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A novel mechanism of enhanced transcription activity and fidelity for influenza A viral RNA-dependent RNA polymerase

Abstract During RNA elongation, the influenza A viral (IAV) RNA-dependent RNA polymerase (RdRp) residues in the active site interact with the triphosphate moiety of nucleoside triphosphate (NTP) for catalysis. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation remain elusive. Here, we demonstrated through enzymology, virology and computational approaches that the R239 and K235 in the PB1 subunit of RdRp are critical to controlling the activity and fidelity of transcription. Contrary to common beliefs that high-fidelity RdRp variants exert a slower incorporation rate, we discovered a first-of-its-kind, single lysine-to-arginine mutation on K235 exhibited enhanced fidelity and activity compared with wild-type. In particular, we employed a single-turnover NTP incorporation assay for the first time on IAV RdRp to show that K235R mutant RdRp possessed a 1.9-fold increase in the transcription activity of the cognate NTP and a 4.6-fold increase in fidelity compared to wild-type. Our all-atom molecular dynamics simulations further elucidated that the higher activity is attributed to the shorter distance between K235R and the triphosphate moiety of NTP compared with wild-type. These results provide novel insights into NTP incorporation and fidelity control mechanisms, which lay the foundation for the rational design of IAV vaccine and antiviral targets.


Cell-based mini-genome luciferase assay
The pcDNA3-WSN-PB1 (wildtype and mutants), PB2, PA, and NP genes were used to express the recombinant IAV polymerase in the HEK-293T cells. The reporter gene plasmid pPolI-NS-Luc plasmid (pBZ81A36) contains the negative-sense influenza vRNA-like firefly luciferase gene flanked by human Pol I and IAV polymerase 3' promoter and 5' terminator. pCMV-Gaussian luciferase plasmid (NEB) was added to serve as transfection control. The plasmids mix were reading was obtained from firefly luciferase and quenched, and the second reading was obtained from gaussian luciferase. Cell lysates were transferred to a 96-well white plate and the signal was measured on the Varioskan LUX Multimode Microplate Reader (Thermofisher) in luminescence mode. The relative polymerase activity was represented as Firefly luciferase intensity / Gaussian luciferase intensity and normalized to the intensity of wild-type polymerase (100%) ( Fig. 2A and   3A). The Gaussian luciferase intensity was normalized to the intensity of polymerase without the PB1 gene (Fig. S3).

Influenza A virus polymerase complex purification
The TAP tag on the PB2 subunit consisting of a TEV protease cleavage site and a protein A domain was used as tandem affinity tags in the purification steps according to previous works (1,2). Prior to plasmid transfection, the HEK-293T cells were passaged at least three times before seeded in 10 ml to a 10 cm dish at 70% confluence. Cells were transfected with 1.6 μg of each of PB1 To quantify the amount of polymerases, we used Bovine Serum Albumin (BSA) as the mass standard to estimate the yield of purified wild-type and mutant IAV RdRp complexes in SDS-PAGE (Fig. S4A). In particular, we prepared the BSA standard by diluting the commercial BSA standard solution (2 mg/mL, Thermofisher) into 30 ng/µl, 20 ng/µl, 10 ng/µl, 5 ng/µl. We loaded 6 µl of each BSA standard in parallel with 10 µl of purified RdRp complexes in 8 % SDS-PAGE.
The gel was stained in InstantBlue (Abcam), and the yield was estimated from the standard curve fitted from various concentrations of BSA. The mass of wild-type and mutant RdRp complex is estimated between 5 to 7 ng/µl.
To verify if mutations made on PB1 affect the correct expression of PB1 subunits and the formation of polymerase complexes, we have conducted the western blot analysis on PB1, PB2, and PA subunits of the purified wild-type and mutant IAV RdRps. The results suggested that all three subunits from wild-type and mutant IAV polymerases were expressed correctly at the expected molecular weight (Fig. S4C). We also stained the SDS-PAGE to visualize the expression and integrity of all three subunits after the purification. As shown in Fig. S4A and S4B, the PB1, PB2-TAP, PA subunits were resolved and visualized in 8 % SDS-PAGE as well as the co-purified Hsp70, similar to a previous protocol publication (1). Altogether, our results from western blot and gel stain indicate that the introduced K235 and R239 mutant systems had well expressed all polymerase subunits as well as maintained the RdRp complex integrity.

Capped RNA initiated in vitro transcription assay
The radioactive capped RNA initiated in vitro transcription of the IAV polymerase was set up in a 10 μl reaction. The mixture contains 5 mM Tris-HCl pH 8, 5 mM MgCl2, 75 mM NaCl, 5% overnight, and the screen was scanned by Sapphire Biomolecular Imager (Azure Biosystems). The gel image was analysed in Image Lab (Biorad).
To ensure that the proper amount of primer was used to best visualize the product completion in the in vitro transcription assay, we conducted an in vitro transcription experiment by gradually increasing the concentration of radioactively labelled primers (Fig. S5). The remaining conditions were kept constant to ensure that the only variable is the primer concentration. When the primer concentration is less than 50 cps, the extension percentages remain at greater than 31%. However, when the primer concentration exceeds 50 cps, the extension percentage drops to 20.0 % and 10.6 %, indicating that an excessive amount of primer would decrease the extension percentage.

in vitro single-turnover NTP incorporation
The method utilizes single-turnover incorporation of one incoming NTP at various concentrations The IAV genome's template sequence starts with 3'-UCGUUUU or 3'-UCGCUUU, which are highly conserved and crucial for transcription efficiency and structural stability of RNA-bound RdRp ( Fig. S7A) (4). Furthermore, to assess the fidelity of IAV transcription in vitro, we employed an 11nt radioisotope-labelled capped primer terminating in AG-3', which mimics the IAV RdRp's unique cap-snatching mechanism in infected host cells (5). To effectively commence transcription, the primer's ending AG'-3 will couple and prime with the 3'-UC in the 3' template's priming region.
Thus, fidelity may be quantified by comparing the NTP incorporation to the G at the template's third position (we use single-turnover NTP integration to assess fidelity, see Fig. S7B). Therefore, in our setup, only CTP and UTP quantification (but not ATP and GTP) is feasible as CTP and UTP will pair with the 3rd position G but not with the subsequent Us in the highly conserved 3'-UCGUUU template sequence and our adopted AG-3' primer ( Fig. S7B). This would yield a single extension product ( Fig. S7C and S7D), and thus satisfy the fidelity measurement condition. On the contrary, if the IAV RdRp is soaked in ATP or GTP, the ATP and GTP will couple with the following Us in the template strand despite the mismatch in the initial incorporation of ATP:G or GTP:G. This results in uncertainty in RNA extension ( Fig. S7E and S7F) and makes it difficult to conduct single-turnover NTP incorporation to measure fidelity.
It is worthy to note that there should be multiple extension products with the AG transcription assays as stated in the previous literature (6)(7)(8). The multiple extension products are mainly due to different priming schemes, including a primary C initiated product by AG-3' priming with 3'-UC, a secondary G initiated product by G-3' priming with 3'-U, and a few realignment products by realigning the primer after an initial addition of several nucleotides (as illustrated in Fig. S8A). As a result, multiple bands can be observed as reported in the previous studies using a 14-nt template (7). However, in this study, we used a 30-nt template which is originated from the 3' end of the PA segment and observed only one compact band, because the multiple extension products from the 30-nt template (e.g., products with a length of 39-nt and 40-nt) could not be resolved clearly in the Urea-PAGE (Fig. S8B). Therefore, we believe that the discrepancy between the multiple extension products observed in the previous literature and 1-nt extension product captured in the current study is mainly attributed to the resolving ability of the Urea-PAGE gel weakens when template length increases from 14-nt to 30-nt. To further verify the different resolving ability of Urea-PAGE gel towards the 14-nt and 30-nt template, we performed the transcription assay on a 30-nt template in parallel with a 14-nt template. Indeed, consistent with previous studies, we observed multiple bands from 14-nt template extension while only one compact band from the 30nt template extension (Fig. S8B). Besides, we found that our extension products from 30-nt template migrate slower than expected even in denaturing environment of 8 M urea. We believe it is due to the strong hybridization formed by product RNA and the complimentary template, as suggested by a recent paper (9).
The above-discussed multiple products shall not impact our single-turnover NTP incorporation assay (Fig. S6), as we conducted it by limiting the NTP pool to ensure only a single NTP extension product occurred (Fig. S8C). In particular, only CTP or UTP was used for the single-turnover assay, which pairs with the G on the template following the initial 3'-UC priming site. Due to the absence of GTP in the NTP pool, the secondary G initiated products will not be extended beyond C on the template when the primer's ending G pairs with the template's initial 3'-U. (Fig. S8D).
Therefore, we only observed 1-nt extension in Fig. S6.

Data fitting
Nonlinear-regression data fitting was performed using GraphPad Prism 8.

Fast and slow phases of NTP substrate incorporation
Single turn-over in vitro transcription was performed under various NTP concentrations. The observed reaction rate (kobs) at each substrate concentration was obtained through the non-linear regression fitting of product formation over time (Fig. S6). The fitting is not optimal when the data are fitted into one phase exponential equation, as the end of the fitted curve does not even out after the initial rapid incorporation. The obtained fast (kfast) and slow (kslow) observed rates in the biphasic exponential equation represented the co-existence of two populations of IAV RdRPs in the conformational equilibrium. kfast represents the fast incorporation of NTP substrate in the reaction contributed by the poised RdRps, while kslow is counted for the catalysis of suboptimal RdRps that requires a rate-limiting step of associated isomerization (e.g., translocation and active site closure). A similar two-phase kinetics has also been observed for the nucleotide incorporation kinetics of yeast RNA polymerase II (3). subsequently derive the discrimination score for cognate over noncognate NTP, which serves as an enzymatic explanation for the fidelity of NTP incorporation.

Exploration of the fitting parameters on the percentage of the fast phase
During the fitting of the biphasic exponential equation, the percentage of fast phase (fast%) describes the percentage of IAV RdRP population that is at optimal conformation for catalyzing substrates, while the percentage of slow phase (slow% = 100% − fast%) represents the suboptimal population in the reaction. To determine the fast% for both wildtype and K235R RdRP in CTP and UTP base pairing, we calculated the sum of squared errors (SSE) for each base pairing scenario for wildtype and K235R and chose the corresponding fast% based on the minimal medium and variation in SSE (Fig. S9A). The fast% for CTP substrate is 58% for wildtype and 53% for K235R RdRP, and the fast% for UTP substrate is 31% for wildtype and 34% for K235R RdRP. The fast% is considerably higher for cognate NTP than noncognate NTP, as the reaction of incorporating cognate NTP is much favored than noncognate NTP for both wildtype and K235R RdRPs.
Nevertheless, we also performed a spectrum scanning to survey if the higher fidelity of K235R mutant depends on the value of fast%. We plotted a 3D diagram for the discrimination (Z-axis) of wildtype and K235R RdRP against the fast% of CTP (X-axis) and UTP (Y-axis) (Fig. S9B). And it is evident that the plane representing the discrimination of K235R mutant is always above that of wildtype, indicating the conclusion of the higher fidelity of K235R mutant is valid regardless of the fast%.

MD simulations of IAV RdRp
The cryo-EM structure of the IAV elongation complex (PDBID: 6SZV (9)) was used as the structural basis to construct the IAV RdRp with the template-product duplex. Missing protein residues were modelled using modeller9.21 (13). To match with the experimental sequence, the UTP analog present in the cryo-EM structure was replaced with a CTP, and its base-paired template nucleotide was also mutated to G by Coot (14). The protonation states of histidine residues were predicted using propka3.0 module (15) in the pdbpqr2.2.1 (16) package, and manual examination was performed to ensure that the coordination with the magnesium ion can be formed as well as the hydrogen bond network can be maintained. The RdRp complex was placed in a dodecahedron box, the size of which was defined to ensure that the box edges at least 12 Å away from the complex surface. The complex was solvated with TIP3P water molecules (17), and sufficient counter ions were added to render the whole system neutral.
Amber99sb-ildn force field (18) was adopted to simulate protein and nucleotides. The default parameters for 3'-cytosine nucleotide were used for the base and sugar of the CTP, while the parameters for the triphosphate tail were taken from those developed by Meagher et al. (19). To reduce the instability caused by the excessive electrostatic repulsion between the two neighboring magnesium ions, dummy atom model (20,21) was used for simulating the magnesium ions in the PA and PB1 domains. Similar procedures as described above were adopted to construct and equilibrate the systems containing K235 or R239 mutants as well as those with UTP binding. Five parallel 100ns simulations with random seeds were performed for each mutant.

Structural analysis
The were used in the distance calculations.

Investigation of K235R mutation on the active site configuration
First, we calculate the effect of the K235R mutation on the critical distances for CTP incorporation catalysis (Fig. S12). Firstly, we computed the distance between the two magnesium ions (Fig.   S12A), which has been suggested to be less than 4.0 Å for the catalysis to occur (26,27). We discovered that the K235R mutant does not perturb this distance, which is 2.1 Å in both wildtype and K235R mutant systems (Fig. S12B). Secondly, we calculated the distance between the P α atom in the incoming CTP and the O3' atom in the nascent strand's 3'-terminal nucleotide (Fig. S12A).
This distance also needs to be smaller than 4.0 Å for the catalysis to occur (28,29). Both the wildtype and K235R mutant systems exhibit an averaged distance of 3.5 Å (Fig. S12B), indicating that CTP can be efficiently incorporated in both systems. The above results indicate that the K235R mutant does not affect the critical distances for the catalysis, which is consistent with the experimental observation that both WT and K235R can efficiently synthesize RNAs.
Next, we examined the effect of the K235R mutant on the orientation of the R239 relative to the P β atom of CTP, which also plays an important role in the catalysis of the phosphoryl transfer reaction. As shown in Fig. S16B and S16E, the K235R mutant does not alter the relative position of R239's side chain to the P β atom of CTP. These results indicate that the mutation of K235R has negligible impact on the orientation of R239. However, we found that the K235R mutant makes itself approach closer to the P β atom than the wildtype K235 ( Fig. S16B and S16E), and this observation is also consistent with the distance analysis in Fig. 5C.
Finally, we investigated the effect of the K235R mutation on the interactions between positively charged residues (R239 and K235/K235R) and the CTP triphosphate moiety. In particular, we discovered that the side-chain of R239 is in close contact (3.5 Å) with the P α group of the incoming CTP in the wildtype system (black curve in Fig. S13A), which may serve to stabilize the transition state during the formation of the phosphodiester bond. The K235R mutant well maintains this interaction, with the mean distance equal to 3.5 Å (red curve in Fig. S13A). Interestingly, we discovered that the K235R mutant form new interactions between K235R and the P α & P γ groups of CTP (red curves in Fig. S13B and S13C), and these interactions are absent in the wildtype system (black curve in Fig. S13B and S13C). These newly formed interactions between K235R/R239 and the triphosphate moiety of CTP well explain the synergistic effects of K235R and R239 to facilitate the NTP incorporation.

Comparison between the K235R mutation in IAV RdRp and K65R mutation in HIV-1 RT
In HIV-I reverse transcription (RT), the side chain of K65 is in contact with the substrate's triphosphate moiety at a minimum distance of 2.4 Å (see vdW representations of K65 and NTP in Fig. S14A). In IAV RdRp, by contrast, the side chain of K235 is located at a distance of 3.7 Å or larger from the substrate's triphosphate moiety (Fig. S14C). As a result, we hypothesize that HIV-1 RT has a more compact active site. To investigate the effect of the Lys-to-Arg mutation on the space between the substrate and the respective protein residue in HIV-1 RT, we also generated structural model by mutating the Lysine residue while retaining its orientation to the substrate. We discovered that the K65R mutation brings it closer to two phosphate atoms of substrate at distances of 2.4 Å and 2.7 Å, respectively (Fig. S14B). The Lys-to-Arg mutation in IAV RdRp, on the other hand, allows the residue to be at least 3.0 Å away from the substrate (Fig. S14D). These findings suggest that the K65R mutation in HIV-1 RT could render the active site too crowded and may significantly reduce the active site's stability. This could also explain why the K65R mutation impairs transcription activity.

Calculations of relative binding free energy
We calculated the relative binding free energy (∆∆G) between the K235R mutant and wildtype  S15B).
We noticed that our relative binding free energy suggests that K235R improves the binding of CTP (ΔΔG=-5.2±0.5 kJ/mol) and UTP (ΔΔG=-1.8±0.7 kJ/mol) to the active site (Fig. S15B), while the experimentally measured Kd,app suggests a higher dissociation constant for K235R (Table S1). We anticipate that this difference is due to the contributions of the NTP loading. In particular, the longer sidechain of Lys-to-Arg mutation in K235R may introduce steric effect to hinder the NTP loading (leading to a larger value of Kd,app), as K235R lies at the exit of the NTP loading channel before entering the active site. This inhibitory effect would exist for all NTP regardless of the base, and thus lead to an increase in Kd,app for both CTP and UTP at a similar magnitude.

Geometric clustering to select representative configurations
To select the representative configurations, we applied the K-Center clustering algorithm (34,35) to divide the MD configurations into five groups based on the geometry similarity of the heavy atoms in NTP and two positively charged protein residues (resids 235 and 239). Notably, we found that the distributions of the K235R-UTP system contain two clusters with significant populations (see these two representative conformations in Fig. S17D). Interestingly, one of these configurations (Right panel of Fig. S17D with population 27%) displays a relatively larger distance between K235R and β-phosphate of the UTP, possibly due to larger fluctuations of the phosphate tail induced by the relatively unstable wobble base pair. In contrast, all the other three systems (WT-CTP, WT-UTP, and K235R-CTP) are dominated by a single cluster (see the representative conformation for each system in Fig. S17A-C). These observations indicate that the conformational flexibility is most pronounced in the K235R mutant RdRp with non-cognate UTP, which is consistent with the observations in Fig. S16.

NGS sequencing of wildtype and PB1-K235R recombinant live viruses
We generated the wild-type and PB1-K235R mutant A/WSN/1933(H1N1) recombinant virus from eight pHW2000 plasmids encoding each of the IAV genome segments, including either the wildtype or mutant K235 PB1 gene. The titres of passage 1 (P1) viruses were determined using the plaque assay, and the P2 viruses were passaged by inoculating P1 viruses into MDCK cells at a multiplicity of infection (MOI) of 0.001. In 10 cm culture dishes, P3 viruses were multiplied by inoculating MDCK cells with P2 viruses at an MOI of 0.001 and subjected to plaque assays to quantify the viral titres (Fig. 6A). 10 mL of media containing P3 wild-type and K235R mutant viruses were collected and centrifuged at 500 ×g for 5 minutes to eliminate floating cell debris.
Amicon Ultra-15 100 K was used to concentrate the supernatant medium to 500 µl at 1500 ×g, wild-type and K235R mutant IAV mediated by the Tn5-tagmentation method (37). To summarize, the viral genome PCR results were processed with Tn5 transposase, resulting in major products of tagged dsDNA fragments of around 300 bp. Twelve cycles of PCR were used to repair and index the dsDNA fragment, which was then fed into the MGI DNBSEQ-G400 platform.
The sequencing reads were first trimmed in paired-end mode with cutadapt (38) using default parameter and mapped into reference IAV genome (GenBank Source Sequence Accession: LC333182 to LC333189) using bwa (39). Reads with Indel and soft-clipped were removed.
Mapped reads (sam file) were sorted and converted to bam files by samtools (40) and the Mpileup files were generated by samtools with the following criteria: 1) Sequencing base with quality scores over 30, 2) Mapping quality over 40. The number of mutation sites was counted by screening the mpileup with the following criteria: 1) Error rate ≥ 1% , 2) Depth ≥ 1000 Error rate(%) .
We analysed and compared wild-type and K235R viral genome from two biological replicates.
The average sequencing depths of both wild-type and K235R are ~10 5 per replicate (Fig. 6B). The number of SNV sites in wildtype live virus is 19.25 ± 0.50 (Mean ± SD), whereas the number of SNV sites in K235R is 12 ± 2.94 (Mean ± SD). Our results suggest that K235R is indeed a highfidelity mutant at the virus level. Our findings imply that K235R is really a virus-level high-fidelity mutant ( Fig. 6C and 6D).       µM, 10 µM CTP or UTP. The CTP:G incorporation was stopped at 0.5s, 2s, 5s, 10s, 30s, 60s, while the UTP:G incorporation was stopped at 5s, 15s, 30s, 60s, 120s, 300s, 600s. The reaction was resolved on 20% denaturing urea polyacrylamide gel. The upper band represents the incorporated nucleotide, and the lower band represents the unincorporated primer. (B) Two-phase exponential fitting of the incorporation products. The quantified and normalized incorporation products were fitted into a biphasic exponential curve and plotted over time.  The capped primer terminating in AG will generate multiple extension products, including those initiated from C and G in the third and second positions on the 3' template, respectively, as well as the re-alignment products. (B) A 14-nt or 30-nt 3' template was used to initiate the in vitro transcription assay of wild-type RdRp. For 1 hour, the reactions were carried out in parallel at 30 °C. In denaturing gel, transcription products were resolved. (C) Illustration of a single-turnover NTP incorporation assay used to determine NTP's specificity. The NTP pairing with G in the third position of the template's 3' end will be evaluated. (D) Only CTP or UTP is used to initiate the single-turnover assay. The wobble pairing priming route between the capped primer's ending G and the template's initial C will not proceed because no GTP is present in the reaction to pair with the template's coming C.           S18. NTP analog incorporation on wild-type and K235R IAV polymerases. 500 µM of 3'dCTP and 2'F-dCTP were mixed with wild-type (WT) and K235R polymerases. The reaction was incubated for one hour and subjected to 20% denaturing urea polyacrylamide gel. The +1 site represents the incorporated NTP analog into the primer.