Fig 2: GRSF1 knockdown reduces cytosolic accumulation and translation efficiency.a Lysates from WT 293 T or 293 T cells stably expressing shRNA for GRSF1 (clone 1–2 and clone 1–16) were used for immunoblotting to determine GRSF1 and β-actin expression. b GRSF1 mRNA expression from total cell lysates was determined by qRT-PCR using the 2^ΔΔ Ct method. c, d WT 293 T or shRNA GRSF1 clone 1–2 or 1–16 cells were fractionated into nuclear and cytosolic fractions. c Percentages of cytosolic GAPDH mRNA and U6 snRNA are shown. d Representative image of immunoblotting analysis for tubulin (cytosolic marker) and lamin (nuclear marker) detected by specific antibodies. e–h WT 293 T or shRNA GRSF1 clone 1–2 or 1–16 cells were transfected with Cal NP, PB1, PB2, pPolI-NP-luc and Nan PA with the indicated mutations. e Luciferase mRNA was quantified by qRT-PCR. f Luciferase activity was determined by the Dual-Luciferase Reporter Assay system. g Ratio of luciferase activity per mRNA, transformed to a normal distribution. h Cells were fractionated into nuclear and cytosolic fractions and mRNA was quantified by qRT-PCR. Percentages of cytosolic luciferase mRNA are shown. Error bars show the means plus/minus standard deviations (n = 3 biological replicates). One-way or Two-way ANOVA followed by Tukey’s multiple comparison test *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: GRSF1 binding regulates cytosolic accumulation and translation efficiency.a A model diagram showing the interaction of GRSF1 with the 10AGGGU14 binding site in the 5' UTR of NP luciferase mRNA. b 293 T cells were transfected with Cal NP and Nan or Cal vRdRp components along with the original pPolI-NP-luc or pPolI-NP-luc CA mutant. Anti-GRSF1 rabbit antibody was used for immunoprecipitation (IP) from whole cell lysates and RNA was extracted from the eluted material. Luciferase mRNAs before and after IP were quantitated by qRT-PCR and percentages of mRNA recovered after IP are shown. c, d 293 T cells were transfected with Cal NP, PB1, PB2, and either Nan PA or Nan PA mutants together with the original pPolI-NP-luc or the pPolI-NP-luc CA mutant c Luciferase mRNA was quantitated by qRT-PCR. d Luciferase activity was determined by the Dual-Luciferase Assay Reporter system. e Ratio of luciferase activity per mRNA, transformed to a normal distribution. f Percentages of cytosolic luciferase mRNA for both templates. Error bars show the means plus/minus standard deviations (n = 3 biological replicates). Two-Way ANOVA followed by Tukey’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001).

Fig 4: Attenuated NPCA virus growth in cultured cells. (a) Schematic of GRSF1 binding site AGGGU and nucleotide identity in pH1N1 Cal WT gene segments. Sequences of 5’UTR of each viral segment are shown as positive sense. Nucleotides at 10–14 of each gene segment are underlined or bolded. (b) A single-step growth curve of indicated viruses in A549 cells infected at an MOI of 0.2. At indicated times, virus in the supernatant was collected and titrated. (c) MDCK cells were infected with the indicated viruses at an MOI of 0.01 and cultured in the presence of acetylated trypsin. At indicated times, virus in the supernatant was collected and titrated. All error bars show means plus/minus the standard deviations (n = 3 biological replicates). Two-way ANOVA followed by Tukey’s multiple comparison test in PRISM (* p < 0.05, ** p < 0.01, *** p < 0.001).