The Host Factor ANP32A Is Required for Influenza A Virus vRNA and cRNA Synthesis

Figures

• 

  FIG 1. Influenza A virus genome replication. (A) Schematic of vRNA-to-cRNA replication by the resident RNA polymerase in vRNPs. Nascent cRNA is encapsidated by a newly synthesized RNA-free polymerase. (B) Schematic of cRNA-to-vRNA replication by the resident RNA polymerase aided by a second regulatory polymerase. Nascent vRNA is being encapsidated by a newly synthesized RNA-free polymerase.

• 

  FIG 2. ANP32A is required for IAV genome replication. (A) Human HEK-293T cells or (B) chicken DF-1 cells were infected with influenza A/WSN/33 (H1N1) virus carrying either a lysine (K; WSN) or glutamic acid (E; WSN-K627E) on residue 627 of the PB2 subunit for 6 h at an MOI of 1. The accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with Dunnett’s post hoc test) as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C-D) HEK-293T cells were transiently transfected with plasmids expressing GFP or chANP32A. At 24 h posttransfection, cells were infected with (C) WSN or (D) WSN-K627E virus at an MOI of 0.01. Infectious viral titers were determined by plaque assay at the indicated time points postinfection from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control…

• 

  FIG 3. The chANP32A LCAR domain is essential for avian RdRp stimulation. (A) Schematic of the wild-type chANP32A protein and the internal and terminal truncations introduced in this study. (B) Western blot analysis of whole cell lysates of HEK-293T cells transiently expressing wild-type or mutant chANP32A or a GFP control. (C) HEK-293T cells were transfected with plasmids expressing NP, PA, PB1, PB2 or PB2-K627E, segment 6 vRNA and either wild-type or truncated chANP32A. At 48h posttransfection, the accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with Dunnett’s post hoc test) as follows: ns, P > 0.05; *, P < 0.05; ****, P < 0.0001.

• 

  FIG 4. The PB2 627-domain does not affect viral RNA binding. (A) HEK-293T cells were transfected with plasmids expressing PA, PB2-627K (K) or PB2-627E (E), catalytically inactive PB1, a 47 nucleotide-long internally truncated segment 6 and where indicated chANP32A. 48 h posttransfection, cells were lysed and the viral RdRp was immunoprecipitated via a TAP-tag on PA. Co-precipitated vRNA was analyzed by primer extension and 12% PAGE. (B) Schematic of the influenza A virus vRNA promoter before and after binding with the viral RdRp. The distance between the Atto647N dye (red) and the Cy3 dye (orange) is indicated. (C) Schematic of the influenza A virus cRNA promoter before and after binding with the viral RdRp. The distance between the Atto647N dye (red) and the Cy3 dye (orange) is indicated. (D) vRNA promoter binding by the influenza A virus RdRp as analyzed by smFRET. Recombinant viral RdRp, either with a PB2 subunit with a lysine (627K) or glutamic acid (627E) at position 627 or with a subunit lacking the entire 627-domain (Δ535-667) was used. The FRET populations were fit with a single Gaussian curve. The RNA only (red) is shown as comparison. The mean FRET signal (E*) is indicated in…

• 

  FIG 5. Avian RdRp restriction in mammalian cells is independent of NP. (A) HEK-293T cells were transfected with plasmids expressing a 76 nucleotide-long internally truncated segment 5 vRNA, PA, PB1, PB2-627K (K) or PB2-627E (E), and where indicated NP and chANP32A. At 48 h posttransfection, the accumulation of viral RNA was analyzed by primer extension and 12% PAGE. (B) HEK-293T cells were transfected with plasmids expressing a 47 nucleotide-long internally truncated segment 6 vRNA, PA, PB1, PB2-627K or PB2-627E, and where indicated chANP32A. At 48 h posttransfection, the accumulation of viral RNA was analyzed by primer extension and 12% PAGE. (C) HEK-293T cells were transfected with plasmids expressing a 30 nucleotide-long internally truncated segment 6 vRNA, PA, PB1, PB2-627K or PB2-627E, and where indicated chANP32A. At 48 h posttransfection, the accumulation of viral RNA was analyzed by primer extension and 12% PAGE. Graphs show the relative mean intensity of viral RNA normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with…

• 

  FIG 6. ANP32A is required for both vRNA and cRNA synthesis. (A) Schematic of the wild-type vRNA promoter or a vRNA promoter carrying a G3A and C8U mutation in the 3′ vRNA promoter (red). (B) HEK-293T cells were transfected with plasmids expressing NP, PA, PB1, PB2-627K (K) or PB2-627E (E), segment 6 vRNA and chANP32A where indicated. 48h posttransfection, the accumulation of viral RNA was analyzed by primer extension and 6% PAGE. The graph shows the relative mean intensity of viral RNA produced from the mutant 3A8U vRNA template normalized to 5S rRNA from three independent biological replicates. Error bars represent the standard deviation of the means and asterisks represent a significant difference from the control group (one-way ANOVA with Dunnett’s post hoc test) as follows: ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (C) Schematic of the vRNA promoter carrying a G5U mutation in the 5′ vRNA promoter with and without the 3A8U 3′ vRNA promoter mutation. (D) HEK-293T cells were transfected with plasmids expressing NP, PA, PB1, PB2 and wild-type or mutant segment 6 vRNA as indicated. At 48 h posttransfection, the accumulation of viral RNA was analyzed by…