Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Polymerase activity and mechanisms of action of nucleotide analogs

<div><p>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 <i>K</i>_<i>app</i> was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.</p></div

Inhibition of influenza virus and crude replication complex.

<p>Inhibition of influenza virus and crude replication complex.</p

Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Endonuclease activity and evaluation of inhibitors

<div><p>Influenza polymerase is a heterotrimer composed of polymerase acidic protein A (PA) and basic proteins 1 (PB1) and 2 (PB2). The endonuclease active site, located in the PA subunit, cleaves host mRNA to prime viral mRNA transcription, and is essential for viral replication. To date, the human influenza A endonuclease activity has only been studied on the truncated active-site containing N-terminal domain of PA (PA_N) or full-length PA in the absence of PB1 or PB2. In this study, we characterized the endonuclease activity of recombinant proteins of influenza A/PR8 containing full length PA, PA/PB1 dimer, and PA/PB1/PB2 trimer, observing 8.3-, 265-, and 142-fold higher activity than PA_N, respectively. Using the PA/PB1/PB2 trimer, we developed a robust endonuclease assay with a synthetic fluorogenic RNA substrate. The observed <i>K</i>_m (150 ± 11 nM) and <i>k</i>_<i>cat</i> [(1.4 ± 0.2) x 10^-3s^-1] values were consistent with previous reports using virion-derived replication complex. Two known influenza endonuclease phenylbutanoic acid inhibitors showed IC₅₀ values of 10–20 nM, demonstrating the utility of this system for future high throughput screening.</p></div

Determination of apparent binding affinity (<i>K</i>_app) of GTP, T-1106-TP, and 4’vinyl-FdGTP during single nucleotide incorporation by the recombinant influenza RdRp.

<p>(A) Diagram illustrating formation of RNA 9-mer product from ApG primer and further elongation to RNA 14-mer product; (B) Summary of <i>K</i>_app and selectivity for GTP and analogs; Incorporation of GTP (panel C), T-1106-TP (panel D), and 4’vinyl-FdGTP (panel E) as a function of NTP analog concentration. The product formation is plotted as a function of compound concentration for GTP (panel F), T-1106-TP (panel G), and 4’vinyl-FdGTP (panel H), respectively. The calculated <i>K</i>_<i>app</i> values for GTP, T-1106-TP, and 4’vinyl-FdGTP are 9 nM, 27 nM, and 29 μM respectively.</p

Mechanism of action for different nucleotide analogs during recombinant RdRp-catalyzed RNA synthesis.

<p>(A) Reaction scheme showing cleavage of the ³³P-m⁷G¹67 primer by influenza polymerase heterotrimer, base pairing with miniHA template, and expected transcription product. (B) Reaction mixtures contained miniHA template, radiolabeled capped RNA primer ³³P-m⁷G¹67, 500 μM GTP or GTP analogs, and 500 μM for each of natural CTP, UTP, and ATP. Products were separated by 25% PAGE and visualized by autoradiography. The top of the gel was illustrated to show the formation of long transcription products. The ladder of nucleotide bases on the side bar demonstrates the correct incoming nucleotide base-pairing with the miniHA template.</p

Activity of RNA replication and transcription by recombinant influenza RdRp heterotrimer.

<p>(A) The rate of ³³P-α-GTP incorporation by cRNP is proportional to the estimated viral particles used to generate cRNP; (B) The rate of ³³P-α-GTP incorporation by recombinant RdRp is proportional to the PA/PB1/PB2 trimer protein concentration; (C and D) The product formation presented by an arbitrary unit on the phosphor imager is linear with time and similar between the replication and transcription rates observed with cRNP (panel C) and recombinant RdRp (panel D) during ApG-primed RNA replication (●) or rabbit globin mRNA-primed RNA transcription (■). (E) Product formation using different RNA templates was analyzed on a 10% polyacrylamide gel. In contract to cRNP-catalyzed RNA synthesis (far left lane) which generated a smeared ladder of long products at the top of the gel, recombinant RdRp generated approximately template length products during ApG-primed RNA replication (D, B). Furthermore, transcripts of PA-30 and mini-HA containing an additional 9–11 nucleotides were generated during rabbit globin mRNA-primed RNA transcription (C, A).</p

RNA substrate and known influenza endonuclease inhibitors used in this study.

<p>(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.</p

Biochemical characterization of purified influenza polymerase proteins.

<p>(A) Diagram showing the interactions between the PA, PB1, and PB2 subunits. (B) Coomassie staining of purified PA proteins. 1 μg of PA_N (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.</p

Inhibition of PA/PB1/PB2 trimer activity by influenza A endonuclease inhibitors.

<p>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).</p

Biophysical characterization of purified influenza polymerase proteins.

<p>(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).</p