Cellular hnRNPAB binding to viral nucleoprotein inhibits flu virus replication by blocking nuclear export of viral mRNA

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  Figure 1. HnRNPAB inhibits the replication of different subtypes of influenza A virus (A) Dynamics of hnRNPAB mRNA in cells infected with influenza A virus. A549 cells were infected with H7N9, H5N1, H9N2, H3N2, or PR8 viruses (MOI = 1.0), respectively. The abundance of viral mRNA at the indicated time was detected using qRT-PCR. Expression was normalized to the mRNA level. (B) Dynamics of hnRNPAB protein in cells infected with influenza A virus. Cell lysates collected from (A) at the indicated time were analyzed by immunoblotting with rabbit anti-hnRNPAB mAb, mouse anti-NP mAb, and rabbit anti-GAPDH pAbs. Quantitation of the immunoreactive protein bands on the blots was performed using ImageJ software, and the hnRNPAB band was normalized using the GAPDH control. (C) Inhibition of H7N9 virus replication in hnRNPAB-overexpressing cells. 293T cells were transfected with myc-hnRNPAB. The myc vector was used as a control. At 24 h after transfection, cells were infected with H7N9, H5N1, H9N2, H3N2, or PR8 viruses (MOI = 1.0), respectively, and cultured for 24 h. The TCID50 was measured by IFA (immunofluorescence assay) with the mouse anti-M2 mAb. (D) Enhancement of H7N9 virus replication in…

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  Figure 2. hnRNPAB interacts with viral protein NP via RNA (A) HnRNPAB colocalization with viral protein NP. A549 cells were infected with H7N9 virus for 12 h and were then fixed with 4% paraformaldehyde. Cells were stained with anti-NP and anti-hnRNPAB antibodies. Nuclei were stained with DAPI. Scale bars, 10 µm. (B) Co-IP assay of viral protein NP and endogenous hnRNPAB in infected cells. 293T cells were infected with H7N9 at MOI = 1.0 for 24 h and immunoprecipitated with anti-mouse IgG and anti-hnRNPAB mAb, followed by western blotting. (C) Co-IP assay of FLAG-NP and myc-hnRNPAB with RNase treatment. 293T cells were co-transfected with myc-hnRNPAB and FLAG-NP or FLAG for 24 h. Cell lysates were treated with or without RNase for 2 h and immunoprecipitated with the mouse anti-FLAG mAb, followed by western blotting with the anti-FLAG mAb and anti-myc pAbs. (D) GST pull-down assay of GST-hnRNPAB and His-NP. The purified GST, GST-hnRNPAB, and His-NP were incubated together as indicated. One group was added with the cellular RNA extract, and another group was treated with RNase. (E) H7N9 virus replication in cells co-expressing hnRNPAB and NP. 293T cells were co-transfected with FLAG-NP and/or…

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  Figure 3. Identification of the Gly motif of hnRNPAB for the inhibition of flu virus replication (A and B) Inhibition of H7N9 virus replication in mouse and chicken hnRNPAB-overexpressing cells. 293T cells were transfected with myc-m-AB (A) or myc-c-AB (B) for 24 h and infected with H7N9 virus at MOI = 1.0 for 16 h. Cell lysates was analyzed using western blotting with the indicated antibody. (C) Schematic representation of the CBFNT, RRM1, RRM2, and Gly domains of hnRNPAB. The numbers indicate amino acid (aa) positions. (D and E) Gly domain of hnRNPAB is critical to inhibit H7N9 virus replication. 293T cells were transfected with myc-tagged hnRNPAB or the hnRNPAB-truncated mutants CBFNT, RRMs, and Gly (D), or hnRNPAB-deleted mutants ΔCBFNT, ΔRRMs, and ΔGly (E) for 24 h, and these cells were infected with H7N9 virus for 16 h. Cell extracts were analyzed by western blotting using anti-NP mAb, anti-NS1 mAb, and anti-GAPDH pAbs. (F) The residues 241–263 aa and 312–332 aa are important for Gly function. 293T cells were transfected with the plasmids myc, myc-tagged hnRNPAB, or hnRNPAB-deleted mutants Δ254–254 aa, Δ241–263 aa, and Δ312–332 aa for 24 h and were infected with H7N9 virus at MOI =…

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  Figure 4. HnRNPAB blocks nuclear export of viral mRNA (A and B) FISH analysis of the distribution of poly(A)+ RNA in cells transfected with hnRNPAB. 293T cells were transfected without (A) or with (B) GFP-hnRNPAB for 12 h and then probed with cy3-oligo(dT)40 for poly(A)+RNA. Nuclei were stained with DAPI. Scale bars, 10 µm. (C) FISH analysis of the distribution of poly(A)+RNA in A549 cells that were transfected with si-hnRNPAB or si-NC as control. Scale bars, 10 µm. (D) Fluorescence intensity for the nuclear/cytoplasmic ratio of poly(A)+RNA in hnRNPAB-overexpressing and knockdown cells was analyzed by countering 100 cells using ImageJ software. (E) FISH analysis of poly(A)+RNA distribution in cells transfected with the Gly domain of hnRNPAB. 293T cells were transfected with GFP-Gly of hnRNPAB for 12 h and probed with cy3-oligo(dT)40. Scale bars, 10 µm. (F) RNA pull-down assay of hnRNPAB. 293T cells were transfected with GFP-tagged hnRNPAB or indicated truncation mutants for 24 h and were then subjected to biotin-oligo(dT) assay and western blotting. (G) Poly(A)+RNA-distributed FISH analysis in cells transfected with the Gly domain-truncated mutants. 293T cells were transfected with…

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  Figure 5. HnRNPAB inhibits viral mRNA binding to NXF1 (A) The impact of NXF1, UAP56, and ALY on H7N9 virus replication. 293T cells were transfected with the indicated siRNAs for 36 h and then infected with H7N9 virus for 12 h. Cell lysates were analyzed with mAbs recognizing ALY, UAP56, NXF1, and NS1 in western blotting. (B) The nucleo-cytoplasmic ratio of H7N9 viral mRNA in NXF1, UAP56, or ALY knockdown cells. Cells from (A) were subjected to nuclear-cytoplasmic separation. RT-qPCR assays were performed for the H7N9 M1 mRNA analysis. (C) Co-IP assay of FLAG-ALY and myc-hnRNPAB. 293T cells were co-transfected with myc-hnRNPAB and FLAG-ALY. Cell lysates were added with or without RNase as indicated and then subjected to immunoprecipitation with anti-FLAG mAb and western blotting with anti-FLAG mAb and anti-myc pAb. FLAG was used as control. (D) Pull-down assay of UAP56 and hnRNPAB. 293T cells were transfected with myc-tagged UAP56 or FLAG-gst-hnRNPAB. Cell lysates were mixed and treated with or without RNase and incubated with glutathione beads. Pull-down and western blotting assays were performed with anti-FLAG mAb and anti-myc pAb. FLAG-gst-vc was used as a control. (E) Co-IP assay of…

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  Figure 6. Viral protein NP interaction with HnRNPAB strengthens inhibition of ALY-viral mRNA binding (A) Detection of viral protein NP interaction with ALY and UAP56. 293T cells were cotransfected with myc-UAP56, FLAG-hnRNPAB, FLAG-NP, or FLAG-ALY for 24 h. Cell lysates were subjected to immunoprecipitation and western blotting with anti-FLAG mAb and anti-myc pAbs. (B) RNA pull-down assay of ALY. 293T cells were infected with H7N9 virus for 12 h and were then transfected with myc tagged hnRNPAB for 24 h. Cell lysates were subjected to RNA pull-down and western blotting with anti-ALY mAb, anti-GAPDH pAbs, and anti-myc pAbs. (C) Inhibition of hnRNPAB's interaction with ALY by viral protein NP. 293T cells were transfected with myc-tagged hnRNPAB, FLAG-tagged NP, or ALY for 24 h. Co-IP and western blotting were conducted with anti-FLAG mAb and Anti-myc pAbs. (D) RNA pull-down assay of NXF1. Cell lysates from (B) were subjected to RNA pull-down and western blotting with anti-NXF1 mAb, anti-GAPDH, and anti-myc pAbs. (E) Inhibition model of viral mRNA nuclear export by hnRNPAB interaction with viral protein NP. To limit RNA nuclear export, hnRNPAB weakens the transfer of poly(A)+RNA from UAP56 to…