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.

On the Recognition of Natural Substrate CTP and Endogenous Inhibitor ddhCTP of SARS-CoV-2 RNA-Dependent RNA Polymerase: A Molecular Dynamics Study

“…The oxygen atoms of P β interacted during the whole simulation with the positively charged Lys551 and Arg553 residues of motif F for both examined ternary complexes, thus contributing to the binding of CTP and ddhCTP. Our finding is in good agreement with the observations on other viral nucleic acid polymerases. ,,,, …”

“…It was suggested that during the reaction, a shrinking of this distance may occur, leading O3′ to become closer to Pα and consequently facilitating the nucleophilic attack. The little longer distance in ddhCTP than that in CTP could affect the O3′–P α alignment for catalysis during the nucleophilic attack, in the reactive Michaelis–Menten complex. − …”

“…Our finding is in good agreement with the observations on other viral nucleic acid polymerases. 11 , 21 , 54 , 59 , 60 …”

“…Our finding is in good agreement with the observations on other viral nucleic acid polymerases. 11,21,54,59,60 V e r y i n t e r e s t i n g l y , t h e R d R p : R N A : C T P a n d RdRp:RNA:ddhCTP analyses of the trajectories of salt-bridges established by Asp618 revealed that it is frequently implicated in the interaction with Lys798, while the same interaction was not frequently observed in the binary complex (Figure 6). This behavior highlights the role played by Lys798 as the "needed counterion" in orienting the P γ toward Mg B .…”

Viral RNA Replication Suppression of SARS-CoV-2: Atomistic Insights into Inhibition Mechanisms of RdRp Machinery by ddhCTP

“…However, k cat ranging from 0.000105 s –1 to 680 s –1 were measured for a number of viruses, corresponding to 22.5 and 13.3 kcal mol –1 , respectively. , These values are, therefore, in reasonable agreement to the calculated energy of TSs presented here, as well as with those calculated for similar polymerases and enzymes catalyzing similar reaction. ,,,,, The reaction is exoenergetic, as expected, due to the cleavage of a highly energetic bond, and the NMP unit has been fully transferred, as reflected by the Pα–Oα distance of 5.641 Å, in CMP, and 4.098 Å, in ddhCMP. Finally, in analogy to other polymerases, ,,− in RdRp:RNA-CMP:PPi and RdRp:RNA-ddhCMP:PPi, the observed formation of pyrophosphate molecule is assisted by the stabilization of Mg B 2+ (see O Pγ –Mg B 2+ distance of 1.960 Å for CMP and 1.955 Å for ddhCMP in Figure ). The PPi is involved in hydrogen bond interaction with Lys621 residue, which resembles the well-known role observed in other systems by histidine residue, implicated in the proton transfer necessary for the completion of the chemical process .…”