METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection

Abstract

It is urgent and important to understand the relationship of the widespread severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2) with host immune response and study the underlining molecular mechanism. N 6 -methylation of adenosine (m6A) in RNA regulates many physiological and disease processes. Here, we investigate m6A modification of SARS-CoV-2 gene in regulating host cell innate immune response. Our data show that SARS-CoV-2 virus has m6A modifications which are enriched in the 3’-end of the viral genome. We find that host cell m6A methyltransferase METTL3 depletion decreases m6A levels in SARS-CoV-2 and host genes, and m6A reduction in viral RNA increases RIG-I binding and subsequently enhances downstream innate immune signaling pathway and inflammatory gene expression. METTL3 expression is reduced and inflammatory genes are induced in severe COVID-19 patients. These findings will aid to understand the COVID-19 pathogenesis and help in designing future studies of regulating innate immunity for COVID-19 treatment.

Direct RNA sequencing reveals SARS-CoV-2 m6A sites and possible differential DRACH motif methylation among variants

“…Sequencing was performed without PCR amplification or any other in vitro synthesis (Figure 1). Mapping of m6A using direct RNA sequencing data was achieved by comparison of raw and basecalled reads and the m6A pattern identified was fully consistent with the nucleocapsid region m6A enrichment observed by liquid chromatography-tandem mass spectrometry and methylated RNA immunoprecipitation sequencing (MeRIP-seq) (Li et al, 2021). The probability of m6A, as calculated by the m6Anet method, was >50% and a minimum coverage of 60x in four positions and the recommended 100x coverage in 11 positions (Chen et al, 2021).…”

“…Among these modified bases, the N 6 -methyladenosine (m6A) is the most frequent internal base modification, and is found in viruses with exclusive cytoplasm replication, such as Zika Virus, Dengue virus and Hepatitis C virus (Gokhale et al, 2016). Methylated base m6A is implicated in SARS-CoV-2 evasion of the host immune response because the methylated viral RNA does not bind to the host protein RIG-I (retinoic acid-inducible gene I), responsible for the type-1 interferon (IFN1) response, an activator of immune pathways (Brocard et al, 2017;Li et al, 2021). The viral RNA is m6A methylated by host's methylases METTL3, METTL14, WATAP and KIAA1429, called "writers", and demethylated by FTO and ALKBH5, called "erasers" which normally methylate the host's RNAs (Gatsiou and Stellos, 2018).…”

“…The viral RNA is m6A methylated by host's methylases METTL3, METTL14, WATAP and KIAA1429, called "writers", and demethylated by FTO and ALKBH5, called "erasers" which normally methylate the host's RNAs (Gatsiou and Stellos, 2018). Knockdown of METTL3 significantly reduces the SARS-CoV-2 methylation and blocks the viral mechanism of RIG-I binding inhibition (Li et al, 2021).…”

“…Although several different technologies have been used for identification of modified bases in mRNAs and viral RNAs they require large quantities of material which precludes single-cell analysis and low abundance samples. Also, the antibody-dependent methylation analysis does not provide nucleotide level accuracy of modified bases (Brocard et al, 2017;Jenjaroenpun et al, 2021;Li et al, 2021).…”

The detection and functions of RNA modification m6A based on m6A writers and erasers

“…4 A ). Consistently, the patients with severe COVID-19 exhibit the decreased METTL3 expression levels and induced inflammatory genes expression ( 130 ). Collectively, these results suggest that m 6 A plays dual functions in SARS-CoV-2 infection: the host uses m 6 A writer to methylate SARS-CoV-2 gRNA for degradation through YTHDF2-mediated mRNA decay; however, m 6 A is also hijacked by SARS-CoV-2 to escape the host RIG-I sensing machinery.…”