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. 2011 Sep;85(17):8646-55.
doi: 10.1128/JVI.02559-10. Epub 2011 Jun 15.

The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection

Christian Wisskirchen  1 , Thomas H Ludersdorfer, Dominik A Müller, Eva Moritz, Jovan Pavlovic
Affiliations

The cellular RNA helicase UAP56 is required for prevention of double-stranded RNA formation during influenza A virus infection

Christian Wisskirchen et al. J Virol. 2011 Sep.

Abstract

The cellular DEAD box RNA helicase UAP56 plays a pivotal role in the efficient transcription/replication of influenza A virus. UAP56 is recruited by the nucleoprotein (NP) of influenza A viruses, and recent data revealed that the RNA helicase is required for the nuclear export of a subset of spliced and unspliced viral mRNAs. The fact that influenza viruses do not produce detectable amounts of double-stranded RNA (dsRNA) intermediates during transcription/replication suggests the involvement of cellular RNA helicases. Hence, we examined whether the RNA-unwinding activity of UAP56 or its paralog URH49 plays a role in preventing the accumulation of dsRNA during infection. First, our data showed that not only UAP56 but also its paralog URH49 can interact with NPs of avian and human influenza A viruses. The small interfering RNA (siRNA)-mediated depletion of either RNA helicase reduced the transport of M1 and hemagglutinin (HA) mRNAs and, to a lesser extent, NP and NS1 mRNAs into the cytoplasm. Moreover, we found that virus infection of UAP56-depleted cells leads to the rapid accumulation of dsRNA in the perinuclear region. In parallel, we observed a robust virus-mediated activation of dsRNA-dependent protein kinase R (PKR), indicating that the cellular RNA helicase UAP56 may be recruited by influenza virus to prevent dsRNA formation. The accumulation of dsRNA was blocked when actinomycin D or cycloheximide was used to inhibit viral transcription/replication or translation, respectively. In summary, we demonstrate that UAP56 is utilized by influenza A viruses to prevent the formation of dsRNA and, hence, the activation of the innate immune response.

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Figures

Fig. 1.
Fig. 1.
Human and avian influenza A virus nucleoproteins interact with the cellular helicases UAP56 and URH49. (A) Coimmunoprecipitation assays were performed with FLAG-tagged UAP56 or URH49 and avian or human influenza A virus nucleoproteins. The arrow indicates the position of the heavy chain of the antibodies used for immunoprecipitation (IP). IB, immunoblot; WCL, whole-cell lysate. (B) Affinity-purified GST-UAP56 or GST-URH49 and His-PR8-NP (A/PR8/34) or His-FPV-NP (A/FPV/Bratislawa/79) were mixed and assayed (30 nM each protein) for interactions in vitro using the Alpha screen technology. As a negative control GST alone was incubated with His-PR8-NP. The interaction of GST-UAP56 and His-PR8-NP was set to 100% as a reference for the other interactions.
Fig. 2.
Fig. 2.
Knockdown of UAP56 or URH49 results in a strong reduction of titers of influenza A virus. (A) A549 cells were treated with the indicated siRNAs for 48 h. Protein levels of UAP56 and URH49 were analyzed by Western blotting. MxA was used to monitor the induction of interferon in siRNA-treated cells. (B to E) A549 cells were treated with the indicated siRNAs for 48 h and subsequently infected with FPV (MOI = 0.1) (B), PR8 (MOI = 0.1) (C), A/Zurich/8665/2009(H1N1) (MOI = 1) (D), and VSV Indiana (MOI = 0.1) (E), and culture supernatants were taken 24 h postinfection and assayed for virus titers by using the TCID50 method. For VSV, virus titers were measured after 12 h. An asterisk indicates a P value of <0.05.
Fig. 3.
Fig. 3.
Reduced export of viral mRNAs in cells treated with UAP56- or URH49-specific siRNA upon infection with influenza A virus. (A) A549 cells were treated with the indicated siRNA for 72 h and subsequently infected with FPV for 4 h (MOI = 5). Cytoplasmic and nuclear RNAs were isolated, and 5 μg of RNA per lane was analyzed by Northern blotting. Methylene blue-stained 18S rRNA was used to normalize the total amounts of RNA in each lane. Precursor rRNA was detectable only in the nuclear RNA fraction. The separated RNAs were hybridized with DIG-labeled probes specific for influenza A virus M and NP segments. (B) Reverse transcription was performed with cytoplasmic and nuclear RNA using an oligo(dT) primer and 1 μg RNA. M1, NP, HA, and NS1 levels in the two subcellular fractions were analyzed by qPCR using specific primer pairs for the indicated viral mRNAs; results are shown as means of data from five independent experiments. (C and D) The nuclear or cytoplasmic localizations of NP (C) and M1 (D) mRNAs in A were quantified by measuring band intensities using Multi Gauge software from Fujifilm. Total mRNA from the cytoplasmic and nuclear fractions of control siRNA-treated cells was set as the reference. (E) Splicing of the influenza A virus M segment was not affected by the knockdown of UAP56 or UARH49. Reverse transcription was performed from cytoplasmic and nuclear RNAs using the oligo(dT) primer and 1 μg RNA. M1 and M2 levels were assessed by real-time qPCR using specific primer pairs for M1 and M2.
Fig. 4.
Fig. 4.
PKR is phosphorylated in UAP56 and URH49 knockdown cells upon infection with influenza A virus. (A) A549 cells were treated with 30 nM the indicated siRNAs for 72 h and subsequently infected with influenza virus PR8 or FPV for 4 h (MOI = 5). Total cell lysates were analyzed by Western blotting using specific antibodies against PKR, phospho-PKR, influenza virus M1, actin, UAP56, and URH49. (B and C) Phospho-PKR levels (B) and influenza virus M1 protein levels (C) were quantified by measuring band intensities using Multi Gauge software from Fujifilm.
Fig. 5.
Fig. 5.
dsRNA accumulates in the perinuclear region of UAP56 knockdown cells infected with human or avian influenza A virus. A549 cells were treated with the indicated siRNAs for 72 h and infected with FPV (A) or PR8 (B) for 4 h or mock infected (C). Cells were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100. Cells were stained by using an anti-influenza virus serum (1:300) and anti-dsRNA antibody (1:3,000). Pictures were taken with a Leica TCS-SP5 confocal microscope.
Fig. 6.
Fig. 6.
Treatment with cycloheximide or actinomycin D prevents accumulation of dsRNA in UAP56-depleted cells infected with influenza A virus. A549 cells were treated with the indicated siRNAs for 72 h. Cells were incubated with actinomycin D (0.5 μg/ml) (A) or cycloheximide (75 μg/ml) (B) 1 h before infection. Cells were infected with FPV (MOI = 5). Inhibitors remained in the medium during and after infection. Cells were fixed at 4 h postinfection and stained by using an anti-influenza virus serum (1:300) and anti-dsRNA antibody (1:3,000). Pictures were taken with a Leica TCS-SP5 confocal microscope.
Fig. 7.
Fig. 7.
Vesicular stomatitis virus induces accumulation of dsRNA in UAP56 knockdown cells. (A) A549 cells were treated with the indicated siRNAs for 72 h and subsequently infected with VSV Indiana for 4 h. Cells were fixed with 4% formaldehyde and permeabilized with 0.5% Triton X-100. Cells were stained by using a polyclonal anti-VSV serum (1:300) and anti-dsRNA antibody (1:3,000). Pictures were taken with a Leica TCS-SP5 confocal microscope.
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