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A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

Hatakeyama D, Shoji M, Yamayoshi S, Hirota T, Nagae M, Yanagisawa S, Nakano M, Ohmi N, Noda T, Kawaoka Y, Kuzuhara T - J. Biol. Chem. (2014)

Bottom Line: In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs).Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity.These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

View Article: PubMed Central - PubMed

Affiliation: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.

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Structural homology between PB2 and eukaryotic acetyltransferases.A, similarity of tertiary structures between the cap-binding domain of the PB2 subunit from the influenza A virus (the red ribbon; PDB code 2vqz) and that of the acetyltransferase from bacterium B. cereus (the blue ribbon; PDB code 1y9w). B, amino acid alignment of cap-binding domains of PB2 across various strains of influenza viruses. Sequences were placed in the order of years when each strain had emerged. Groups of influenza A, B, and C viruses were separated by dotted lines. The VRG site was highly conserved in strains of influenza A and B viruses. This site was not present in any strains of influenza C viruses. C, amino acid alignment of GCN5 acetyltransferases across various eukaryotic species. The VRG site and its corresponding sequence, the VKG site, are conserved in various GCN5 eukaryotic acetyltransferases. These amino acid sequences were aligned in a multiple-sequence alignment program, ClustalX. Orange, glycine; yellow, proline; cyan, hydrophobic neutral amino acids; green, hydrophilic neutral amino acids; red, acidic amino acids; magenta, basic amino acids.
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Figure 1: Structural homology between PB2 and eukaryotic acetyltransferases.A, similarity of tertiary structures between the cap-binding domain of the PB2 subunit from the influenza A virus (the red ribbon; PDB code 2vqz) and that of the acetyltransferase from bacterium B. cereus (the blue ribbon; PDB code 1y9w). B, amino acid alignment of cap-binding domains of PB2 across various strains of influenza viruses. Sequences were placed in the order of years when each strain had emerged. Groups of influenza A, B, and C viruses were separated by dotted lines. The VRG site was highly conserved in strains of influenza A and B viruses. This site was not present in any strains of influenza C viruses. C, amino acid alignment of GCN5 acetyltransferases across various eukaryotic species. The VRG site and its corresponding sequence, the VKG site, are conserved in various GCN5 eukaryotic acetyltransferases. These amino acid sequences were aligned in a multiple-sequence alignment program, ClustalX. Orange, glycine; yellow, proline; cyan, hydrophobic neutral amino acids; green, hydrophilic neutral amino acids; red, acidic amino acids; magenta, basic amino acids.

Mentions: In this study, we investigated the central region of the PB2 subunit, which includes the cap-binding domain, and searched for proteins containing a similar tertiary structure using the MATRAS evolution program to compare the three-dimensional structures of proteins (42). Using this approach, we identified many types of prokaryotic acetyltransferases (27 of the 87 molecules shown in supplemental Table S1). Comparison of the tertiary structures of the PB2 cap-binding domain (PDB code 2vqz; amino acids 318 to 483) and acetyltransferase of Bacillus cereus (PDB code 1y9w) revealed similarities in the Cα-traces of loops, α-helices, and β-sheets (Fig. 1A). In addition, we investigated functional domains involved in acetyltransferase activity of the cap-binding domain of PB2. Amino acid sequences around this domain were almost completely conserved across the various strains of influenza A viruses from 1918 to 2013 and influenza B viruses from 1972 to 2012; the VRG site was conserved in all strains of influenza A and B viruses (Fig. 1B). However, the VRG site was not found in any strains of influenza C viruses (Fig. 1B). This functional domain (VRG and its corresponding sequence, VKG) is widely conserved in eukaryotic GCN5 acetyltransferases (Fig. 1C), and is essential for the interaction between acetyltransferase and its ligand, acetyl-CoA (22). Therefore, the results derived from these structural analyses suggest that the cap-binding domain of PB2 interacts with acetyl-CoA and exhibits acetyltransferase activity.


A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

Hatakeyama D, Shoji M, Yamayoshi S, Hirota T, Nagae M, Yanagisawa S, Nakano M, Ohmi N, Noda T, Kawaoka Y, Kuzuhara T - J. Biol. Chem. (2014)

Structural homology between PB2 and eukaryotic acetyltransferases.A, similarity of tertiary structures between the cap-binding domain of the PB2 subunit from the influenza A virus (the red ribbon; PDB code 2vqz) and that of the acetyltransferase from bacterium B. cereus (the blue ribbon; PDB code 1y9w). B, amino acid alignment of cap-binding domains of PB2 across various strains of influenza viruses. Sequences were placed in the order of years when each strain had emerged. Groups of influenza A, B, and C viruses were separated by dotted lines. The VRG site was highly conserved in strains of influenza A and B viruses. This site was not present in any strains of influenza C viruses. C, amino acid alignment of GCN5 acetyltransferases across various eukaryotic species. The VRG site and its corresponding sequence, the VKG site, are conserved in various GCN5 eukaryotic acetyltransferases. These amino acid sequences were aligned in a multiple-sequence alignment program, ClustalX. Orange, glycine; yellow, proline; cyan, hydrophobic neutral amino acids; green, hydrophilic neutral amino acids; red, acidic amino acids; magenta, basic amino acids.
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Figure 1: Structural homology between PB2 and eukaryotic acetyltransferases.A, similarity of tertiary structures between the cap-binding domain of the PB2 subunit from the influenza A virus (the red ribbon; PDB code 2vqz) and that of the acetyltransferase from bacterium B. cereus (the blue ribbon; PDB code 1y9w). B, amino acid alignment of cap-binding domains of PB2 across various strains of influenza viruses. Sequences were placed in the order of years when each strain had emerged. Groups of influenza A, B, and C viruses were separated by dotted lines. The VRG site was highly conserved in strains of influenza A and B viruses. This site was not present in any strains of influenza C viruses. C, amino acid alignment of GCN5 acetyltransferases across various eukaryotic species. The VRG site and its corresponding sequence, the VKG site, are conserved in various GCN5 eukaryotic acetyltransferases. These amino acid sequences were aligned in a multiple-sequence alignment program, ClustalX. Orange, glycine; yellow, proline; cyan, hydrophobic neutral amino acids; green, hydrophilic neutral amino acids; red, acidic amino acids; magenta, basic amino acids.
Mentions: In this study, we investigated the central region of the PB2 subunit, which includes the cap-binding domain, and searched for proteins containing a similar tertiary structure using the MATRAS evolution program to compare the three-dimensional structures of proteins (42). Using this approach, we identified many types of prokaryotic acetyltransferases (27 of the 87 molecules shown in supplemental Table S1). Comparison of the tertiary structures of the PB2 cap-binding domain (PDB code 2vqz; amino acids 318 to 483) and acetyltransferase of Bacillus cereus (PDB code 1y9w) revealed similarities in the Cα-traces of loops, α-helices, and β-sheets (Fig. 1A). In addition, we investigated functional domains involved in acetyltransferase activity of the cap-binding domain of PB2. Amino acid sequences around this domain were almost completely conserved across the various strains of influenza A viruses from 1918 to 2013 and influenza B viruses from 1972 to 2012; the VRG site was conserved in all strains of influenza A and B viruses (Fig. 1B). However, the VRG site was not found in any strains of influenza C viruses (Fig. 1B). This functional domain (VRG and its corresponding sequence, VKG) is widely conserved in eukaryotic GCN5 acetyltransferases (Fig. 1C), and is essential for the interaction between acetyltransferase and its ligand, acetyl-CoA (22). Therefore, the results derived from these structural analyses suggest that the cap-binding domain of PB2 interacts with acetyl-CoA and exhibits acetyltransferase activity.

Bottom Line: In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs).Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity.These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.

View Article: PubMed Central - PubMed

Affiliation: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.

Show MeSH
Related in: MedlinePlus