Snapshots of a Non-Canonical RdRP in Action.

Diego S Ferrero,Michela Falqui,Nuria Verdaguer

doi:10.3390/v13071260

Diego S Ferrero, Michela Falqui + Show 1 more

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https://doi.org/10.3390/v13071260

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Journal: Viruses	Publication Date: Jun 28, 2021
Citations: 5	License type: CC BY 4.0

Affiliation: Institut de Biologia Molecular de Barcelona

Abstract

RNA viruses typically encode their own RNA-dependent RNA polymerase (RdRP) to ensure genome replication and transcription. The closed “right hand” architecture of RdRPs encircles seven conserved structural motifs (A to G) that regulate the polymerization activity. The four palm motifs, arranged in the sequential order A to D, are common to all known template dependent polynucleotide polymerases, with motifs A and C containing the catalytic aspartic acid residues. Exceptions to this design have been reported in members of the Permutotetraviridae and Birnaviridae families of positive single stranded (+ss) and double-stranded (ds) RNA viruses, respectively. In these enzymes, motif C is located upstream of motif A, displaying a permuted C–A–B–D connectivity. Here we study the details of the replication elongation process in the non-canonical RdRP of the Thosea asigna virus (TaV), an insect virus from the Permutatetraviridae family. We report the X-ray structures of three replicative complexes of the TaV polymerase obtained with an RNA template-primer in the absence and in the presence of incoming rNTPs. The structures captured different replication events and allowed to define the critical interactions involved in: (i) the positioning of the acceptor base of the template strand, (ii) the positioning of the 3’-OH group of the primer nucleotide during RNA replication and (iii) the recognition and positioning of the incoming nucleotide. Structural comparisons unveiled a closure of the active site on the RNA template-primer binding, before rNTP entry. This conformational rearrangement that also includes the repositioning of the motif A aspartate for the catalytic reaction to take place is maintained on rNTP and metal ion binding and after nucleotide incorporation, before translocation.

Highlights

IntroductionRNA viruses include the most abundant group of human, animal and insect pathogens
RNA viruses include the most abundant group of human, animal and insect pathogens.These viruses encode a specialized enzyme, the RNA-dependent RNA polymerase (RdRP), responsible for RNA genome replication and transcription
The Thosea asigna virus (TaV) RdRP domain was previously structurally and biochemically characterized, showing two essential elements controlling polymerization activity: the N-terminal arm that invades the template binding channel of the neighboring RdRP molecule and the λ6 loop that protrudes towards the catalytic cavity, providing a platform for initial rNTP

Summary

Introduction

RNA viruses include the most abundant group of human, animal and insect pathogens These viruses encode a specialized enzyme, the RNA-dependent RNA polymerase (RdRP), responsible for RNA genome replication and transcription. All known RdRPs share a similar closed right-hand architecture, with the fingers, palm and thumb subdomains and use the same mechanism of catalysis [1]. This has led to the hypothesis that all these enzymes share a common origin [2]. RdRPs function either as single polypeptides or in a complex with other viral or host components to transcribe and replicate the viral RNA genome. They use a variety of strategies to recognize substrates and coordinate the different steps of RNA synthesis [3]

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