Mechanism of action of T-705 ribosyl triphosphate against influenza virus RNA polymerase

Antimicrob Agents Chemother. 2013 Nov;57(11):5202-8. doi: 10.1128/AAC.00649-13. Epub 2013 Aug 5.


T-705 (favipiravir; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) selectively and strongly inhibits replication of the influenza virus in vitro and in vivo. T-705 has been shown to be converted to T-705-4-ribofuranosyl-5-triphosphate (T-705RTP) by intracellular enzymes and then functions as a nucleotide analog to selectively inhibit RNA-dependent RNA polymerase (RdRp) of the influenza virus. To elucidate these inhibitory mechanisms, we analyzed the enzyme kinetics of inhibition using Lineweaver-Burk plots of four natural nucleoside triphosphates and conducted polyacrylamide gel electrophoresis of the primer extension products initiated from (32)P-radiolabeled 5'Cap1 RNA. Enzyme kinetic analysis demonstrated that T-705RTP inhibited the incorporation of ATP and GTP in a competitive manner, which suggests that T-705RTP is recognized as a purine nucleotide by influenza virus RdRp and inhibited the incorporation of UTP and CTP in noncompetitive and mixed-type manners, respectively. Primer extension analysis demonstrated that a single molecule of T-705RTP was incorporated into the nascent RNA strand of the influenza virus and inhibited the subsequent incorporation of nucleotides. These results suggest that a single molecule of T-705RTP is incorporated into the nascent RNA strand as a purine nucleotide analog and inhibits strand extension, even though the natural ribose of T-705RTP has a 3'-OH group, which is essential for forming a covalent bond with the phosphate group.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amides / pharmacology*
  • Animals
  • Antiviral Agents / pharmacology*
  • Binding, Competitive
  • Cytidine Triphosphate / metabolism
  • Dogs
  • Enzyme Assays
  • Guanosine Triphosphate / metabolism
  • Influenza A Virus, H1N1 Subtype / drug effects*
  • Influenza A Virus, H1N1 Subtype / genetics
  • Kinetics
  • Madin Darby Canine Kidney Cells
  • Protein Binding
  • Pyrazines / pharmacology*
  • RNA, Viral / antagonists & inhibitors*
  • RNA, Viral / biosynthesis
  • RNA-Dependent RNA Polymerase / antagonists & inhibitors*
  • RNA-Dependent RNA Polymerase / genetics
  • RNA-Dependent RNA Polymerase / metabolism
  • Uridine Triphosphate / metabolism
  • Viral Proteins / antagonists & inhibitors*
  • Viral Proteins / genetics
  • Viral Proteins / metabolism


  • Amides
  • Antiviral Agents
  • Pyrazines
  • RNA, Viral
  • Viral Proteins
  • Cytidine Triphosphate
  • Guanosine Triphosphate
  • Adenosine Triphosphate
  • RNA-Dependent RNA Polymerase
  • favipiravir
  • Uridine Triphosphate