Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo

Abstract

Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stoppedflow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg 2؉ ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe 364 and Pro 357 , which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe 364 to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.The RNA-dependent RNA polymerases (RdRPs) 2 from positive strand RNA viruses close their active sites for catalysis via a subtle NTP-induced conformational change within conserved motifs A and C (1). This palm domain-based closure mechanism differs from what is observed in other classes of replicative polymerases where the palm is fully structured prior to NTP binding, and the nascent base pair is delivered into the active site for catalysis via molecular motions originating in the finger domain (2, 3). Despite these different molecular motions, the structural end points are essentially equivalent, and the RdRPs retain the highly conserved polymerase active site geometry with aspartate residues and a magnesium-catalyzed twometal reaction mechanism (4, 5). The origin of the palm-based movement in the viral RdRPs is likely the conserved molecular contact between the finger and thumb domains that stabilizes the protein structure at the expense of reducing finger domain flexibility (6).One key characteristic of the viral RdRPs is their relatively low replication fidelity with mutation frequencies of 10 Ϫ4 -10 Ϫ5 that result in a heterogeneous virus population often referred to as a quasispecies (7,8). The population consensus sequence defines a particular virus and strain, but closer inspection of individual genomes reveals that they each contain a few random point mutations relative to the consensus. This poo...

A nucleobase-binding pocket in a viral RNA-dependent RNA polymerase contributes to elongation complex stability

“…Fluorescence excitation wavelength was 492 nm (monochromator, bandwidth 2 nm) and fluorescent signal was detected using a 515-nm high band pass filter. ECs were pre-assembled at 22.5 • C for 90 min using 1 M Data analysis and fitting were done as described previously (39)(40)(41). The data of rapid signal increase observed at the end of the elongation reaction were fitted into a single exponential rise curve.…”

Picornaviral polymerase structure, function, and fidelity modulation

“…residue 363 in PV) (McDonald et al, 2016). The idea came from a comparison of the open and closed active structures that revealed Phe364 undergoing a sliding movement atop the motif D helix and underneath a conserved proline in an interaction that likely helps the active site “lock” into the closed state for catalysis (Figure 8E).…”

“…This did prove to be the case, and the F364W mutation increased CTP-vs-dCTP discrimination factor in vitro , decreased mutation rates 2-fold in vivo based on sequencing progeny from infectious virus studies, and attenuated pathogenesis and tissue specific titers in mice. None of the experiments so far have resulted in compensatory mutations or reversion, likely due to several key features of this specific mutation design; 1) Trp364 is on the 3D pol surface and has limited interactions with other 3D pol residues, 2) the Pro357 interaction partner is unlikely to change because of its unique structure and backbone conformation, and 3) the tryptophan is encoded with a TGG codon where the more common transition mutations will results in stop codons or small non-planar amino acids that do not support virus growth (McDonald et al, 2016). …”

“…In effect, a tryptophan can act as a molecular poison pill to curtail the replication of pseudo-revertant viruses. This likely explains the genetic stability of F364W in CV 3D pol (McDonald et al, 2016); the native phenylalanine is well conserved in the picornaviral 3D pol enzymes and there is no precedent for a tryptophan, but that may in fact be because it is difficult to mutate to a Trp due the very same codon restrictions that make it difficult to mutate away from a Trp.…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…Indeed, even a point mutation may increase the accuracy of these enzymes severalfold (8)(9)(10)(11)(12); also see the other references in this paragraph). However, a more faithful replication may result in a fitness loss by preventing accumulation of adaptive mutations (13)(14)(15)(16)(17)(18)(19)(20)(21)(22), although the relationships between fidelity and fitness may differ between virus-host systems, being influenced in part by the conditions under which the fitness assay is performed (23)(24)(25)(26)(27)(28)(29). Moreover, a decrease in replicative fidelity may also lead to fitness loss, suggesting that the accuracy of replication of RNA viruses is naturally fine-tuned (29).…”