Analysis of SARS-CoV-2 known and novel subgenomic mRNAs in cell culture, animal model, and clinical samples using LeTRS, a bioinformatic tool to identify unique sequence identifiers

Xiaofeng Dong, Rebekah Penrice-Randal, Hannah Goldswain, Tessa Prince, Nadine Randle, I'ah Donovan-Banfield, Francisco J. Salguero, Julia Tree, Ecaterina Vamos, Charlotte Nelson, Jordan Clark, Yan Ryan, James P. Stewart, Malcolm G. Semple, J. Kenneth Baillie, Peter J.M. Openshaw, Lance Turtle, David A. Matthews, Miles W. Carroll, Alistair C. DarbyJulian A. Hiscox*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a complex strategy for the transcription of viral subgenomic mRNAs (sgmRNAs), which are targets for nucleic acid diagnostics. Each of these sgmRNAs has a unique 5′ sequence, the leader-transcriptional regulatory sequence gene junction (leader-TRS junction), that can be identified using sequencing. High-resolution sequencing has been used to investigate the biology of SARS-CoV-2 and the host response in cell culture and animal models and from clinical samples. LeTRS, a bioinformatics tool, was developed to identify leader-TRS junctions and can be used as a proxy to quantify sgmRNAs for understanding virus biology. LeTRS is readily adaptable for other coronaviruses such as Middle East respiratory syndrome coronavirus or a future newly discovered coronavirus. LeTRS was tested on published data sets and novel clinical samples from patients and longitudinal samples from animal models with coronavirus disease 2019. LeTRS identified known leader-TRS junctions and identified putative novel sgmRNAs that were common across different mammalian species. This may be indicative of an evolutionary mechanism where plasticity in transcription generates novel open reading frames, which can then subject to selection pressure. The data indicated multiphasic abundance of sgmRNAs in two different animal models. This recapitulates the relative sgmRNA abundance observed in cells at early points in infection but not at late points. This pattern is reflected in some human nasopharyngeal samples and therefore has implications for transmission models and nucleic acid-based diagnostics. LeTRS provides a quantitative measure of sgmRNA abundance from sequencing data. This can be used to assess the biology of SARS-CoV-2 (or other coronaviruses) in clinical and nonclinical samples, especially to evaluate different variants and medical countermeasures that may influence viral RNA synthesis.

Original languageEnglish
Article numbergiac045
JournalGigaScience
Volume11
DOIs
Publication statusPublished - 2022

Bibliographical note

Publisher Copyright:
© 2022 The Author(s) 2022

Keywords

  • COVID-19
  • RNA modification
  • SARS-CoV-2
  • coronavirus
  • direct RNA sequencing
  • nanopore
  • sgmRNA
  • subgenomic mRNA
  • transcriptional regulatory sequences

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