Picornaviral polymerase domain exchanges reveal a modular basis for distinct biochemical activities of viral RNA-dependent RNA polymerases

• Published in: The Journal of biological chemistry Vol. 295; no. 31; pp. 10624 - 10637
• Main Authors: Watkins, Colleen L., Kempf, Brian J., Beaucourt, Stéphanie, Barton, David J., Peersen, Olve B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 31.07.2020; American Society for Biochemistry and Molecular Biology
• Subjects: Biochemistry, Molecular Biology; Chemical Sciences; conformational change; coxsackievirus; Cristallography; Enterovirus B, Human; Enterovirus B, Human - enzymology; Enterovirus B, Human - genetics; enzyme kinetics; HeLa Cells; Humans; Life Sciences; Microbiology and Parasitology; Poliovirus; Poliovirus - enzymology; Poliovirus - genetics; Protein Domains; protein engineering; Protein Structure and Folding; RNA, Viral; RNA, Viral - chemistry; RNA, Viral - genetics; RNA, Viral - metabolism; RNA-Dependent RNA Polymerase; RNA-dependent RNA polymerase (RdRP); RNA-Dependent RNA Polymerase - chemistry; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - metabolism; RNA–protein interaction; Structural Biology; viral polymerase; Viral Proteins; Viral Proteins - chemistry; Viral Proteins - genetics; Viral Proteins - metabolism; viral replication; Virology; virus; viral replication; RNA-dependent RNA polymerase (RdRP); protein engineering; viral polymerase; coxsackievirus; RNA–protein interaction; conformational change; virus; enzyme kinetics; poliovirus
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA120.013906

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.