Dataset Information

The Curious Case of the Nidovirus Exoribonuclease: Its Role in RNA Synthesis and Replication Fidelity.

ABSTRACT: Among RNA viruses, the order Nidovirales stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3'-to-5' exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors made by the RNA-dependent RNA polymerase. Here, we review our current understanding of ExoN evolution, structure, and function. Most experimental data are derived from studies of the ExoN domain of coronaviruses (CoVs), which were triggered by the bioinformatics-based identification of ExoN in the genome of severe acute respiratory syndrome coronavirus (SARS-CoV) and its relatives in 2003. Although convincing data supporting the proofreading hypothesis have been obtained, from biochemical assays and studies with CoV mutants lacking ExoN functionality, the features of ExoN from most other nidovirus families remain to be characterized. Remarkably, viable ExoN knockout mutants were obtained only for two CoVs, mouse hepatitis virus (MHV) and SARS-CoV, whose RNA synthesis and replication kinetics were mildly affected by the lack of ExoN function. In several other CoV species, ExoN inactivation was not tolerated, and knockout mutants could not be rescued when launched using a reverse genetics system. This suggests that ExoN is also critical for primary viral RNA synthesis, a property that poorly matches the profile of an enzyme that would merely boost long-term replication fidelity. In CoVs, ExoN resides in a bifunctional replicase subunit (nsp14) whose C-terminal part has (N7-guanine)-methyltransferase activity. The crystal structure of SARS-CoV nsp14 has shed light on the interplay between these two domains, and on nsp14's interactions with nsp10, a co-factor that strongly enhances ExoN activity in vitro assays. Further elucidation of the structure-function relationships of ExoN and its interactions with other (viral and/or host) members of the CoV replication machinery will be key to understanding the enzyme's role in viral RNA synthesis and pathogenesis, and may contribute to the design of new approaches to combat emerging nidoviruses.

SUBMITTER: Ogando NS

PROVIDER: S-EPMC6693484 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Curious Case of the Nidovirus Exoribonuclease: Its Role in RNA Synthesis and Replication Fidelity.

Ogando Natacha S NS Ferron Francois F Decroly Etienne E Canard Bruno B Posthuma Clara C CC Snijder Eric J EJ

Frontiers in microbiology 20190807

Among RNA viruses, the order <i>Nidovirales</i> stands out for including viruses with the largest RNA genomes currently known. Nidoviruses employ a complex RNA-synthesizing machinery comprising a variety of non-structural proteins (nsps). One of the postulated drivers of the expansion of nidovirus genomes is the presence of a proofreading 3'-to-5' exoribonuclease (ExoN) belonging to the DEDDh family. ExoN may enhance the fidelity of RNA synthesis by correcting nucleotide incorporation errors mad ...[more]

PMID: 31440227

Similar Datasets

Project description:UnlabelledViral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity.ImportancePositive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

Dataset Information

The Curious Case of the Nidovirus Exoribonuclease: Its Role in RNA Synthesis and Replication Fidelity.

Publications

The Curious Case of the Nidovirus Exoribonuclease: Its Role in RNA Synthesis and Replication Fidelity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets