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Piece of the puzzle: Remdesivir disassembles the multimeric SARS-CoV-2 RNA-dependent RNA polymerase complex.

ABSTRACT: The recently emerged SARS-like coronavirus (SARS-CoV-2) has continued to spread rapidly among humans with alarming upsurges in global mortality rates. A major key to tackling this virus is to disrupt its RNA replication process as previously reported for Remdesivir (Rem-P₃). In this study, we theorize, using computational simulations, novel mechanisms that may underlie the binding of Rem-P₃ to SARS-CoV-2 RdRp-NSPs complex; a multimeric assembly that drives viral RNA replication in human hosts. Findings revealed that while ATP-binding stabilized the replicative tripartite, Rem-P₃ disintegrated the RdRp-NSP complex, starting with the detachment of the NSP7-NSP8 heterodimer followed by minimal displacement of the second NSP8 subunit (NSP8_II). More so, Rem-P₃ interacted with a relatively higher affinity (ΔG_bind) while inducing high perturbations across the RdRp-NSP domains. D452, T556, V557, S682, and D760 were identified for their crucial roles in stacking the cyano-adenosine and 3,4-dihydroxyoxolan rings of Rem-P₃ while its flexible P₃ tail extended towards the palm domain blocking D618 and K798; a residue-pair identified for essential roles in RNA replication. However, ATP folded away from D618 indicative of a more coordinated binding favorable for nucleotide polymerization. We believe findings from this study will significantly contribute to the structure-based design of novel disruptors of the SARS-CoV-2 RNA replicative machinery.

SUBMITTER: Olotu FA

PROVIDER: S-EPMC8014903 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCEIn vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.

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Piece of the puzzle: Remdesivir disassembles the multimeric SARS-CoV-2 RNA-dependent RNA polymerase complex.

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