Abstract
Picornaviral RNA-dependent RNA polymerases (RdRPs) have low replication fidelity that is essential for viral fitness and evolution. Their global fold consists of the classical "cupped right hand" structure with palm, fingers, and thumb domains, and these RdRPs also possess a unique contact between the fingers and thumb domains. This interaction restricts movements of the fingers, and RdRPs use a subtle conformational change within the palm domain to close their active sites for catalysis. We have previously shown that this core RdRP structure and mechanism provide a platform for polymerases to fine-tune replication rates and fidelity to optimize virus fitness. Here, we further elucidated the structural basis for differences in replication rates and fidelity among different viruses by generating chimeric RdRPs from poliovirus and coxsackievirus B3. We designed these chimeric polymerases by exchanging the fingers, pinky finger, or thumb domains. The results of biochemical, rapid-quench, and stopped-flow assays revealed that differences in biochemical activity map to individual modular domains of this polymerase. We found that the pinky finger subdomain is a major regulator of initiation and that the palm domain is the major determinant of catalytic rate and nucleotide discrimination. We further noted that thumb domain interactions with product RNA regulate translocation and that the palm and thumb domains coordinately control elongation complex stability. Several RdRP chimeras supported the growth of infectious poliovirus, providing insights into enterovirus species-specific protein-protein interactions required for virus replication.
Highlights
Picornaviruses are a family of positive-strand RNA viruses responsible for a multitude of human and animal diseases
Positive-strand RNA virus RNA-dependent RNA polymerases (RdRPs) with their thumb-tethered fingers domain cannot undergo such motions, and they instead use a subtle movement of motifs A and D within the palm domain to fully structure the active site for chemistry only upon NTP binding [9, 10]
Polymerase domains for chimeric RdRPs were identified by inspection of superposed PV and coxsackievirus B3 (CV) 3Dpol structures [3, 30] and involve four distinct folding domains; the pinky finger subdomain, the fingers domain, the palm domain, and the thumb domain (Fig. 1A)
Summary
Picornaviruses are a family of positive-strand RNA viruses responsible for a multitude of human and animal diseases. Positive-strand RNA virus RdRPs with their thumb-tethered fingers domain cannot undergo such motions, and they instead use a subtle movement of motifs A and D within the palm domain to fully structure the active site for chemistry only upon NTP binding [9, 10] Their fingers domains remain essentially stationary during the nucleotide addition cycle, and their mechanism for translocation is not yet well-understood beyond an initial half base-step product strand movement seen in enterovirus 71 polymerase structures [11].
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