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. Author manuscript; available in PMC: 2019 Jul 24.
Published in final edited form as: Cell Rep. 2018 Jul 24;24(4):851–860. doi: 10.1016/j.celrep.2018.06.078

N-terminal acetylation by NatB is required for the shutoff activity of influenza A virus PA-X

Kohei Oishi 1, Seiya Yamayoshi 1,#, Hiroko Kozuka-Hata 2, Masaaki Oyama 2, Yoshihiro Kawaoka 1,3,4,5,#
PMCID: PMC6296758  NIHMSID: NIHMS989458  PMID: 30044982

Summary

N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of influenza virus PA-X. The shutoff activity of PA-X was suppressed in NatB-deficient cells, and PA-X mutants that are not acetylated by NatB showed reduced shutoff activities. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells. Moreover, mutant PAs that are not acetylated by NatB lost their function in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation is required for the shutoff activity of PA-X and for viral polymerase activity.

Introduction

Posttranslational modifications, such as phosphorylation, methylation, ubiquitination, N-terminal acetylation, and lysine acetylation, modulate cellular biological activities. Phosphorylation, methylation, ubiquitination, and lysine acetylation modify the target protein to switch on and off of its function after protein biosynthesis (Hunter, 1995; Pickart, 2001; Yang and Seto, 2008). Unlike these posttranslational modifications, N-terminal acetylation of target proteins occurs during protein biosynthesis (Aksnes et al., 2016). N-terminal acetyltransferases (NATs), which include NatA through NatF, preferentially acetylate the N-terminal amino acid of approximately 80% of cellular proteins (Aksnes et al., 2016; Starheim et al., 2012). Therefore, N-terminal acetylation appears to be one of the major posttranslational modifications in cells. Despite its prevalence among proteins, N-terminal acetylation has not been extensively studied, and its importance has been evaluated in only a limited number of proteins such as Ubc12 (Scott et al., 2017; Scott et al., 2011), Rgs2 (Park et al., 2015), α-synuclein (Kang et al., 2012; Miotto et al., 2015), Hsp90 (Oh et al., 2017), tropomyosin (Singer and Shaw, 2003), and Arl3p (Behnia et al., 2004). Consequently, the roles of N-terminal acetylation are not fully understood.

Influenza A virus suppresses host gene expression (Garfinkel and Katze, 1992; Jagger et al., 2012; Nemeroff et al., 1998), and influenza virus PA-X plays central roles in this suppressive or shutoff activity (Jagger et al., 2012; Khaperskyy and McCormick, 2015). PA-X, which is expressed from the PA mRNA via a ribosomal frameshift (Firth et al., 2012), cleaves host mRNAs via its endonuclease activity to suppress host protein expression (Jagger et al., 2012). The shutoff activity of PA-X inhibits IFN-β production and antiviral antibody production in vivo and modulates virus pathogenicity in mice (Gao et al., 2015; Hayashi et al., 2015; Hu et al., 2016; Jagger et al., 2012). The N-terminal endonuclease active site, which is common to both PA and PA-X, and the C-terminal basic amino acids that are unique to PA-X are important for the shutoff activity of PA-X (Desmet et al., 2013; Jagger et al., 2012; Oishi et al., 2015). PA-X selectively cleaves RNA transcripts derived from the cellular Pol II promotor (Khaperskyy et al., 2016). Moreover, PA-X, purified from an E. coli expression system, preferentially digested single-stranded RNA in vitro, depending on the metal ion cofactor present (Bavagnoli et al., 2015).

Here, to further our knowledge about PA-X, we attempted to identify host proteins that contribute to the shutoff activity of PA-X. Using a single-gene deletion yeast library, we identified Nat3p (NAA20) and Mdm20p (NAA25), the components of NatB, as host proteins involved in the shutoff activity of PA-X.

Results

N-terminal acetylation by NatB is essential for the shutoff activity of PA-X in yeast.

Many viruses, including influenza A virus, encode viral proteins that interfere with host protein expression (Glaunsinger and Ganem, 2004; Nemeroff et al., 1998; Sadek and Read, 2016; Terada et al., 2017). Although a number of host proteins have been linked to the shutoff activity of various viruses (Gaglia et al., 2012; Kamitani et al., 2006; Nemeroff et al., 1998), the host protein or proteins that contribute to the shutoff activity of PA-X remain unknown. Saccharomyces cerevisiae (yeast) is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. Therefore, it has been broadly utilized as a model for aging, regulation of gene expression, signal transduction, the cell cycle, metabolism, and neurodegenerative disorders (Draetta and Beach, 1988; Ferretti et al., 2017; Graf et al., 2017; Peli-Gulli et al., 2017; Roychoudhury et al., 2017; Zuehlke et al., 2017). By using yeast single-gene deletion libraries, several groups have revealed host-pathogen interactions (Matsuda et al., 2012; Naito et al., 2007; Panavas et al., 2005; Tomita et al., 2011). To examine whether a yeast single-gene knockout library could be used to identify host genes that contribute to the shutoff activity of PA-X, we attempted to express PA-X in yeast. Wild-type yeast strain BY4347 was transformed with an empty plasmid, or a plasmid encoding wild-type PA-X, or PA-X K134A, which lacks shutoff activity (Hayashi et al., 2015). Transformed yeast harboring the empty or PA-X K134A-expressing plasmid formed colonies. In contrast, yeast transformed with the plasmid encoding wild-type PA-X hardly formed colonies (Fig. 1A). These results suggest that PA-X suppresses yeast growth via its shutoff activity. Moreover, these results demonstrated that we could use a yeast single-gene deletion library, containing 4,653 knockout yeast strains, to screen for host genes that are required for the shutoff activity of PA-X. To screen the yeast knockout library, each knockout yeast strain was transformed with a plasmid encoding wild-type PA-X or an empty plasmid, which served as a transformation control (Fig. 1B). When a knockout yeast strain that was transformed with the plasmid encoding PA-X formed a similar number of colonies to that transformed with the empty plasmid, the host gene lacking in the yeast strain would be required for the shutoff activity of PA-X (Fig. 1B). Using this screening approach, two knockout yeast strains, ΔNat3 and ΔMdm20, were selected as candidates because many colonies emerged. One or two colonies were found in 125 knockout yeast strains transformed with the plasmid encoding wild-type PA-X, whereas more than 1000 colonies were found in the ΔNat3 and ΔMdm20 yeast strains. Upon examination of the PA-X sequences recovered from the single colonies of the 125 knockout yeast strains, we found that 96 clones did not express PA-X and 29 clones harbored plasmids encoding PA-X with an unintended mutation that reduced the shutoff activity of PA-X (Oishi et al., 2018). To confirm the result, wild-type, ΔNat3, and ΔMdm20 yeast strains were again transformed with the empty plasmid or the plasmid encoding wild-type PA-X or PA-X K134A, and then tested for their growth on a selection plate. As expected, ΔNat3 and ΔMdm20 grew even when they were transformed with the PA-X-expressing plasmid, whereas the wild-type yeast did not grow when it was transformed with a wild-type PA-X, but did grow when transformed with the PA-X K134A-expressing plasmid (Fig. 1C, left). To exclude the possibility that the growth was caused by a defect in PA-X expression, the expression of wild-type PA-X in ΔNat3 and ΔMdm20 was examined by western blotting. The expression of PA-X in ΔNat3 was slightly lower than that of PA-X K134A, whereas in ΔMdm20, the expression of PA-X was comparable to that of PA-X K134A (Fig. 1C). We consistently detected double bands in yeast that expressed wild-type or mutant PA-X for an unknown reason. These data indicate that Nat3 and Mdm20 individually play roles in the shutoff activity of PA-X in yeast.

Figure 1. N-terminal acetylation by NatB is essential for the shutoff activity of PA-X in yeast.

Figure 1.

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(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–D) Wild-type, ΔNat3, or ΔMdm20 yeast was transformed with an empty plasmid or a plasmid encoding wild-type or the indicated PA-X mutant. 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.

Nat3p and Mdm20p form a heterodimer, named NatB, which is a member of the NAT family that includes NatA, NatC, NatD, NatE, and NatF (Arnesen et al., 2009; Starheim et al., 2012). Each NAT family member preferentially catalyzes the N-terminal acetylation of the N-terminal amino acid of its target proteins. NatB prefers to acetylate proteins beginning with Met-Asp, Met-Glu, and Met-Asn in yeast and mammals (Aksnes et al., 2016; Van Damme et al., 2012). PA-X starts with Met-Glu. Therefore, we hypothesized that PA-X must be N-terminally acetylated by NatB for its shutoff activity. To test this hypothesis, we prepared a series of plasmids encoding PA-X mutants, which possessed a NatB-permissive substitution at the second amino acid of E to D (PA-X E2D), to N (PA-X E2N), or to A (PA-X E2A), which is recognized by NatA, but not NatB, or to P (PA-X E2P), which should be unacetylated based on a previous report (Starheim et al., 2012). Wild-type, ΔNat3, or ΔMdm20 yeast strains were transformed with an empty plasmid or the series of PA-X plasmids and spotted onto the selection plate. The wild-type yeast strain transformed with the plasmid encoding wild-type PA-X, PA-X E2D, or PA-X E2N barely formed any colonies, whereas wild-type yeast transformed with the plasmid encoding PA-X E2A or PA-X E2P formed many colonies (Fig. 1D, top panel, left side), indicating that PA-X E2A and PA-X E2P have lower shutoff activity compared with wild-type PA-X. The knockout yeast strains, ΔNat3 and ΔMdm20, efficiently grew regardless of the plasmid with which they were transformed (Fig. 1D, middle and bottom panels, left side). Western blotting revealed that the expression of the PA-X mutants in ΔNat3 or ΔMdm20 was comparable to that of wild-type PA-X (Fig. 1D). These data demonstrate that N-terminal acetylation by the NatB complex of Nat3p and Mdm20p is essential for the shutoff activity of PA-X in yeast.

N-terminal acetylation by NatB is important for shutoff activity of PA-X in mammalian cells.

Since N-terminal acetylation by NatB is essential for the shutoff activity of PA-X in yeast, we asked whether NatB has a similar role in mammalian cells. To this end, we attempted to establish NAA20-knockout (NAA20-KO) and NAA25-knockout (NAA25-KO) cell lines by using HAP1 cells (we used HAP1 because it is a human haploid cell line from which it is easy to generate complete knockout cells) and the CRISPR/Cas9 approach: NAA20 and NAA25 are the human homologs of yeast Nat3 and Mdm20, respectively. We obtained nine NAA20-KO and 11 NAA25-KO clones; we arbitrarily chose one clone from each group and used those clones for most of the experiments. NAA20 was not detected in NAA20-KO cells (Fig. 2A). In NAA25-KO cells, neither NAA25 or NAA20 was detected (Fig. 2A), as previously described (Starheim et al., 2008). Using these cells, together with their parental cells, we compared the shutoff activity of wild-type PA-X by using a luciferase-based shutoff assay (Desmet et al., 2013; Hayashi et al., 2016; Hu et al., 2015; Nogales et al., 2017; Oishi et al., 2015). Briefly, wild-type, NAA20-KO, or NAA25-KO cells were transfected with a plasmid encoding firefly luciferase together with an empty plasmid or a plasmid encoding wild-type PA-X, and firefly luciferase activities were then measured at 24 h post-transfection. The shutoff activity was calculated as the ratio of luciferase activity in the empty plasmid-transfected samples to that in the PA-X-expressing samples, and is shown as relative values. The shutoff activities of wild-type PA-X in NAA20-KO and NAA25-KO cells were lower than that in wild-type HAP1 cells (Fig. 2B). Usually, the expression of wild-type PA-X is suppressed by its own shutoff activity (Oishi et al., 2015). However, when the shutoff activity of PA-X was decreased, the expression of PA-X conversely increased. To evaluate the shutoff activity from this aspect, we compared the expression of PA-X in transfected cells. The expression of PA-X in NAA20-KO and NAA25-KO cells was higher than that in wild-type HAP1 cells (Fig. 2B). Similar results were also obtained by using the other eight NAA20-KO and 10 NAA25-KO clones. Together, these results indicate that NAA20 and NAA25 are important for the shutoff activity of PA-X in mammalian cells. To further confirm that the reduction in PA-X shutoff activity was due to the loss of NAA20 or NAA25 expression, NAA20-KO or NAA25-KO cells were transfected with plasmids expressing firefly luciferase and wild-type PA-X together with NAA20 or NAA25. In NAA20-KO or NAA25-KO cells, low levels of NAA20 or NAA25, respectively, were detected when the expression plasmid for each protein was co-transfected; under these conditions, the shutoff activity of wild-type PA-X increased (Fig. 2C). In wild-type HAP1 cells, the shutoff activity of wild-type PA-X was not affected by co-expression with NAA20 or NAA25 (Fig. 2C). These results confirm that the human NatB components, NAA20 and NAA25, are important for the shutoff activity of PA-X in mammalian cells.

Figure 2. N-terminal acetylation by NatB is required for the shutoff activity of PA-X in mammalian cells.

Figure 2.

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(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. ** indicates 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.

To directly demonstrate N-terminal acetylation of PA-X, wild-type PA-X with a C-terminal FLAG-tag was expressed in HEK293 cells, purified by using the affinity tag, and analyzed for N-terminal modification by means of nanoLC-MS/MS. The mass spectrometry revealed that all detected N-terminal peptides of wild-type PA-X were N-terminally acetylated (Fig. 2D). NatB acetylates the N-terminal first methionine (Starheim et al., 2012); consistent with this report, the first methionine was acetylated in all detected N-terminal peptides of PA-X (Table S1). To confirm that NatB acetylated the N-terminus of PA-X, wild-type PA-X was expressed in NAA20-KO or NAA25-KO cells and the N-terminal modification of purified PA-X was analyzed by mass spectrometry. N-terminal acetylation was detected in 50% and 44% of N-terminal peptides of PA-X derived from NAA20-KO and NAA25-KO cells, respectively (Fig. 2D and Table S2 and S3). These data suggest that NatB is involved in the N-terminal acetylation of PA-X.

The second amino acid of PA-X, recognized by NatB, is important for the NatB-dependent shutoff activity of PA-X.

To elucidate whether the second N-terminal amino acid affects the shutoff activity of PA-X in mammalian cells, as it did in yeast, we compared the shutoff activity of wild-type PA-X with that of the PA-X mutants PA-X E2D, PA-X E2N, PA-X E2A, and PA-X E2P in wild-type HAP1 cells. PA-X E2D and PA-X E2N, which are recognized by NatB, showed somewhat reduced shutoff activity (Fig. 3A). In contrast, PA-X E2A, which is recognized by NatA, but not by NatB, and PA-X E2P, which should not be recognized by NATs (Starheim et al., 2012), possessed lower shutoff activity than that of wild-type PA-X, PA-X E2D, or PA-X E2N (Fig. 3A). Similar results were observed with both 293 and A549 cells (Fig. 3B and 3C). To determine whether the second amino acid of PA-X affects shutoff activity in the absence of the NatB, the shutoff activity of PA-X mutants in wild-type cells was compared to that in NAA20-KO or NAA25-KO cells. The shutoff activities of PA-X E2D and PA-X E2N in NAA20-KO and NAA25-KO cells were lower than that in wild-type cells (Fig. 3D and 3E). In contrast, the shutoff activities of PA-X E2A and PA-X E2P in NAA20-KO or NAA-25-KO cells were comparable to that in wild-type cells (Fig. 3F and 3G). These results indicate that the shutoff activity of PA-X possessing the second residue, recognized by NatB, is NatB-dependent.

Figure 3. The second amino acid, recognized by NatB, is required for the shutoff activity of PA-X in a NatB-dependent manner.

Figure 3.

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(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 the indicated PA-X mutants possessing an N-terminal second amino acid that is recognized and acetylated by NatB or other NAT family members 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.

To examine whether PA-X E2A and PA-X E2P are N-terminally acetylated, their N-terminal modifications were analyzed by nanoLC-MS/MS. We found that all detected N-terminal peptides of PA-X E2A were N-terminally acetylated. Although it has been reported that a protein with proline at the second position is not recognized by NATs (Starheim et al., 2012), we found that an N-terminal peptide of PA-X E2P was acetylated (Fig. 3H). Moreover, the N-terminal first methionine was removed from all detected N-terminal peptides of both PA-X E2A and PA-X E2P, and each alanine or proline in the second amino acid position was acetylated (Table S4 and S5). PA-X E2A, thus, showed reduced shutoff activity even though it was N-terminally acetylated, indicating that N-terminal acetylation alone is not sufficient for PA-X shutoff activity.

The importance of N-terminal acetylation by NatB to the shutoff activity of PA-X from other influenza A virus subtypes.

We next asked whether the second amino acid E of PA-X is conserved among influenza isolates. We analyzed the amino acid sequences of PA deposited in the Influenza Research Databases in April 2017 and found that 99.7% of H1N1pdm09 viruses (10,235/10,265 isolates), 99.9% of H3N2 viruses (12,899/12,904 isolates), and all H5N1 (2,382/2,382 isolates) and H7N9 (737/737 isolates) viruses have glutamic acid (E) as the second amino acid (Fig. 4A). This high conservation supports the concept that N-terminal acetylation of PA-X by NatB is required for its shutoff activity. To experimentally demonstrate the importance of N-terminal acetylation by NatB in PA-X derived from H1N1pdm09, H3N2, H5N1, and H7N9 viruses, we performed shutoff assays in NAA20-KO and NAA25-KO cells. The shutoff activities of all tested PA-Xs in NAA20-KO or NAA25-KO cells were lower than those in wild-type cells (Fig. 4B). These findings indicate that N-terminal acetylation is also important for the shutoff activity of PA-X derived from other subtypes of influenza virus.

Figure 4. Importance of N-terminal acetylation by NatB to the shutoff activity of PA-X derived from other virus subtypes.

Figure 4.

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(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).

NatB contributes to viral polymerase activity and virus growth.

Since PA-X is expressed from the PA mRNA via a ribosomal frameshift, PA-X and PA contain a common N-terminal sequence. PA, which is a subunit of the viral polymerase protein, thus has the potential to be modified by NatB. A high proportion of the PB2 (99.2% among 990 isolates) and PB1 (99.9% among 991 isolates) proteins (the other two subunits of the viral polymerase) also contains either D or E as the second amino acid and N-terminal acetylation of PB1 and PA in virions has been reported (Hutchinson et al., 2012), whereas the NP protein always starts with M-A (100% of 1,000 isolates). Therefore, we assessed whether N-terminal acetylation by NatB affects viral polymerase activity in NAA20-KO and NAA25-KO cells by using a minigenome assay. Wild-type, NAA20-KO, or NAA25-KO cells were transfected with plasmids for the expression of PB2, PB1, PA, and NP, a plasmid expressing viral RNA encoding firefly luciferase (which contains E as the second residue), and pGL4.74[hRluc/TK], which expresses Renilla luciferase (containing T as the second residue), as a transfection control. The expression and enzymatic activity of both the firefly and Renilla luciferases were not downregulated in NAA20-KO and NAA25-KO cells (Supplemental Fig. 1A and 1B). At 24 hours post-transfection (hpi), a dual luciferase assay was performed to calculate viral polymerase activities on the basis of the ratio of firefly luciferase activity and Renilla luciferase activity. Under conditions where the expression of wild-type PA and Renilla luciferase in the three tested cell types were comparable, viral polymerase activities in NAA20-KO or NAA25-KO cells were significantly reduced compared with that in wild-type cells (Fig. 5A), demonstrating that NatB also plays a role in the viral polymerase activity. Since PA-X is expressed via ribosomal frameshift from the plasmid encoding wild-type PA used in the minigenome assay, we examined its effect on the minigenome assay by using a plasmid that encodes mutant PA (PA FS); this plasmid expresses wild-type PA but the expression of PA-X via the ribosomal frameshift is decreased to one-seventh of wild-type PA levels (Jagger et al., 2012). The polymerase activity of PA FS in wild-type cells was comparable to that of wild-type PA (Supplementary Fig. 2A). In NAA20-KO and NAA25-KO cells, the polymerase activity of the PA FS mutant was reduced similarly to wild-type PA (Supplementary Fig. 2B). These results suggest that our minigenome assay is not affected by PA-X expressed via the ribosomal frameshift.

Figure 5. Contribution of N-terminal acetylation by NatB to viral polymerase activity and virus growth in mammalian cells.

Figure 5.

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(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%. (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). (A and C) 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). (A and C) β-actin served as a loading control.

We next compared the viral growth kinetics in wild-type, NAA20-KO, and NAA25-KO cells. These cells were infected with A/WSN/33 (H1N1) virus at a multiplicity of infection (MOI) of 0.0001, and virus titers in the cell culture supernatant were assessed at the indicated times. Virus growth in NAA20-KO and NAA25-KO cells was severely delayed (Fig. 5B). Specifically, the virus titers in NAA20-KO and NAA25-KO cells were at least 2 logs lower than those in wild-type cells at 12–48 hpi. At 72 hpi, the virus titers in NAA20-KO and NAA25-KO cells were comparable to those in wild-type cells. To examine the growth kinetics of different subtypes of influenza A viruses in NAA20-KO or NAA25-KO cells, these cells together with wild-type cells were infected with A/California/04/2009 (H1N1pdm09) or A/Victoria/361/2011 (H3N2) virus. The replication of both viruses in each KO cell line was reduced relative to that in wild-type cells (Supplementary Fig. 3A and 3B). To verify that PA-X expressed from PA mRNA affects viral growth, we compared the virus growth kinetics of wild-type virus and mutant virus possessing PA FS in wild-type, NAA20-KO, and NAA25-KO cells. The growth of PA FS in each cell line was comparable to that of wild-type virus (Supplementary Fig. 2C). These results suggest that PA-X expressed via the ribosomal frameshift does not affect virus growth in cell culture. Taken together, these data show the contribution of NatB to virus replication.

The role of N-terminal acetylation by NatB in viral polymerase proteins.

To clarify the role of N-terminal acetylation by NatB for polymerase activity, we evaluated the expression, intracellular localization, and PB1 binding of PA in infected cells. The expression of PA and NP, intracellular localization of PA, and interaction of PA with PB1 in NAA20-KO and NAA25-KO cells were similar to those in wild-type cells (Supplementary Fig. 4A–C). These results indicate that these PA properties are not affected by N-terminus acetylation by NatB.

The second amino acid of PA, recognized by NatB, is important for viral polymerase activity.

To evaluate the importance of the second amino acid of PA for viral polymerase activity, we constructed plasmids encoding PA E2D, PA E2N, PA E2A, and PA E2P, and assessed their viral polymerase activities in wild-type cells. Under conditions where the expression of PA and Renilla luciferase was comparable among the wild-type and mutant PAs, both PA E2D and PA E2N supported viral polymerase activity, although the level of viral polymerase activity differed substantially from that of wild-type PA. Viral polymerase activity of PA E2A which is recognized by NatA was lower than that of wild-type PA, PA E2D or PA E2N, viral polymerase activity was not detectable for PA E2P, which is not recognized by NATs (Fig. 5C). Similar results were observed in 293 and A549 cells (Supplementary Fig. 5A).

To further examine whether the polymerase activity of PA possessing the second amino acid that is recognized by NatB is affected in NatB-depleted cells, we compared the polymerase activities of PA mutants in NAA20-KO or NAA25-KO cells with that in wild-type cells. The polymerase activities of PA E2D and PA E2N in NAA20-KO and NAA25-KO cells were lower than that in wild-type cells, whereas that of PA E2A in each KO cell line was comparable to that in wild-type cells (Supplementary Fig. 5B). These results indicate that NatB is involved in the viral polymerase activity of PA in which the second residue is recognized by NatB.

Discussion

Here, we showed that NatB, which is a heterodimer of NAA20 (Nat3p) and NAA25 (Mdm20p), is required for the shutoff activity of influenza virus PA-X. The NatB acetylates N-terminal amino acids in proteins that start with M-E, -D, or –N (Starheim et al., 2012). Phosphorylation, methylation, ubiquitination, and lysine acetylation modify target proteins to regulate biological activities, whereas N-terminal acetylation stably modifies the target during protein biosynthesis (Aksnes et al., 2016). Over 80% of proteins are acetylated at the N-terminus by NAT family members, including NatA through NatF, which preferentially acetylate the N-terminal amino acid (Aksnes et al., 2016; Starheim et al., 2012). Although N-terminal acetylation is a fundamental modification, it is not fully understood, except for its functions in a small number of proteins, for example the binding of the ubiquitin enzyme E2 Ubc12 to scaffold-type E3 ligase Dcn1 (Scott et al., 2017; Scott et al., 2011), stabilization of Rgs2 (Park et al., 2015), and maintenance of actomyosin fiber (Van Damme et al., 2012). Even less is known about the N-terminal acetylation of viral proteins. In the present study, we not only showed the importance of N-terminal acetylation by NatB to PA-X function, but we also showed that N-terminal acetylation by NatB plays roles in viral polymerase activity and virus growth. Since all influenza viral polymerase proteins (PB2, PB1, and PA) could be N-terminally acetylated by the NatB, N-terminal acetylation could affect multiple steps of vRNA, cRNA, and mRNA synthesis during influenza virus replication. Moreover, N-terminal acetylation of host proteins by NatB may be involved in viral polymerase activity, virus growth, and shutoff activity since this activity was diminished in NAA20-KO and NAA25-KO cells. Further analysis is required to clarify the importance of NatB especially in virus replication since NatB also contributes to a wide range of host functions. Nevertheless, our findings contribute to a deeper understanding of N-terminal acetylation in virology as well as cell biology.

Approximately 50% of N-terminal peptides of PA-X expressed in NAA20-KO or NAA25-KO cells were N-terminally acetylated. This result suggests that other Nat family members N-terminally acetylate PA-X. Furthermore, we found that one N-terminal peptide of PA-X E2P was in fact acetylated, although it has been reported that a protein with proline at the second position is not recognized by NATs (Starheim et al., 2012). Also, the monomer NAA10, which is a component of human NatA, functions as an N-terminal acetyltransferase with a substrate preference that differs from that of the NatA complex (Van Damme et al., 2011). Taken together, these results suggest that the substrate specificity of Nat family members in mammalian cells is regulated in a more complex fashion than we currently understand. The substrate specificity profile of each Nat family member in mammals warrants further evaluation.

We showed that PA-X and PA with the second amino acid of alanine or proline, which is not recognized by NatB (Aksnes et al., 2016), possessed reduced shutoff activity and no viral polymerase activity, whereas PA-X and PA mutants with the second amino acid of aspartic acid or asparagine, which is recognized by NatB (Aksnes et al., 2016), maintained these activities. Although the shutoff activity of PA-X E2N was lower than that of wild-type PA-X, the polymerase activity of PA E2N was higher than that of wild-type PA (Fig. 3A and Fig. 5C), indicating that the effect of the second amino acid on the shutoff activity of PA-X could be different from its effect on the polymerase activity. Further analysis is needed to elucidate the mechanism of the effects of this amino acid on the polymerase and shutoff activities. In these mutagenesis analyses, PA-X E2A showed an interesting phenotype: it showed reduced shutoff activity, but was recognized by NatA and was N-terminally acetylated at the second amino acid residue after removal of the first methionine. These results indicate that the shutoff activity of PA-X is not solely regulated by N-terminal acetylation. Our data suggest that both the second amino acid residue of PA-X and N-terminal acetylation by NatB play important roles in the shutoff activity of PA-X. PA-X has an acetylated first methionine at its N-terminus (Table S1). However, although the N-terminus of PA-X E2A and PA-X E2P is acetylated, their first methionine is removed and the second residue of alanine and proline, respectively, is acetylated. Thus, although both PA-X and PA-X E2A are N-terminally acetylated, their N-terminal structures may be different, which may affect the shutoff activity.

Many viruses encode a protein that has shutoff activity for escape from host innate defenses (Rivas et al., 2016). Although the suppression mechanisms are varied among viral proteins, the viral proteins of herpes simplex, SARS corona, Epstein-Barr, and Kaposi’s sarcoma-associated herpes viruses directly cleave mRNA via their endonuclease activity (Gaglia et al., 2012). Their N-terminal sequences do not match the N-terminal sequences recognized by NatB. Therefore, N-terminal acetylation by NatB is unlikely to be responsible for the shutoff activity of these viruses. However, L proteins of bunya, arena, and hanta viruses, which also have endonuclease activity for cap-snatching, all contain conserved N-terminal sequences that are recognized by NatB (Morin et al., 2010; Reguera et al., 2010), implying that the L proteins of these viruses may also be N-terminally acetylated by NatB and that their endonuclease activity may be regulated by the NatB. Further analysis is required to determine the importance of N-terminal acetylation by NatB and other NATs in the polymerase and shutoff activities of viruses other than influenza virus.

In summary, here we discovered that the NatB, comprising NAA20 and NAA25, is involved in the shutoff activity of PA-X and the viral polymerase activity of PA. Although N-terminal acetylation by NatB is required for the shutoff activity of PA-X, further functional analyses are needed to elucidate the detailed mechanism of how N-terminal acetylation contributes to PA-X shutoff activity. A crystal structure of PA-X and structure–activity correlation analyses may provide further insights.

Experimental Procedures

Yeast screening.

Each knockout yeast strain was transformed with pKT10 (Ura) encoding PA-X with the C-FLAG or empty plasmid by using S. cerevisiae Direct Transformation Kit Wako (Wako). Transformed yeast were spotted onto SD/-Ura plates containing 200 μg/ml G418 and incubated for 3–5 days at 30 °C.

Shutoff assay.

Wild-type, NAA20-KO, or NAA25-KO cells in four wells of a 24-well plate were transfected with a plasmid encoding firefly luciferase together with an empty plasmid, or the plasmid encoding wild-type or mutant PA-X with C-FLAG or three N-terminal prolines. In some cases, a plasmid encoding NAA20, NAA25, or GFP was co-transfected to restore expression of NAA20 or NAA25. The transfected cells in three of the wells were analyzed for firefly luciferase activity by using the Bright-Glo luciferase assay system (Promega) at 24 h post-transfection. The shutoff activity of PA-X was calculated by dividing the luminescence value of firefly luciferase co-transfected with an empty plasmid by that co-transfected with PA-X-expressing plasmids. Data are shown as the average of the relative shutoff activity ± standard deviation (n=3). To analyze the expression of PA-X, NAA20, and NAA25, the transfected cells in the fourth well were lysed in 2×SDS sample buffer at 24 h post-transfection. The cell lysates were sonicated, incubated for 10 min at 95 °C, and then loaded onto an Any KD Mini-PROTEAN TGX Gel (Bio-Rad). Separated proteins were transferred to Immobilon-P PVDF membrane (Millipore) and detected by using anti-NAA20 antibody clone 2C6, the anti-NAA25 antibody, anti-DYKDDDDK (FLAG) tag antibody clone 1E6, anti-PA antibody clone 55/2 (Hatta et al., 2000), or anti-β-actin antibody clone AC-74 (Sigma), followed by donkey anti-rabbit IgG-HRP (GE Healthcare) or sheep anti-mouse IgG-HRP (GE Healthcare).

Purification of PA-X and mass spectrometry.

HEK293 cells, NAA20-KO, or NAA25-KO HAP1 cells were transfected with a plasmid encoding wild-type or mutant PA-X with C-FLAG by using TransIT 293. At 24 h post-transfection, cells were lysed in lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5 % Nonidet P-40, and protease inhibitor Complete Mini cocktail (Roche)] for 1 h at 4 °C. After centrifugation to remove cellular debris, the supernatants were incubated with anti-FLAG M2 magnetic beads (Sigma) overnight at 4 °C. The magnetic beads were washed three times with the lysis buffer, and then washed twice with wash buffer [50 mM Tris-HCl (pH 7.5) and 150 mM NaCl]. Proteins bound to the magnetic beads were released by incubation with elution buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.5 mg/ml FLAG peptide (Sigma)] for 1 h at 4 °C or by incubation in 2×SDS sample buffer (Invitrogen) for 10 min at 95 °C. The eluted samples in the former case were mixed with 2×SDS sample buffer, and incubated for 10 min at 95 °C. The denatured samples were then subjected to SDS-PAGE using 10% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad). The gels were stained with a silver stain MS kit (Wako), and bands corresponding to PA-X were excised from the gels. The PA-X-containing gel was digested with endoproteinase Lys-C, and then subjected to nanoLC-MS/MS analysis to detect the N-terminal peptides of PA-X.

Supplementary Material

Supplemental procedure
Supplemental tables
3

Acknowledgements

We thank Shuhei Sammaibashi and Yuriko Tomita for technical assistance, and Susan Watson for editing the manuscript. This research was supported by Strategic Basic Research Programs from the Japan Science and Technology Agency (JST), by Leading Advanced Projects for medical innovation (LEAP) from the Japan Agency for Medical Research and Development (AMED) (JP17am001007), by a Grant-in-Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Science, Sports, and Technology (MEXT) of Japan (No. 16H06429, 16K21723, and 16H06434), by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from the MEXT of Japan, and from AMED (JP17fm0108006), by e-ASIA Joint Research Program from AMED (JP17jm0210042), and by NIH Functional Genomics award, “Characterization of novel genes encoded by RNA and DNA viruses” (U19 AI 107810) from the NIAID.

Declaration of interests

Y.K. has received speaker’s honoraria from Toyama Chemical, has received grant support from Chugai Pharmaceuticals, Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Tsumura and Co, and Denka Seiken Co., Ltd., and is a co-founder of FluGen.

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Supplementary Materials

Supplemental procedure
NIHMS989458-supplement-Supplemental_procedure.docx (49.1KB, docx)
Supplemental tables
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3
NIHMS989458-supplement-3.pdf (230.6KB, pdf)

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