The SARS-CoV nsp12 Polymerase Active Site Is Tuned for Large-Genome Replication

Autor: Grace Campagnola, Vishnu Govindarajan, Annelise Pelletier, Bruno Canard, Olve B. Peersen
Přispěvatelé: Colorado State University [Fort Collins] (CSU), Architecture et fonction des macromolécules biologiques (AFMB), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
Rok vydání: 2022
Předmět:
Zdroj: Journal of Virology
Journal of Virology, 2022, 96 (16), ⟨10.1128/jvi.00671-22⟩
ISSN: 1098-5514
0022-538X
DOI: 10.1128/jvi.00671-22
Popis: Positive-strand RNA viruses replicate their genomes using virally encoded RNA-dependent RNA polymerases (RdRP) with a common active-site structure and closure mechanism upon which replication speed and fidelity can evolve to optimize virus fitness. Coronaviruses (CoV) form large multicomponent RNA replication-transcription complexes containing a core RNA synthesis machine made of the nsp12 RdRP protein with one nsp7 and two nsp8 proteins as essential subunits required for activity. We show that assembly of this complex can be accelerated 5-fold by preincubation of nsp12 with nsp8 and further optimized with the use of a novel nsp8L7 heterodimer fusion protein construct. Using rapid kinetics methods, we measure elongation rates of up to 260 nucleotides (nt)/s for the core replicase, a rate that is unusually fast for a viral polymerase. To address the origin of this fast rate, we examined the roles of two CoV-specific residues in the RdRP active site: Ala547, which replaces a conserved glutamate above the bound NTP, and Ser759, which mutates the palm domain GDD sequence to SDD. Our data show that Ala547 allows for a doubling of replication rate, but this comes at a fidelity cost that is mitigated by using a SDD sequence in the palm domain. Our biochemical data suggest that fixation of mutations in polymerase motifs F and C played a key role in nidovirus evolution by tuning replication rate and fidelity to accommodate their large genomes.
Databáze: OpenAIRE
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