Influenza A virus nucleoprotein is acetylated by histone acetyltransferases PCAF and GCN5

Abstract

Histone acetylation plays crucial roles in transcriptional regulation and chromatin organization. Viral RNA of the influenza virus interacts with its nucleoprotein (NP), whose function corresponds to that of eukaryotic histones. NP regulates viral replication and has been shown to undergo acetylation by the cAMP-response element (CRE)-binding protein (CBP) from the host. However, whether NP is the target of other host acetyltransferases is unknown. Here, we show that influenza virus NP undergoes acetylation by the two host acetyltransferases GCN5 and P300/CBP-associated factor (PCAF) and that this modification affects viral polymerase activities. Western blot analysis with anti-acetyl-lysine antibody on cultured A549 human lung adenocarcinoma epithelial cells infected with different influenza virus strains indicated acetylation of the viral NP. A series of biochemical analyses disclosed that the host lysine acetyltransferases GCN5 and PCAF acetylate NP in vitro MS experiments identified three lysine residues as acetylation targets in the host cells and suggested that Lys-31 and Lys-90 are acetylated by PCAF and GCN5, respectively. RNAi-mediated silencing of GCN5 and PCAF did not change acetylation levels of NP. However, interestingly, viral polymerase activities were increased by the PCAF silencing and were decreased by the GCN5 silencing, suggesting that acetylation of the Lys-31 and Lys-90 residues has opposing effects on viral replication. Our findings suggest that epigenetic control of NP via acetylation by host acetyltransferases contributes to regulation of polymerase activity in the influenza A virus.

Keywords: GCN5; HAT; NP; PCAF; acetylation; acetyltransferase; histone acetylase; influenza virus; influenza virus A; nucleoprotein; viral protein.

Figure 1.

Biochemical analyses using anti–acetyl-lysine antibody showed the acetylation of NP in the H1N1 (A/Puerto Rico/8/34) and H3N2 (A/Uruguay/716/2007) strains of influenza A virus. A and F, a simple Western blot analysis of homogenized cultured cells using anti–acetyl-lysine antibody showed positive bands of around 50 kDa 8 h after infection with the H1N1 strain. The same bands were observed in cells infected by the H3N2 strain, together with bands at around 25 kDa. B and G, the positive bands at 50 kDa shown by anti–acetyl-lysine antibody in (A) and (F) were also detected by anti-NP antibody, suggesting that the acetylated protein was NP. C and H, Western blotting using anti-NS1 antibody suggested that the positive bands at 25 kDa shown by anti–acetyl-lysine antibody in (F) were NS1. This anti-NS1 antibody exhibited cross-reactions with NP. D and I, the molecular weight of PB2 did not correspond to the protein positively detected by anti–acetyl-lysine antibody. E and J, Western blotting using anti-actin antibody showed no differences in the loaded amount among samples. K, combined experiments of immunoprecipitation by anti-NP antibody and Western blotting by anti–acetyl-lysine antibody showed that the acetylated protein was NP (arrowhead). L, NP and other viral proteins were overexpressed in cultured cells. NP (arrowheads) individually overexpressed in cells (lane 2) was acetylated, suggesting that acetylation of NP occurred independently of other viral proteins.

Figure 2.

Eukaryotic HATs acetylated NP in vitro. A, the recombinant protein of NP derived from E. coli was acetylated by PCAF and GCN5, but not by the nuclear extract or by P300/CBP. Histone H1 was used as the positive control for acetylation. B, acetylation levels on the recombinant protein of the NP depended on incubation duration with the enzymes. The extra bands, from contamination of E. coli proteins, are indicated by blank arrowheads. C, RNP purified from virions of A/Puerto Rico/8/34 (PR8, H1N1) was incubated with PCAF and GCN5. NP constructing RNP was acetylated by PCAF and GCN5. Autoacetylation on the partial recombinant proteins of PCAF and GCN5 was also detected around the mass of 20 kDa. Arrowheads show the bands of NP. Upper and lower pictures of both panels show the results of Coomassie Brilliant Blue (CBB) staining and autoradiography, respectively.

Figure 3.

Inhibition of acetylation of NP by HAT blockers in vitro. A, chemical formulas of the HAT inhibitors anacardic acid, embelin, and garcinol. B, these chemicals blocked acetylation of NP in a concentration-dependent manner. Concentrations of anacardic acid greater than 25 μm inhibited acetylation. Embelin and garcinol showed acetylation inhibition at concentrations greater than 50 μm. Arrowheads show the bands of NP. CBB and ARG correspond to Coomassie Brilliant Blue staining and autoradiography, respectively.

Figure 4.

LC-MS/MS analysis of influenza NP isolated from infected cells by immunoprecipitation. A–C, base peak ion chromatogram derived from a 12-min separation of the digested NP peptides containing Lys-31 (A), Lys-90 (B), and Lys-184 (C). The elution time and representative m/z of the eluted peptides are indicated at the top of each peak. Formulas to calculate the molecular weights of acetylated lysine residues are shown in each panel. The observed y and b ions and fragment map are shown. Ac and Me mean an acetyl and a methyl group, respectively. K31, Lys-31; K90, Lys-90; K184, Lys-184.

Figure 5.

LC-MS/MS analysis of recombinant protein of influenza NP incubated with PCAF and GCN5 in vitro. A–D, base peak, ion chromatogram for a 12-min separation of the digested NP peptides containing Lys-31 (A) and Lys-184 (B) acetylated by PCAF and that contained Lys-90 (C) and Lys-184 (D) acetylated by GCN5. The elution time and representative m/z of the eluted peptides are indicated at the top of each peak. Formulas to calculate the molecular weights of acetylated lysine residues were shown in each panel. The observed y and b ions and fragment map are shown. K31, Lys-31; K90, Lys-90; K184, Lys-184.

Figure 6.

Acetylated lysine residues in the tertiary structure of NP. A–C, tertiary structure of influenza NP dimer (PDB ID: 4IRY) showed that candidate lysine residues for acetylation were concentrated on the surface of the RNA-binding groove. Ribbon structures (A) and surface structures (B and C) show the positions of target lysine residues using the MOE software. A, structures of the helix and strand are shown in red and yellow, respectively. The atoms of carbon and nitrogen in the side chains of the acetylated lysines are highlighted as green and blue spheres, respectively. B and C, the atoms of hydrogen, carbon, nitrogen, oxygen, and sulfur are shown in cyan, gray, blue, red, and yellow, respectively. The carbon atoms in the side chains of the acetylated lysines are shown in green. Panel C is the 90 degree–rotated structure of panel B. K31, Lys-31; K90, Lys-90; K184, Lys-184.

Figure 7.

Inhibition of expression of GCN5 and PCAF altered the polymerase activity of viral RdRp. A, treatment of cultured A549 cells with the siRNAs of GCN5 and PCAF decreased the expression levels of these proteins. B, ratios of band intensities (AcK/NP) were calculated. There were no significant differences among the four groups. The data are represented as mean ± S.D. of all the individual data points and are indicated by gray-colored circles, diamonds, triangles, and inverted triangles. C, minigenome assays were performed using siRNA-treated cells. Transfection of random siRNA increased the relative polymerase activity significantly. Interestingly, compared with the cells injected with random siRNA, viral transcription increased with addition of PCAF siRNA, but decreased with GCN5 siRNA. The data are represented as mean ± S.D. of all the individual data points and are indicated by gray-colored circles, squares, and triangles. Relative polymerase activity was analyzed by one-way analysis of variance (ANOVA) followed by a Tukey's post hoc test (*, p < 0.05; **, p < 0.01).