Fig 1.
Biochemical characterization of purified influenza polymerase proteins.
(A) Diagram showing the interactions between the PA, PB1, and PB2 subunits. (B) Coomassie staining of purified PA proteins. 1 μg of PAN (lane 1), PA (lane 2), PA/PB1 dimer (lane 3) and PA/PB1/PB2 trimer (lane 4) were analyzed by Coomassie Blue-stained SDS-PAGE. Molecular weights of protein standards are indicated on the left. (C) Western blot detection of purified proteins. 100 ng of PA, PA/PB1 dimer, and PA/PB1/PB2 trimer were subjected to SDS-PAGE and transferred to PVDF membrane. The membrane was blotted with anti-FLAG, anti-PB1 and anti-PB2 antibodies for detection of PA, PB1 and PB2 proteins respectively.
Fig 2.
RNA substrate and known influenza endonuclease inhibitors used in this study.
(A) Sequence of the RNA-FRET substrate with FAM fluorophore and Dabcyl quencher; (B) Structures and systematic names of compounds A and B tested in IC50 assays.
Fig 3.
Biophysical characterization of purified influenza polymerase proteins.
(A) Analytical ultracentrifugation analysis of purified proteins. For PA, PA/PB1 dimer and PA/PB1/PB2 trimer, analytical ultracentrifugation was performed in a buffer containing 25 mM HEPES (pH 7.6), 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and 0.01% C12E8. Sedimentation velocity analysis was performed at 42,000 rpm. Representative traces for each protein are shown. (B-D) SEC-MALS analysis of purified PA (B) PA/PB1 (C), and PA/PB1/PB2 in the presence (black) or absence (green) of detergent during SEC purification (D). The normalized UV absorbance trace (280 nm) is plotted on the left axis. The horizontal gray lines correspond to the calculated mass by SEC-MALS (right axis).
Fig 4.
Enzymatic characterization of purified proteins.
(A) The endonuclease activity of PA/PB1/PB2 trimer as a function of time and increasing protein concentration. Dilutions of the trimer were prepared in buffer containing 50 mM HEPES (pH 7.5), 100 mM KCl, 1 mM DTT, and 1 mM MnCl2. Reactions were initiated with 1 μM RNA-FRET substrate at room temperature and fluorescence was measured with excitation at 495 nm and emission at 516 nm at 30 second intervals. (B) Different endonuclease activity of the PA/PB1/PB2 trimer in the presence of manganese or magnesium. Dilutions of the trimer were prepared and assayed as described previously using either 1 mM MnCl2 or 5 mM MgCl2. The average reaction rates were calculated from the linear portion of each curve over a 30-minute reaction time and plotted as a function of the trimer enzyme concentration. (C) Determination of kcat for the endonuclease activity of the PA/PB1/PB2 trimer and binding affinity (Km) for the RNA-FRET substrate. 10 nM PA/PB1/Pb2 was incubated in a buffer containing 1 mM MnCl2 as described previously. Two-fold serial dilutions of the RNA-FRET probe from 1 μM were prepared in buffer containing DMSO to 5% final reaction volume. Reactions were initiated with RNA dilutions at room temperature and fluorescence emission was monitored with excitation 495 nm and emission at 516 nm at 30 second intervals. Average rates were calculated from the linear portion of each curve and plotted as a function of RNA concentration. Total hydrolysis of the substrate was achieved by incubation for 10 minutes with 10 U/mL RNAse I and used to generate a standard curve used in the calculation of kcat. The Km and kcat values obtained were 150 ± 11 nM and (1.4 ± 0.2) x 10-3s-1, respectively.
Fig 5.
Inhibition of PA/PB1/PB2 trimer activity by influenza A endonuclease inhibitors.
5 nM trimer was pre-incubated for 15 minutes with three fold serial dilutions of compound A (A) or compound B (B) from 1000–2000 nM at room temperature in a buffer containing 50 mM HEPES (pH 7.5), 100 mM KCl, 1 mM DTT, 1 mM MnCl2, and DMSO (2% v/v). Reactions were initiated by addition of 200 nM RNA-FRET substrate and monitored as described above. Average reaction rates were calculated over the 30-minute reaction time. IC50 curves were fitted with a four parameter dose response with variable slope equation (GraphPad Prism 6).