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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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Inhibition of the binding activity of recombinant PB2 with acetyl-CoA by HAT blockers.A, HAT inhibitors were mixed in the reaction solution prior to incubation. Following addition of 1 mg of recombinant PB2 cap-binding domain proteins, incubation was performed for 30 min at 30 °C. Anacardic acid and garcinol showed strong inhibition of the binding activity of recombinant PB2 with [14C]acetyl-CoA. CBB, Coomassie Brilliant Blue staining; ARG, autoradiography. B, anti-influenza activity of HAT inhibitors was examined by means of a plaque assay using cultured MDCK cells. DMSO and ribavirin were used as negative and positive controls, respectively. The data are shown as mean ± S.E. (n = 4) and are representative of 3 independent experiments. Differences between the effect of each drug and the effect of DMSO were analyzed for statistical significance using analysis of variance. *, p < 0.05 and **, p < 0.01; DMSO versus ribavirin. ##, p < 0.01; DMSO versus anacardic acid. †, p < 0.05 and ††, p < 0.01; DMSO versus garcinol.
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Figure 4: Inhibition of the binding activity of recombinant PB2 with acetyl-CoA by HAT blockers.A, HAT inhibitors were mixed in the reaction solution prior to incubation. Following addition of 1 mg of recombinant PB2 cap-binding domain proteins, incubation was performed for 30 min at 30 °C. Anacardic acid and garcinol showed strong inhibition of the binding activity of recombinant PB2 with [14C]acetyl-CoA. CBB, Coomassie Brilliant Blue staining; ARG, autoradiography. B, anti-influenza activity of HAT inhibitors was examined by means of a plaque assay using cultured MDCK cells. DMSO and ribavirin were used as negative and positive controls, respectively. The data are shown as mean ± S.E. (n = 4) and are representative of 3 independent experiments. Differences between the effect of each drug and the effect of DMSO were analyzed for statistical significance using analysis of variance. *, p < 0.05 and **, p < 0.01; DMSO versus ribavirin. ##, p < 0.01; DMSO versus anacardic acid. †, p < 0.05 and ††, p < 0.01; DMSO versus garcinol.

Mentions: One HAT, p300/cAMP-response element-binding protein-associated factor, contains a Val/Lys/Gly (VKG) site that corresponds to the VRG site in GCN5 and the PB2 cap-binding domain, which is required for acetyl-CoA interaction (53). Results from a recent study, in which molecular docking simulation was used, suggest that anacardic acid competes with acetyl-CoA for the VKG site in p300/cAMP-response element-binding protein-associated factor (54). Analogues of anacardic acid are also known to target the acetyl-CoA binding site of other HATs, such as Tip60 (55). In addition to anacardic acid and its derivatives (56, 57), several types of natural chemicals have been reported to bind HATs, such as epigallocatechin-3-gallate (58), plumbagin (59), curcumin (60), and garcinol (61). Therefore, we investigated whether these chemicals can inhibit the interaction between the PB2 cap-binding domain and acetyl-CoA (Fig. 4A). Low concentrations (5 μm) of epigallocatechin-3-gallate slightly reduced the interaction between the PB2 cap-binding domain and acetyl-CoA, increasing the epigallocatechin-3-gallate concentration to 25 and 100 μm and resulted in similar, but not greater, levels of inhibition as observed with the 5 μm treatment. Plumbagin and curcumin at concentrations of 5 and 25 μm did not block the interaction, although concentrations of 100 μm did result in a slight attenuation of interaction activity. In contrast, anacardic acid and garcinol drastically inhibited the interaction, and concentrations of anacardic acid greater than 25 μm completely eliminated interaction, whereas only faint activity was detected by adding 25 μm garcinol; however, no interaction was detected with the addition of 100 μm garcinol. These results demonstrated that several HAT inhibitors also blocked the interaction between the PB2 cap-binding domain and acetyl-CoA.


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)

Inhibition of the binding activity of recombinant PB2 with acetyl-CoA by HAT blockers.A, HAT inhibitors were mixed in the reaction solution prior to incubation. Following addition of 1 mg of recombinant PB2 cap-binding domain proteins, incubation was performed for 30 min at 30 °C. Anacardic acid and garcinol showed strong inhibition of the binding activity of recombinant PB2 with [14C]acetyl-CoA. CBB, Coomassie Brilliant Blue staining; ARG, autoradiography. B, anti-influenza activity of HAT inhibitors was examined by means of a plaque assay using cultured MDCK cells. DMSO and ribavirin were used as negative and positive controls, respectively. The data are shown as mean ± S.E. (n = 4) and are representative of 3 independent experiments. Differences between the effect of each drug and the effect of DMSO were analyzed for statistical significance using analysis of variance. *, p < 0.05 and **, p < 0.01; DMSO versus ribavirin. ##, p < 0.01; DMSO versus anacardic acid. †, p < 0.05 and ††, p < 0.01; DMSO versus garcinol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Inhibition of the binding activity of recombinant PB2 with acetyl-CoA by HAT blockers.A, HAT inhibitors were mixed in the reaction solution prior to incubation. Following addition of 1 mg of recombinant PB2 cap-binding domain proteins, incubation was performed for 30 min at 30 °C. Anacardic acid and garcinol showed strong inhibition of the binding activity of recombinant PB2 with [14C]acetyl-CoA. CBB, Coomassie Brilliant Blue staining; ARG, autoradiography. B, anti-influenza activity of HAT inhibitors was examined by means of a plaque assay using cultured MDCK cells. DMSO and ribavirin were used as negative and positive controls, respectively. The data are shown as mean ± S.E. (n = 4) and are representative of 3 independent experiments. Differences between the effect of each drug and the effect of DMSO were analyzed for statistical significance using analysis of variance. *, p < 0.05 and **, p < 0.01; DMSO versus ribavirin. ##, p < 0.01; DMSO versus anacardic acid. †, p < 0.05 and ††, p < 0.01; DMSO versus garcinol.
Mentions: One HAT, p300/cAMP-response element-binding protein-associated factor, contains a Val/Lys/Gly (VKG) site that corresponds to the VRG site in GCN5 and the PB2 cap-binding domain, which is required for acetyl-CoA interaction (53). Results from a recent study, in which molecular docking simulation was used, suggest that anacardic acid competes with acetyl-CoA for the VKG site in p300/cAMP-response element-binding protein-associated factor (54). Analogues of anacardic acid are also known to target the acetyl-CoA binding site of other HATs, such as Tip60 (55). In addition to anacardic acid and its derivatives (56, 57), several types of natural chemicals have been reported to bind HATs, such as epigallocatechin-3-gallate (58), plumbagin (59), curcumin (60), and garcinol (61). Therefore, we investigated whether these chemicals can inhibit the interaction between the PB2 cap-binding domain and acetyl-CoA (Fig. 4A). Low concentrations (5 μm) of epigallocatechin-3-gallate slightly reduced the interaction between the PB2 cap-binding domain and acetyl-CoA, increasing the epigallocatechin-3-gallate concentration to 25 and 100 μm and resulted in similar, but not greater, levels of inhibition as observed with the 5 μm treatment. Plumbagin and curcumin at concentrations of 5 and 25 μm did not block the interaction, although concentrations of 100 μm did result in a slight attenuation of interaction activity. In contrast, anacardic acid and garcinol drastically inhibited the interaction, and concentrations of anacardic acid greater than 25 μm completely eliminated interaction, whereas only faint activity was detected by adding 25 μm garcinol; however, no interaction was detected with the addition of 100 μm garcinol. These results demonstrated that several HAT inhibitors also blocked the interaction between the PB2 cap-binding domain and acetyl-CoA.

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