Volume 24, Issue 4, Pages 851-860 (July 2018) N-Terminal Acetylation by NatB Is Required for the Shutoff Activity of Influenza A Virus PA-X  Kohei Oishi, Seiya Yamayoshi, Hiroko Kozuka-Hata, Masaaki Oyama, Yoshihiro Kawaoka  Cell Reports  Volume 24, Issue 4, Pages 851-860 (July 2018) DOI: 10.1016/j.celrep.2018.06.078 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 24, 851-860DOI: (10.1016/j.celrep.2018.06.078) Copyright © 2018 The Authors Terms and Conditions

Figure 1 N-Terminal Acetylation by NatB Is Essential for the Shutoff Activity of PA-X in Yeast (A) Equal amounts of wild-type yeast strain BY4743 that was transformed with an empty plasmid or a plasmid encoding wild-type PA-X or PA-X K134A were spotted onto SD/-Ura plates. (B) Schematic diagram of the screening approach used to identify a host gene that is required for the shutoff activity of PA-X. ΔX or ΔY means the knockout yeast strain of gene X or Y. (C and D) Wild-type, ΔNat3, or ΔMdm20 yeast was transformed with an empty plasmid or a plasmid encoding wild-type PA-X, PA-X K134A (C), PA-X E2D, PA-X E2N, PA-X E2A, or PA-X E2P (D). Equal amounts of transformed wild-type, ΔNat3, or ΔMdm20 yeast were spotted onto SD/-Ura plates. PA-X in whole cell extracts prepared from each yeast transformant was analyzed by western blotting using the anti-FLAG monoclonal antibody. Cell Reports 2018 24, 851-860DOI: (10.1016/j.celrep.2018.06.078) Copyright © 2018 The Authors Terms and Conditions

Figure 2 N-Terminal Acetylation by NatB Is Required for the Shutoff Activity of PA-X in Mammalian Cells (A) Expression of NAA20 and NAA25 in wild-type, NAA20-KO, or NAA25-KO cells was analyzed by western blotting using the anti-NAA20 antibody or the anti-NAA25 antibody. (B) Shutoff activity and expression of wild-type PA-X in wild-type, NAA20-KO, or NAA25-KO cells were analyzed. Shutoff activity of wild-type PA-X in wild-type HAP1 cells was set to 100%. The shutoff activities are mean values ± SD (n = 3 biological replicates, n = 3 technical replicates). Average data from three independent experiments are shown. ∗∗p < 0.01 (one-way ANOVA followed by Dunnett’s test). (C) Shutoff activity of wild-type PA-X in wild-type, NAA20-KO, or NAA25-KO cells co-expressed with GFP, NAA20, or NAA25 was analyzed. Shutoff activity of wild-type PA-X co-expressed with GFP in wild-type HAP1 cells was set to 100%. Expression of PA-X in wild-type, NAA20-KO, or NAA25-KO cells was analyzed by western blotting using an anti-PA antibody. The shutoff activities are mean values ± SD (n = 3 biological replicates, n = 3 technical replicates). Average data from three independent experiments are shown. ∗∗p < 0.01 according a two-tailed unpaired Student’s t test. (D) Assessment of N-terminal acetylation of PA-X. N-terminal acetylation of wild-type PA-X expressed in wild-type, NAA20-KO, or NAA25-KO cells was examined by mass spectrometry. Differences in N-terminal acetylation frequencies between wild-type cells and each KO cell line were statistically analyzed by the prop.test. (A–C) β-actin served as a loading control. Cell Reports 2018 24, 851-860DOI: (10.1016/j.celrep.2018.06.078) Copyright © 2018 The Authors Terms and Conditions

Figure 3 The Second Amino Acid, Recognized by NatB, Is Required for the Shutoff Activity of PA-X in a NatB-Dependent Manner (A‒C) Shutoff activity of wild-type or the indicated PA-X mutant possessing an N-terminal second amino acid that is recognized and acetylated by NatB or other NAT family members in HAP1 (A), 293 (B), or A549 (C) cells. Average data from three independent experiments are shown. Shutoff activity of wild-type PA-X was set to 100%. ∗∗p < 0.01 (one-way ANOVA followed by Dunnett’s test). (A) Expression of wild-type PA-X or each PA-X mutant was detected by western blotting using the anti-FLAG antibody. (D‒G) Shutoff activity of PA-X E2D (D), PA-X E2N (E), PA-X E2A (F), or PA-X E2P (G) in wild-type, NAA20-KO, or NAA25-KO cells. Shutoff activity of wild-type PA-X was set to 100%. The shutoff activities are mean values ± SD (n = 3 biological replicates, n = 3 technical replicates). ∗∗p < 0.01 (one-way ANOVA followed by Dunnett’s test). (H) Assessment of N-terminal acetylation of PA-X mutants. N-terminal acetylation of PA-X E2A and PA-X E2P was examined by mass spectrometry. Cell Reports 2018 24, 851-860DOI: (10.1016/j.celrep.2018.06.078) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Importance of N-Terminal Acetylation by NatB to the Shutoff Activity of PA-X Derived from Other Virus Subtypes (A) Alignment of the N-terminal amino acid sequences of PA-X among representative isolates of several subtypes of influenza A virus. The percentages of isolates that have glutamic acid (E) as the second amino acid of PA-X are shown in pie charts. The total numbers of isolates (N) are also indicated. (B) Shutoff activity of PA-X derived from H1N1pdm09, H3N2, H5N1, and H7N9 viruses in wild-type, NAA20-KO, or NAA25-KO cells. Shutoff activity of each wild-type PA-X in wild-type HAP1 cells was set to 100%. The shutoff activity shown is the mean values ± SD (n = 3 biological replicates, n = 3 technical replicates). ∗∗p < 0.01 (one-way ANOVA followed by Dunnett’s test). Cell Reports 2018 24, 851-860DOI: (10.1016/j.celrep.2018.06.078) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Contribution of N-Terminal Acetylation by NatB to Viral Polymerase Activity and Virus Growth in Mammalian Cells (A) Viral polymerase activity in wild-type, NAA20-KO, or NAA25-KO cells. Wild-type, NAA20-KO, or NAA25-KO cells were transfected with plasmids encoding PB2, PB1, PA, and NP, with a plasmid for the expression of viral RNA encoding the firefly luciferase, and with a plasmid encoding Renilla luciferase as a transfection control. Firefly and Renilla luciferase activities were measured by using a dual-luciferase assay. Viral polymerase activity was calculated by normalization of the firefly luciferase activity to the Renilla luciferase activity. The expression of PA, Renilla luciferase, and β-actin was analyzed by western blotting using anti-PA, anti-Renilla luciferase, and anti-β-actin antibodies. The polymerase activity in wild-type HAP1 cells was set to 100%. The data are shown as mean relative polymerase activities ± SD (n = 3 biological replicates, n = 3 technical replicates). ∗∗p < 0.01 (one-way ANOVA followed by Dunnett’s test). β-actin served as a loading control. (B) Growth kinetics of A/WSN/33 (H1N1) virus in wild-type, NAA20-KO, or NAA25-KO cells. A/WSN/33 virus was inoculated at an MOI = 0.0001 and virus titers were assessed at the indicated time points by means of plaque assays. The data are shown as mean virus titers ± SD (n = 2 technical replicates). Representative data from two individual experiments are shown. ∗p < 0.05, ∗∗p < 0.01, respectively (two-way ANOVA followed by Turkey’s test). (C) Viral polymerase activity of each mutant PA. Wild-type HAP1 cells were transfected with plasmids encoding PB2, PB1, NP, and wild-type or mutant PA, with a plasmid for the expression of viral RNA encoding the firefly luciferase, and with a plasmid encoding Renilla luciferase as a transfection control. The expression of each mutant PA and Renilla luciferase was analyzed by western blotting. Polymerase activity was calculated as described in (A). The data are shown as mean relative polymerase activities ± SD (n = 3 biological replicates, n = 3 technical replicates). ∗∗p < 0.01 (one-way ANOVA followed by Dunnett’s test). β-actin served as a loading control. Cell Reports 2018 24, 851-860DOI: (10.1016/j.celrep.2018.06.078) Copyright © 2018 The Authors Terms and Conditions