Key result
Mutating Phe364 to tryptophan in the coxsackievirus B3 polymerase resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice.
Engineering the viral polymerase of coxsackievirus B3 by mutating Phe(364) to tryptophan creates a high-fidelity variant that attenuates viral growth and pathogenesis in vivo.
Attenuated high-fidelity coxsackievirus B3 in mice is hypothesis-generating; leaves open translation of polymerase mutations to human therapies.
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.
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McDonald et al. (2016) studied Coxsackievirus B3 infection. Engineered mutations in coxsackievirus B3 polymerase (Phe364 to tryptophan) vs. Wild-type virus was evaluated on Virus growth and pathogenesis in mice. Mutating Phe364 to tryptophan in the coxsackievirus B3 polymerase resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice.
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