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, stopped-flow 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.
Highlights
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]
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
Superpositioning of the mutant and wild type 3Dpol structures clearly delineates them into two groups, one with the native conformations for the motif D loop and active site (Fig. 2A) and the other with various motif D loop distortions that are accompanied by a movement of Pro357 and closure of the active site (Fig. 2B)
Summary
Protein Expression and Purification—CVB3 polymerases were expressed in Escherichia coli from ubiquitin fusion constructs to generate the native N terminus required for activity [19] and purified as described previously [13]. Preinitiated elongation complexes were generated as in the processive elongation experiments but with the 2AP prepositioned in the ϩ2 site such that CTP incorporation and subsequent translocation will move the 2AP into the templating ϩ1 site where the fluorescence is quenched due to stacking on the nascent duplex These reactions were diluted to a final RNA concentration of 10 nM in the reaction cell, and maximal single nucleotide incorporation rates (kpol) and apparent Km values were determined by titrating CTP and analyzing the observed 2-aminopurine translocation rates (Fig. 3E). Tissue-specific viral titers were determined by TCID50 assay after harvesting whole organs that were homogenized in PBS using a Precellys 24 tissue homogenizer (Bertin Technologies)
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