Eukaryotic RNA binding protein hnRNPH1 suppresses influenza A virus replication through interaction with virus NS1 protein

Figure 1.. hnRNPH1 interacts with IAV NS1 protein. (A) The flowchart of pull-down assay. Lysates of PR8-infected A549 cells were collected and incubated with NS1 antibody at 4°C overnight, followed by incubation with protein A/G. SDS-PAGE was performed and the sample was analyzed using LC-MS. (B) Protein A/G were incubated with NS1 antibody, and then incubated with lysate of A549 cells infected with PR8 virus (MOI = 11). SDS-PAGE was performed. (C) The cellular abundance of hnRNPH1 was examined in A549, 293 T and MDCK, and HeLa was used as a tumour cell control. (D) NS1-Flag was first enriched in 293 T using anti-Flag beads and then incubated with MDCK lysate for co-IP. (E and F) hnRNPH1 levels in PR8 infected A549 cells (MOI = 0.1) at different moments were detected. hnRNPH1 protein levels were revealed by western blot. The expression level of hnRNPH1 was quantified using Image J as fold change after normalized to tubulin. Values are means ± S.D. (n = 3). *p < 0.05, **p < 0.01 vs. Mock (E). The RNA levels of hnRNPH1 were revealed by qPCR. The relative amounts of hnRNPH1 and NS1 mRNAs were determined using the comparative (2-ΔΔCt) method. Values are means ± S.D. (n = 3). P values vs. Mock…

Figure 2.. hnRNPH1 overexpression can inhibit IAV proliferation. (A and B) hnRNPH1-NS1 interaction was validated of by Co-IP. NS1 and hnRNPH1 were co-transfected into 293 T. hnRNPH1-Flag was fished using anti-Myc magnetic beads binding NS1-Myc (A). And NS1-Myc was fished using anti-Flag magnetic beads combined with hnRNPH1-Flag (B). (C) NS1-Myc and hnRNPH1-Flag were co-transfected into 293 T cells. All the cell lysates were treated without or with RNase (10 and 100 μg/mL) at 37°C for 15 min, and then the hnRNPH1-Flag was fished using anti-Myc magnetic beads binding NS1-Myc. (D and E) The effects on IAV proteins after overexpression of hnRNPH1 was investigated. A549 cells were transfected with hnRNPH1-Flag. At 36 h post-transfection, cells were infected with PR8 (MOI = 0.1). The protein level of hnRNPH1, IAV HA and NS1 was revealed by western blot. The expression level of HA and NS1 was quantified using Image J as fold change after normalized to β-actin. Values are means ± S.D. (n = 3). P values vs. Negative Control are shown in the figure (D). The RNA level of hnRNPH1, IAV NA and NS1 was detected by qPCR. The relative amounts of mRNAs were determined using the comparative (2-ΔΔCt) method.…

Figure 3.. Interference of hnRNPH1 can promote IAV proliferation. (A) A549 cells were transfected with different siRNAs. The RNA level of hnRNPH1 was detected by qPCR. The relative amounts of mRNAs were normalized using the comparative (2-ΔΔCt) method. Values are means ± S.D. (n = 3). P values vs. Negative Control are shown in the figure. (B and C) The effects on IAV proteins after interference of hnRNPH1 were investigated. A549 cells were transfected with si-hnRNPH1. At 36 h interference, cells were infected with PR8 (MOI = 0.1). The protein level of hnRNPH1, IAV HA and NS1 was revealed by western blot. The expression level of HA and NS1 was quantified using ImageJ as fold change after normalized to β-actin. Values are means ± S.D. (n = 3). P values vs. Negative Control are shown in the figure (B). The RNA level of hnRNPH1, IAV NA and NS1 was detected by qPCR. The relative amounts of mRNAs were determined using the comparative (2-ΔΔCt) method. Values are means ± S.D. (n = 3). P values vs. Negative Control are shown in the figure (C). (D) siRNA interfered A549 was infected with PR8 (MOI = 0.1) at 36 h post-transfection, and supernatants were collected for plaque assay. Values are means ±…

Figure 4.. Endogenous and exogenous hnRNPH1 co-localize with NS1. (A) The excitation and emission wavelengths of BiFC fluorescent proteins. BiFC fluorescence is excited at 515 nm and detected at 527 nm. (B) A549 cells were transfected with VC155-NS1 and VN173-hnRNPH1. At 36 h post-transfection, cells were fixed and the co-localization of NS1 with hnRNPH1 was detected by fluorescence microcopy. Scale bars represent 25 and 10 μm. (C) A549 cells were transfected with NS1-Myc and hnRNPH1-Flag. Cells were fixed and the co-localization of NS1 with hnRNPH1 was detected by fluorescence microcopy after 36 h. Scale bars represent 25 and 10 μm. Co-localization was analyzed by Image J. The person’s correlation coefficient is shown in the figure. (D) PR8 (MOI = 10) infected A549 cells were fixed at 4, 6, 8 h.p.i.. The co-localization of NS1 with hnRNPH1 was detected by fluorescence microcopy. Scale bars represent 25 and 10 μm. (E) PR8 (MOI = 0.1) infected A549 cells were lysated at 4, 6, 8 h.p.i. Nucleus and cytopastic mRNA were separated, and the mRNA level of hnRNPH1 and NS1 was detected using qPCR. The relative amounts of Nucleus/cytoplasm mRNA were determined using the comparative (2-ΔΔCt) method.…

Figure 5.. hnRNPH1 affects NS1 function by regulating IFN-β, apoptosis and NS1 splicing. (A-C) PR8 (MOI = 10) infected A549 cells were were fixed at 0.5 h.p.i. (A), 1.5 h.p.i. (B), and 2–4 h.p.i. (C), then the virus NP protein or M1 protein were detected by fluorescence microcopy. Scale bars represent 25 μm. (D) The flowchart of NS1 interferon inhibition assay. 293 T cells were transfected with NS1, IFN-β-Luc and hnRNPH1-Flag or si-hnRNPH1. Poly (I:C) was transfected after 16 h. And the expression of luciferase was examined after 16 h. (E) All groups were transfected with IFN-β-Luc and plasmids labelled on the figure. Values are means ± S.D. (n = 3). P values vs. (IFN-β-Luc + poly (I:C) + NS1) are shown in the figure. (F) Different proteins of the apoptotic pathway after PR8 infection (MOI = 0.1) were detected at different moments. The protein level was revealed by western blot. The relative amounts of proteins were determined using Image J as fold change after normalized to tubulin. Values are means ± S.D. (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, #p < 0.05, ##p < 0.01 vs. Negative Control. (G and H) The effects on apoptosis after overexpression or interference of hnRNPH1 were…

Figure 6.. GYR-NLS domain of hnRNPH1 interact with the RBD domain of NS1. (A) Three different fragments of hnRNPH1. (B) A549 cells were transfected with VC155-NS1 and VN173-hnRNPH1-ΔGY, ΔR3GY, R1R2. At 36 h post-transfection, cells were fixed and the co-localization of NS1 with hnRNPH1 was detected by fluorescence microcopy. Scale bar represents 5 μm. (C) The molecular docking results for PR8-NS1 and hnRNPH1. Docking sites of NS1 dimer with hnRNPH1 full length are shown in the figure. (D) Different hnRNPH1 fragments and NS1 interaction sites were validated of by Co-IP. NS1 and hnRNPH1 fragments were co-transfected into 293 T. Then the hnRNPH1-Flag was fished using anti-Myc magnetic beads binding NS1-Myc. (E and F) Validation of the interaction sites between NS1 and hnRNPH1 using Co-IP assay with the different point mutant plasmids of NS1-Myc (E) and hnRNPH1-Flag (F), respectively. (G) A549 cells were transfected with plasmids expressing different point mutants of hnRNPH1. At 36 h post-transfection, cells were infected with PR8 (MOI = 0.1). Then the protein levels of hnRNPH1, virus PB1 and NS1 was detected by western blot.

Figure 7.. Hypothetical mechanistic map about the effects of hnRNPH1 on IAV proliferation. The yellow stars are hnRNPH1, a newly identified NS1-interacting protein. After apoptotic signalling is picked up by EGFR receptor on cell membrane, the signal is then transmitted to the PI3 K/Akt pathway, followed by activation of downstream Bcl-2 and MDM2 expression, which activates apoptosis and inhibits downstream Bax and p53, as well as activating the degradation of p53 by MDM2. hnRNPH1 may be able to affect viral proliferation by regulating p53 levels and also capable of disrupting the transcriptional replication of IAV by affecting the splicing of NS1 to NS2, thereby affecting viral proliferation.