The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif

Kaouther Ben Ouirane , Yves Boulard (1, 2, 3) , Stéphane Bressanelli (1, 3, 2)

1 I2BC - Institut de Biologie Intégrative de la Cellule
2 B3S - Département Biochimie, Biophysique et Biologie Structurale
3 IMAPP - Interactions et mécanismes d’assemblage des protéines et des peptides

RNA viruses synthesize new genomes in the infected host thanks to dedicated, virally-encoded RNA-dependent RNA polymerases (RdRps). As such, these enzymes are prime targets for antiviral therapy, as has recently been demonstrated for hepatitis C virus (HCV). However, peculiarities in the architecture and dynamics of RdRps raise fundamental questions about access to their active site during RNA polymerization. Here, we used molecular modelling and molecular dynamics simulations, starting from the available crystal structures of HCV NS5B in ternary complex with template-primer duplexes and nucleotides, to address the question of ribonucleotide entry into the active site of viral RdRp. Tracing the possible passage of incoming UTP or GTP through the RdRp-specific entry tunnel, we found two successive checkpoints that regulate nucleotide traffic to the active site. We observed that a magnesium-bound nucleotide first binds next to the tunnel entry, and interactions with the triphosphate moiety orient it such that its base moiety enters first. Dynamics of the RdRp motifs F1 + F3 then allow the nucleotide to interrogate the RNA template base prior to nucleotide insertion into the active site. These dynamics are finely regulated by a second magnesium dication, thus coordinating the entry of a magnesium-bound nucleotide with shuttling of the second magnesium necessary for the two-metal ion catalysis. The findings of our work suggest that some at least of these features are general to viral RdRps and provide further details on the original nucleotide selection mechanism operating in RdRps of RNA viruses.

Mots clés: complex; molecular dynamics; structural biology; structural basis; crystal-structure; mechanism; catalysis; simulations; nucleoside/nucleotide transport; protein motif; RNA virus; RNA-dependent RNA polymerase (RdRp); Single-stranded; positive-sense RNA virus; viral polymerase; insights; fidelity; nucleoside; nucleotide transport

Domaines: Sciences du Vivant [q-bio]

Kaouther Ben Ouirane, Yves Boulard, Stéphane Bressanelli. The hepatitis C virus RNA-dependent RNA polymerase directs incoming nucleotides to its active site through magnesium-dependent dynamics within its F motif. Journal of Biological Chemistry, 2019, 294 (19), pp.7573--7587. ⟨10.1074/jbc.RA118.005209⟩. ⟨hal-02169568⟩