CpG and UpA dinucleotides in both coding and non-coding regions of echovirus 7 inhibit replication initiation post-entry.

Jelke Jan Fros,Peter Simmonds,Tim Casper Passchier,David John Evans,Isabelle Dietrich,Kinda Alshaikhahmed

doi:10.7554/elife.29112

Jelke Jan Fros, Peter Simmonds + Show 4 more

Open Access

https://doi.org/10.7554/elife.29112

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Abstract

Most vertebrate and plant RNA and small DNA viruses suppress genomic CpG and UpA dinucleotide frequencies, apparently mimicking host mRNA composition. Artificially increasing CpG/UpA dinucleotides attenuates viruses through an entirely unknown mechanism. Using the echovirus 7 (E7) model in several cell types, we show that the restriction in E7 replication in mutants with increased CpG/UpA dinucleotides occurred immediately after viral entry, with incoming virions failing to form replication complexes. Sequences of CpG/UpA-high virus stocks showed no evidence of increased mutational errors that would render them replication defective, these viral RNAs were not differentially sequestered in cytoplasmic stress granules nor did they induce a systemic antiviral state. Importantly, restriction was not mediated through effects on translation efficiency since replicons with high CpG/UpA sequences inserted into a non-coding region were similarly replication defective. Host-cells thus possess intrinsic defence pathways that prevent replication of viruses with increased CpG/UpA frequencies independently of codon usage.

Highlights

A primary function of the genomes of RNA viruses is to code for viral genes that replicate and package genomic RNA for new rounds of infection
Attenuation of viruses containing UpA-high and CpG-high sequences was evident in RD and A549 cell lines, but whether the restriction in replication extended to cell lines of different tissue origins was not determined, nor whether the restriction was related to host-cell susceptibility to echovirus 7 (E7) infection
CpG and UpA dinucleotides are under-represented in the genomes of most RNA viruses infecting vertebrates and plants (Karlin et al, 1994; Rima and McFerran, 1997; Simmonds et al, 2013) but the host factors or mutational mechanisms that impose this suppression in viral genomic nucleotide compositions are still unknown

Summary

Introduction

A primary function of the genomes of RNA viruses is to code for viral genes that replicate and package genomic RNA for new rounds of infection. Fros et al confirmed that these changes did not affect the ability of the virus’ genetic material to make the proteins it needs to multiply and make its coat This suggests that the host prevents the virus genetic material from being copied, solely based on the order of the bases in the viral genetic material. Because of the compact nature of RNA virus genomes, alteration of CpG and UpA frequencies in viruses in these previous studies inevitably involved extensive modification of coding regions Such changes may have the secondary effect of introducing normally unfavoured codons or codon pairs in. Viral genomes, reducing translation rates that may cause further virus attenuation (Martınez et al, 2016) It is possible, to modify sequences in such a way that limited changes in CpG/UpA dinucleotide frequencies can be introduced while keeping codon pair bias (as measured by the summary metric, codon pair bias) constant, and vice versa. Using a wide range of pathway knockouts and inhibitors and direct observation of the fate of viral RNA post-entry, we were able to determine where in the viral replication cycle attenuation by unfavoured dinucleotides occurred and what components of the cellular antiviral response were responsible for virus attenuation

Results

Discussion

Materials and methods

Funding Funder Wellcome