tetano
Editor, Senior Moderator
J Biol Chem. 2014 Jul 25. pii: jbc.M114.559708. [Epub ahead of print]
A Novel Functional Site in the PB2 Subunit of Influenza A Virus Essential for Acetyl-CoA Interaction, RNA Polymerase Activity, and Viral Replication.
Hatakeyama D1, Shoji M1, Yamayoshi S2, Hirota T1, Nagae M1, Yanagisawa S1, Nakano M2, Ohmi N1, Noda T2, Kawaoka Y3, Kuzuhara T4.
Author information
Abstract
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate [m7GTP]) and supports the endonuclease activity of PA to snatch the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. 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). In-vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino-acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced PB2s binding ability to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. 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.
Copyright ? 2014, The American Society for Biochemistry and Molecular Biology.
KEYWORDS:
RNA polymerase; acetyl coenzyme A (acetyl-CoA); influenza virus; negative-strand RNA virus; recombinant protein expression
PMID:
25063805
[PubMed - as supplied by publisher]
Free full text
http://www.ncbi.nlm.nih.gov/pubmed/25063805
A Novel Functional Site in the PB2 Subunit of Influenza A Virus Essential for Acetyl-CoA Interaction, RNA Polymerase Activity, and Viral Replication.
Hatakeyama D1, Shoji M1, Yamayoshi S2, Hirota T1, Nagae M1, Yanagisawa S1, Nakano M2, Ohmi N1, Noda T2, Kawaoka Y3, Kuzuhara T4.
Author information
Abstract
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate [m7GTP]) and supports the endonuclease activity of PA to snatch the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. 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). In-vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino-acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced PB2s binding ability to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. 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.
Copyright ? 2014, The American Society for Biochemistry and Molecular Biology.
KEYWORDS:
RNA polymerase; acetyl coenzyme A (acetyl-CoA); influenza virus; negative-strand RNA virus; recombinant protein expression
PMID:
25063805
[PubMed - as supplied by publisher]
Free full text
http://www.ncbi.nlm.nih.gov/pubmed/25063805