Emergence of SARS-CoV-2 Subgenomic RNAs that Enhance Viral Fitness and Immune Evasion
Harriet V. Mears, George R. Young, Theo Sanderson, Ruth Harvey, Jamie Barrett-Rodger, Rebecca Penn, Vanessa Cowton, Wilhelm Furnon, Giuditta De Lorenzo, Margaret Crawford, Daniel M. Snell, Ashley S. Fowler, Anob M. Chakrabarti, Saira Hussain, Ciarán Gilbride, Edward Emmott, Katja Finsterbusch, Jakub Luptak, Thomas P. Peacock, Jérôme Nicod, Arvind H. Patel, Massimo Palmarini, Emma Wall, Bryan Williams, Sonia Gandhi, Charles Swanton, David L. V. Bauer.
Abstract
Coronaviruses express their structural and accessory genes via a set of subgenomic RNAs, whose synthesis is directed by transcription regulatory sequences (TRSs) in the 5′ genomic leader and upstream of each body open reading frame. In SARS-CoV-2, the TRS has the consensus AAACGAAC; upon searching for emergence of this motif in the global SARS-CoV-2 sequences, we find that it evolves frequently, especially in the 3′ end of the genome.
Introduction
SARS-CoV-2 has continued to evolve since its emergence in the human population. An important emphasis throughout the pandemic has been on characterising the amino acid substitutions in new variants, particularly within the Spike glycoprotein, which contribute to increased transmission and immune evasion. However, there has also been substantial evolution at the nucleotide level in both coding and non-coding regions of the genome.
Materials and Methods:
Vero E6 (Pasteur), Vero V1 (a gift from Stephen Goodbourn), and A549 ACE-TMPRSS2 and VeroE6 ACE2-TMPRSS2 cells (gifts from Suzannah Rihn) were maintained in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% foetal calf serum and penicillin-streptomycin (100 U/mL each). A549-dual hACE2-TMPRSS2, A549-dual KO MDA5 hACE2-TMPRSS2 and A549-dual KO RIG-I hACE2-TMPRSS2 cells were purchased from Invivogen, and were maintained in DMEM as above, with addition of 100 µg/mL Normicin, 10 µg/mL Blasticidin, 100 µg/mL Hygromycin, 0.5 µg/mL Puromycin and 100 µg/mL Zeocin. Forty-eight hours before infection selection antibiotics were removed to avoid off-target pressure on viral growth.
Discussion
Since its emergence in late 2019, SARS-CoV-2 has continued to adapt to the human host. Many of these adaptations have been within Spike, the major glycoprotein on the virion surface, to increase affinity for its receptor, ACE2, or to evade recognition by the adaptive immune system. However, there have also been numerous changes to genes outside of Spike and in non-coding regions. Here, we have focussed on the emergence of novel TRSs during SARS-CoV-2 evolution.
Acknowledgments
We thank Simon Caidan, Robert Goldstone, Maria Greco, and Michael Bennett for technical assistance; and Steve Gamblin, George Kassiotis, Wendy Barclay, and Ervin Fodor for helpful discussions. We thank Wendy Barclay, ‘Assessment of Transmission and Contagiousness of COVID-19 in Contacts’ (ATACCC), the NIHR Health Protection Research Unit in Respiratory Infections, Imperial College London (NIHR200927), Public Health England, Leo James, Steve Goodbourn, Tulio de Oliveira, Alex Sigal, Khadija Kahn, Thushan de Silva, Gavin Screaton, and the G2P-UK National Virology Consortium for support and rapid sharing of reagents and samples, as well as the participants of the Crick SARS-CoV-2 Longitudinal Study: Understanding Susceptibility, Transmission and Disease Severity (Legacy Study). We thank all researchers who have submitted SARS-CoV-2 genomes to the GISAID database: an acknowledgement table for the specific set analysed here can be found in S5 Table. The phylogenetic reconstructions depicted in Figs 2B and S2A are derivatives of those produced by nextstrain.org, used under CC BY. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.
Citation: Mears HV, Young GR, Sanderson T, Harvey R, Barrett-Rodger J, Penn R, et al. (2025) Emergence of SARS-CoV-2 subgenomic RNAs that enhance viral fitness and immune evasion. PLoS Biol 23(1): e3002982. https://doi.org/10.1371/journal.pbio.3002982
Editor: Jason T. Ladner, Northern Arizona University, UNITED STATES OF AMERICA
Received: July 29, 2022; Accepted: December 11, 2024; Published: January 21, 2025.
Copyright: © 2025 Mears et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files, or within the following repositories: Gel and blot images are available on Figshare (https://doi.org/10.25418/crick.27953013), numerical data are available on Figshare (https://doi.org/10.25418/crick.27952842), nanopore sequencing data analysis code is available on Figshare (https://doi.org/10.25418/crick.27959910), and amplicon sequencing data analysis code is available on Zenodo (https://zenodo.org/records/14277568). All nanopore and amplicon sequencing data files are available from EBI ArrayExpress (accession numbers E-MTAB-14681 and E-MTAB-14680).
Funding: This work was supported by the Francis Crick Institute which receives its core funding from Cancer Research UK (CC2166, CC1283), the UK Medical Research Council (CC2166, CC1283), and the Wellcome Trust (CC2166, CC1283). This work was also supported by the UK Medical Research Council (MR/W005611/1, MR/Y004205/1 to DLVB) and by UK Research and Innovation (to JL) and the Wellcome Trust (210918/Z/18/Z to TS). The Legacy Study is supported by the NIHR University College London Hospitals Biomedical Research Centre. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: While the authors declare no competing interests directly related to this work, CS receives grants from Bristol Myers Squibb, Ono Pharmaceuticals, Boehringer Ingelheim, Roche-Ventana, Pfizer, and Archer Dx; receives personal fees from Genentech, the Sarah Canon Research Institute, Medicxi, Bicycle Therapeutics, GRAIL, Amgen, AstraZeneca, Bristol Myers Squibb, Illumina, GlaxoSmithKline, MSD, and Roche-Ventana; holds stock options in Apogen Biotech, Epic Biosciences, GRAIL, and Achilles Therapeutics; is a member of a scientific advisory board for Bicycle Therapeutics, GRAIL, Relay Therapeutics, SAGA Diagnostics, and Achilles Therapeutics; is a co-founder of Achilles Therapeutics; receives consulting fees from Genentech, Medicxi, MetaboMed, Novartis, the China Innovation Centre of Roche, and the Sarah Cannon Research Institute; and receives honoraria from Amgen, AstraZeneca, Bristol Myers Squibb, Illumina, and Incyte. DLVB receives grants, paid to their institution, from AstraZeneca and GSK related to COVID-19, and is a member of the UK Genotype-to-Phenotype 2 Consortium. All other authors have declared that no competing interests exist.
