RESUMO
The post-translational acetylation of lysine residues is found in many nonhistone proteins and is involved in a wide range of biological processes. Recently, we showed that the nucleoprotein of the influenza A virus is acetylated by histone acetyltransferases (HATs), a phenomenon that affects viral transcription. Here, we report that the PA subunit of influenza A virus RNA-dependent RNA polymerase is acetylated by the HATs, P300/CREB-binding protein-associated factor (PCAF), and general control nonderepressible 5 (GCN5), resulting in accelerated endonuclease activity. Specifically, the full-length PA subunit expressed in cultured 293T cells was found to be strongly acetylated. Moreover, the partial recombinant protein of the PA N-terminal region containing the endonuclease domain was also acetylated by PCAF and GCN5 in vitro, which facilitated its endonuclease activity. Mass spectrometry analyses identified K19 as a candidate acetylation target in the PA N-terminal region. Notably, the substitution of the lysine residue at position 19 with glutamine, a mimic of the acetyl-lysine residue, enhanced its endonuclease activity in vitro; this point mutation also accelerated influenza A virus RNA-dependent RNA polymerase activity in the cell. Our findings suggest that PA acetylation is important for the regulation of the endonuclease and RNA polymerase activities of the influenza A virus.
Assuntos
Histona Acetiltransferases/genética , Vírus da Influenza A/genética , Influenza Humana/genética , RNA Polimerase Dependente de RNA/genética , Fatores de Transcrição de p300-CBP/genética , Acetilação , Sequência de Aminoácidos/genética , Humanos , Influenza Humana/virologia , Nucleoproteínas/genética , Ligação Proteica/genética , Processamento de Proteína Pós-Traducional/genética , RNA Viral/genética , Proteínas Virais/genética , Transcrição Viral/genéticaRESUMO
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.
