The tRNA epitranscriptome: a novel player in viral infections

Project Information

EpiCoVs

Grant agreement ID: 101065094

DOI

10.3030/101065094

Project closed

EC signature date 20 June 2022

Start date 1 May 2023

End date 30 April 2025

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 181 152,96

Coordinated by

UNIVERSIDAD POMPEU FABRA
Spain

Periodic Reporting for period 1 - EpiCoVs (The tRNA epitranscriptome: a novel player in viral infections)

Reporting period: 2023-05-01 to 2025-04-30

Summary of the context and overall objectives of the project

A long-standing question in virology is how viruses with suboptimal codon usage can efficiently produce high levels of proteins during infection. The EpiCoVs project set out to address this paradox using coronaviruses as a model, since their genomes are significantly enriched in A/U-ending codons, which are considered rare in the host G/C-rich translational environment.
The central hypothesis of the project was that coronaviruses manipulate the host tRNA epitranscriptome, specifically by inducing targeted tRNA modifications, to reprogram translation in a codon-specific manner that favors viral protein synthesis. We proposed that this strategy is a conserved mechanism among multiple coronavirus species, enabling them to fine-tune host translation machinery to their own codon usage. Importantly, this mechanism could represent a novel target for the development of broad-spectrum antiviral therapies.

This hypothesis was supported by:
1. The observation that coronavirus genomes are consistently enriched in codons typically considered rare in mammalian cells.
2. Coronaviruses replicate very efficiently despite their enrichment in suboptimal codons.
2. Preliminary results from the laboratory showing that at least one tRNA-modifying enzyme, KIAA1456, is strongly upregulated in DENV and CHIKV infected cells, that are positive strand RNA viruses like coronaviruses.

In the broader context of the COVID-19 pandemic and the EU strategic focus on pandemic preparedness, this research addressed a critical and previously underexplored aspect of host-virus interactions. EpiCoVs aimed to identify conserved, coronavirus-induced changes in tRNA modifications and the enzymes responsible, to assess whether these changes support viral protein synthesis and contribute to pathogenicity. To achieve this, the project focused on both highly pathogenic viruses (using SARS-CoV-2) and mildly pathogenic strains (such as HCoV-OC43).

Overall, the findings establish a new conceptual framework for virus-host interactions and lay the foundation for innovative antiviral strategies with broad applicability.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the fellowship, the EpiCoVs project systematically mapped the impact of coronavirus infection on the host tRNA epitranscriptome, demonstrating that both SARS-CoV-2 and HCoV-OC43 reprogram tRNA modifications in a codon-specific manner. By combining bioinformatic analyses with molecular biology techniques (e.g. western blotting, qPCR), Liquid Chromatography–Tandem Mass Spectrometry (LC-MS/MS), and misincorporation tRNA sequencing (mim-tRNAseq), the project uncovered several key findings:
- Using relative synonymous codon usage (RSCU) analysis across all alpha- and beta-coronaviruses, we discovered that these viruses are enriched in codons whose efficient decoding depends on specific tRNA modifications, suggesting a conserved viral strategy to exploit host translation via tRNA epitranscriptomic modulation.
- Through LC-MS/MS, we found that both pathogenic (SARS-CoV-2) and non-pathogenic (HCoV-OC43) coronaviruses significantly remodel the host tRNA epitranscriptome. The modified tRNAs favor the decoding of codons preferentially used by these viruses, confirming a functional correlation between viral codon usage and host tRNA modification remodeling.
- These changes are driven by virus-induced upregulation of the corresponding tRNA-modifying enzymes, as confirmed by protein-level analyses in infected cells.
- Changes in tRNA modification levels are not due to altered tRNA gene expression, but rather to modification of existing tRNAs. Mim-tRNAseq analysis showed no significant changes in tRNA abundance for the affected isoacceptors. Instead, we observed that coronavirus infection activates a DNA damage response, which drives epitranscriptomic reprogramming through modification of pre-existing tRNAs, highlighting a stress-response-mediated mechanism.
- Coronavirus infection also causes downregulation of a specific group of tRNAs—notably Leucine and Serine tRNAs—in an interferon-dependent manner, suggesting a regulatory interaction between the innate immune response and the host tRNA pool.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our findings establish a direct link between the enrichment of specific tRNA modification-dependent codons and virus-induced changes in the levels of the corresponding tRNA modifications. This has two key implications:
1. These codons, though predicted to be suboptimal, are in fact well-adapted to the tRNA modification landscape under infection conditions, supporting a positive selection pressure;
2. Codon usage may predict viral pathogenicity, as some of the observed tRNA modifications are triggered by virus-induced stress pathways, and the corresponding codons are differentially enriched in mild and highly pathogenic coronavirus strains.
Beyond basic virology, our results have broader implications.
1. Since the decoding of the most enriched codons across coronavirus genera depends on the identified tRNA modifications, their regulation presents a promising target for the development of pan-coronavirus antiviral strategies.
2. EpiCoVs findings imply viral gene expression is finely tuned to infection-modified tRNAome, indicating that recoding may be necessary when expressing viral proteins individually for biotechnology applications.

The interplay of viruses with the host tRNA epitranscriptome