Article Figures & Data
Figures
- Fig 1
Chemical structures of T-705 (A) and T-705RTP (B). (A) Prodrug; (B) active form.
- Fig 2
Inhibition of influenza virus RNA polymerase by T-705RTP. The percentage inhibition of influenza virus RNA polymerase at different concentrations of T-705RTP was determined as described in Materials and Methods. All samples were in triplicate, and means ± the SD values are shown. All samples were incubated at 30°C for 1 h and counted in each sample with [α-32P]GTP (○) or [5,6-3H] UTP (▲) as labeled precursors.
- Fig 3
Inhibitory activity of T-705RTP versus the incorporation of ATP, GTP, CTP, or UTP. The activity of influenza virus RNA polymerase in the presence of T-705RTP was determined at different concentrations of NTPs. The incorporation of [α-32P]GTP was measured when the UTP concentration was varied, and the incorporation of [5,6-3H] UTP was determined when the concentration of ATP, GTP, and CTP was varied. The results are presented as Lineweaver-Burk plots. All samples were incubated at 30°C for 1 h, and the nucleotide concentrations were indicated in Table 1. (A) Incorporation at different ATP concentrations with 0 (○), 5 (▲), and 10 (□) μM T-705RTP. (B) Incorporation at different GTP concentrations with 0 (○), 2.5 (▲), and 5 (□) μM T-705RTP. (C) Incorporation at different CTP concentrations with 0 (○), 5 (▲), and 10 (□) μM T-705RTP. (D) Incorporation at different UTP concentrations with 0 (○), 0.1 (▲), and 0.3 (□) μM T-705RTP. The results represent the means ± the SD of triplicate determinations.
- Fig 4
Incorporation of T-705RTP or various nucleotides into a nascent RNA strand. (A) Incorporation of CTP, GTP, and T-705RTP at the position of G11+1. The 32P-labeled pGEM-7zf(+) DNA runoff transcript with a 5′Cap1 structure (Cap1-pGEM-mRNA), crude influenza virus RdRp containing a viral genome, and nucleotides including T-705RTP were incubated. Reaction products were then electrophoresed. Lane 1, Cap1-pGEM-mRNA; lanes 2 to 6, Cap1-pGEM-mRNA + crude enzyme solution; lane 3, conditions of lane 2 + 50 μM CTP; lane 4, conditions of lane 2 + 50 μM GTP; lanes 8 and 9, conditions of lane 2 + 100 or 1,000 μM T-705RTP. (B) Incorporation of GTP, T-705RTP, 2FdGTP, and dGTP at the position of G11+2. The 32P-labeled pGEM-7zf(+) DNA runoff transcript with a 5′Cap1 structure (Cap1-pGEM-mRNA), crude influenza virus RdRp containing a viral genome, and nucleotides including T-705RTP were incubated. Reaction products were then electrophoresed. Lanes 1 to 7, Cap1-pGEM-mRNA + crude enzyme solution + 50 μM CTP; lanes 2 and 3, conditions of lane 1 + 100 or 1,000 μM GTP; lanes 4 and 5, conditions of lane 2 + 100 or 1,000 μM T-705RTP; lane 6, conditions of lane 1 + 1,000 μM 2FdGTP; lane 7, conditions of lane 1 + 1,000 μM dGTP.
- Fig 5
Inhibition of T-705RTP against influenza virus RdRp. The 32P-labeled pGEM-7zf(+) DNA runoff transcript with a 5′Cap1 structure (Cap1-pGEM-mRNA), crude influenza virus RdRp containing a viral genome, and nucleotides including T-705RTP were incubated. Reaction products were then electrophoresed. Lane 1, Cap1-pGEM-mRNA; lanes 2 to 6, Cap1-pGEM-mRNA + crude enzyme solution; lanes 3 to 6, conditions of lane 2 + 50 μM CTP, 100 μM ATP, or 50 μM GTP; lanes 4 to 6, conditions of lane 3 + 10, 100, or 1,000 μM T-705RTP. *, Elongated RNA was detected when GTP, ATP, and CTP were added to the reaction mixture.
Tables
- Table 2
Sequences of nascent RNA transcripts transcribed from the 3′ ends of genome segments
Segment no. Segment name Primer Incorporated nucleotide at position: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 PB2 G11+ C G A A A G C A G G U 2 PB1 G11+ C G A A A G C A G G C A A A C C A U 3 PA G11+ C G A A A G C A G G U 4 HA G11+ C A A A A G C A G G G G A A A A U 5 NP G11+ C A A A A G C A G G G U 6 NA G11+ C A A A A G C A G G G G U 7 M G11+ C A A A A G C A G G U 8 NS G11+ C A A A A G C A G G G U
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Figure S1, G11 primer synthesis and transcription initiation for analysis of the incorporation of T-705RTP to the influenza virus RNA chain by the primer extension method.
PDF, 8.8K
- Supplemental file 1 -
