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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

The earliest steps of IAV replication are not affected by DDX19 depletion.A549 cells treated with control (C in a and b, dark grey bars in c and d) or DDX19 (19 in a and b, light grey bars in c and d) siRNAs were infected with WSN (5 pfu/cell). (a) Total extracts from control cells treated with CHX (+CHX) or not (−CHX) were prepared at 6 hpi and analyzed by immunoblots using antibodies directed against the indicated proteins. Cropped blots are shown. The corresponding full-length blots are shown in Figure S4. (b) Cytoplasmic and nuclear fractions were prepared at 4 hpi. Aliquots of the indicated subcellular fractions were analyzed by immunoblots with antibodies directed against MEK1/2 kinase (cytoplasmic marker), TBP (nuclear marker) and NP. Alternatively, total RNAs were extracted and the levels of GAPDH pre-mRNA, a nuclear marker, were determined by real-time RT-PCR. Results are expressed as the mean of two determinations of the crossing point value (Cp). Cropped blots are shown. The corresponding full-length blots are shown in Figure S5. (c,d) Infection was carried out for 6 h in the presence of CHX (100 µg/mL). Total RNAs were isolated from cytoplasmic (solid bars) and nuclear (hatched bars) fractions, and the levels of NP and NA vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean percentages ± SEM of cytoplasmic and nuclear vRNAs levels determined in three independent experiments (c). Total RNAs were extracted and the levels of NP or NA mRNAs and vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean ratios of mRNA/vRNA ± SEM determined in three independent experiments (d).
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f3: The earliest steps of IAV replication are not affected by DDX19 depletion.A549 cells treated with control (C in a and b, dark grey bars in c and d) or DDX19 (19 in a and b, light grey bars in c and d) siRNAs were infected with WSN (5 pfu/cell). (a) Total extracts from control cells treated with CHX (+CHX) or not (−CHX) were prepared at 6 hpi and analyzed by immunoblots using antibodies directed against the indicated proteins. Cropped blots are shown. The corresponding full-length blots are shown in Figure S4. (b) Cytoplasmic and nuclear fractions were prepared at 4 hpi. Aliquots of the indicated subcellular fractions were analyzed by immunoblots with antibodies directed against MEK1/2 kinase (cytoplasmic marker), TBP (nuclear marker) and NP. Alternatively, total RNAs were extracted and the levels of GAPDH pre-mRNA, a nuclear marker, were determined by real-time RT-PCR. Results are expressed as the mean of two determinations of the crossing point value (Cp). Cropped blots are shown. The corresponding full-length blots are shown in Figure S5. (c,d) Infection was carried out for 6 h in the presence of CHX (100 µg/mL). Total RNAs were isolated from cytoplasmic (solid bars) and nuclear (hatched bars) fractions, and the levels of NP and NA vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean percentages ± SEM of cytoplasmic and nuclear vRNAs levels determined in three independent experiments (c). Total RNAs were extracted and the levels of NP or NA mRNAs and vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean ratios of mRNA/vRNA ± SEM determined in three independent experiments (d).

Mentions: To test whether the nuclear import of parental vRNAs is affected by DDX19 silencing, A549 cells were treated with cycloheximide (CHX) during infection to inhibit de novo viral protein expression, and incoming vRNAs were monitored in subcellular fractions. As shown in Fig. 3a, no viral protein accumulation was detected at 6 hpi in the presence of CHX, demonstrating the efficiency of CHX treatment. The quality of subcellular fractionation was controlled in each experiment by immunoblotting using antibodies against the MEK1/2 kinase, a cytoplasmic marker, and the TATA Binding Protein (TBP), a nuclear marker. In addition, the levels of GAPDH pre-mRNAs were evaluated by real-time RT-PCR and used as a nuclear marker. Representative data are shown in Fig. 3b. The quality of subcellular fractionation was not affected by viral infection (Fig. 3b).


Influenza A Virus Polymerase Recruits the RNA Helicase DDX19 to Promote the Nuclear Export of Viral mRNAs
The earliest steps of IAV replication are not affected by DDX19 depletion.A549 cells treated with control (C in a and b, dark grey bars in c and d) or DDX19 (19 in a and b, light grey bars in c and d) siRNAs were infected with WSN (5 pfu/cell). (a) Total extracts from control cells treated with CHX (+CHX) or not (−CHX) were prepared at 6 hpi and analyzed by immunoblots using antibodies directed against the indicated proteins. Cropped blots are shown. The corresponding full-length blots are shown in Figure S4. (b) Cytoplasmic and nuclear fractions were prepared at 4 hpi. Aliquots of the indicated subcellular fractions were analyzed by immunoblots with antibodies directed against MEK1/2 kinase (cytoplasmic marker), TBP (nuclear marker) and NP. Alternatively, total RNAs were extracted and the levels of GAPDH pre-mRNA, a nuclear marker, were determined by real-time RT-PCR. Results are expressed as the mean of two determinations of the crossing point value (Cp). Cropped blots are shown. The corresponding full-length blots are shown in Figure S5. (c,d) Infection was carried out for 6 h in the presence of CHX (100 µg/mL). Total RNAs were isolated from cytoplasmic (solid bars) and nuclear (hatched bars) fractions, and the levels of NP and NA vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean percentages ± SEM of cytoplasmic and nuclear vRNAs levels determined in three independent experiments (c). Total RNAs were extracted and the levels of NP or NA mRNAs and vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean ratios of mRNA/vRNA ± SEM determined in three independent experiments (d).
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f3: The earliest steps of IAV replication are not affected by DDX19 depletion.A549 cells treated with control (C in a and b, dark grey bars in c and d) or DDX19 (19 in a and b, light grey bars in c and d) siRNAs were infected with WSN (5 pfu/cell). (a) Total extracts from control cells treated with CHX (+CHX) or not (−CHX) were prepared at 6 hpi and analyzed by immunoblots using antibodies directed against the indicated proteins. Cropped blots are shown. The corresponding full-length blots are shown in Figure S4. (b) Cytoplasmic and nuclear fractions were prepared at 4 hpi. Aliquots of the indicated subcellular fractions were analyzed by immunoblots with antibodies directed against MEK1/2 kinase (cytoplasmic marker), TBP (nuclear marker) and NP. Alternatively, total RNAs were extracted and the levels of GAPDH pre-mRNA, a nuclear marker, were determined by real-time RT-PCR. Results are expressed as the mean of two determinations of the crossing point value (Cp). Cropped blots are shown. The corresponding full-length blots are shown in Figure S5. (c,d) Infection was carried out for 6 h in the presence of CHX (100 µg/mL). Total RNAs were isolated from cytoplasmic (solid bars) and nuclear (hatched bars) fractions, and the levels of NP and NA vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean percentages ± SEM of cytoplasmic and nuclear vRNAs levels determined in three independent experiments (c). Total RNAs were extracted and the levels of NP or NA mRNAs and vRNAs were determined by strand specific RT-qPCR. The results are expressed as the mean ratios of mRNA/vRNA ± SEM determined in three independent experiments (d).
Mentions: To test whether the nuclear import of parental vRNAs is affected by DDX19 silencing, A549 cells were treated with cycloheximide (CHX) during infection to inhibit de novo viral protein expression, and incoming vRNAs were monitored in subcellular fractions. As shown in Fig. 3a, no viral protein accumulation was detected at 6 hpi in the presence of CHX, demonstrating the efficiency of CHX treatment. The quality of subcellular fractionation was controlled in each experiment by immunoblotting using antibodies against the MEK1/2 kinase, a cytoplasmic marker, and the TATA Binding Protein (TBP), a nuclear marker. In addition, the levels of GAPDH pre-mRNAs were evaluated by real-time RT-PCR and used as a nuclear marker. Representative data are shown in Fig. 3b. The quality of subcellular fractionation was not affected by viral infection (Fig. 3b).

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