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PLoS One. 2017 Oct 11;12(10):e0185998. doi: 10.1371/journal.pone.0185998. eCollection 2017.
Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Polymerase activity and mechanisms of action of nucleotide analogs.
Barauskas O1, Xing W1, Aguayo E1, Willkom M1, Sapre A1, Clarke M1, Birkus G1, Schultz BE1, Sakowicz R1, Kwon H1, Feng JY1.
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Abstract
Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity Kapp was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.
PMID: 29020100 DOI: 10.1371/journal.pone.0185998
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Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Polymerase activity and mechanisms of action of nucleotide analogs.
Barauskas O1, Xing W1, Aguayo E1, Willkom M1, Sapre A1, Clarke M1, Birkus G1, Schultz BE1, Sakowicz R1, Kwon H1, Feng JY1.
Author information
Abstract
Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity Kapp was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.
PMID: 29020100 DOI: 10.1371/journal.pone.0185998
Free full text