP or NA mRNAs and vRNAs (b or c and d or e, respectively) were determined at the indicated times post-infection by strand specific RT-qPCR and were normalized to the level of the same RNA species at 3 hpi in cells treated with the control siRNAs. The results are expressed as the mean ± SEM of three independent experiments and the significance was tested with a one-sample t test using GraphPad Prism Software (*p < 0.05; **p < 0.01; ***p < 0.001). Dashed lines were used to indicate that the Y-axes have been segmented. Different scales were used for the mRNA and vRNA graphs.

Mentions: To further document the role of DDX19 in IAV multiplication, the temporal accumulation of viral components was analyzed in DDX19-silenced cells during single-cycle infection with WSN. As shown in Fig. 2a, in control A549 cells, the accumulation of HA, NA, NP, M1 and NS1 viral proteins was detectable from 6 hpi. In DDX19-silenced cells, the accumulation of viral proteins was no longer (HA, NA, M1) or weakly (NP, NS1) detectable at 6 hpi, and was strongly reduced at 9 hpi compared to control cells.


Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs
The accumulation of viral proteins and RNAs is reduced in DDX19-depleted cells.A549 cells were treated with control (C) or DDX19 (19) siRNAs and infected with WSN (5 pfu/cell). (a) Total extracts were prepared at the indicated times post-infection and analyzed by immunoblots using antibodies directed against the indicated proteins. Results representative of 3 independent experiments are shown. Cropped blots are shown. The corresponding full-length blots are shown in Figure S3. (b–e) The levels of NP or NA mRNAs and vRNAs (b or c and d or e, respectively) were determined at the indicated times post-infection by strand specific RT-qPCR and were normalized to the level of the same RNA species at 3 hpi in cells treated with the control siRNAs. The results are expressed as the mean ± SEM of three independent experiments and the significance was tested with a one-sample t test using GraphPad Prism Software (*p < 0.05; **p < 0.01; ***p < 0.001). Dashed lines were used to indicate that the Y-axes have been segmented. Different scales were used for the mRNA and vRNA graphs.
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f2: The accumulation of viral proteins and RNAs is reduced in DDX19-depleted cells.A549 cells were treated with control (C) or DDX19 (19) siRNAs and infected with WSN (5 pfu/cell). (a) Total extracts were prepared at the indicated times post-infection and analyzed by immunoblots using antibodies directed against the indicated proteins. Results representative of 3 independent experiments are shown. Cropped blots are shown. The corresponding full-length blots are shown in Figure S3. (b–e) The levels of NP or NA mRNAs and vRNAs (b or c and d or e, respectively) were determined at the indicated times post-infection by strand specific RT-qPCR and were normalized to the level of the same RNA species at 3 hpi in cells treated with the control siRNAs. The results are expressed as the mean ± SEM of three independent experiments and the significance was tested with a one-sample t test using GraphPad Prism Software (*p < 0.05; **p < 0.01; ***p < 0.001). Dashed lines were used to indicate that the Y-axes have been segmented. Different scales were used for the mRNA and vRNA graphs.
Mentions: To further document the role of DDX19 in IAV multiplication, the temporal accumulation of viral components was analyzed in DDX19-silenced cells during single-cycle infection with WSN. As shown in Fig. 2a, in control A549 cells, the accumulation of HA, NA, NP, M1 and NS1 viral proteins was detectable from 6 hpi. In DDX19-silenced cells, the accumulation of viral proteins was no longer (HA, NA, M1) or weakly (NP, NS1) detectable at 6 hpi, and was strongly reduced at 9 hpi compared to control cells.

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.