Dataset Information

Targeting the m⁶A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication.

ABSTRACT: N⁶-methyladenosine (m⁶A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m⁶A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m⁶A-modified. How the cellular m⁶A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human β-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m⁶A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m⁶A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m⁶A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m⁶A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m⁶A-modified and host m⁶A pathway components control β-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m⁶A pathway to restrict coronavirus reproduction.

SUBMITTER: Burgess HM

PROVIDER: S-EPMC8247602 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Publications

Targeting the m6A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication.

Burgess Hannah M HM Depledge Daniel P DP Thompson Letitia L Srinivas Kalanghad Puthankalam KP Grande Rebecca C RC Vink Elizabeth I EI Abebe Jonathan S JS Blackaby Wesley P WP Hendrick Alan A Albertella Mark R MR Kouzarides Tony T Stapleford Kenneth A KA Wilson Angus C AC Mohr Ian I

Genes & development 20210624 13-14

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknow ...[more]

PMID: 34168039

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has renewed interest in human coronaviruses that cause the common cold, particularly as research with them at biosafety level (BSL)-2 avoids the added costs and biosafety concerns that accompany work with SARS-CoV-2, BSL-3 research. One of these, human coronavirus OC43 (HCoV-OC43), is a well-matched surrogate for SARS-CoV-2 because it is also a Betacoronavirus, targets the human respiratory system, is transmitted via respiratory aerosols and droplets and is relatively resistant to disinfectants. Unfortunately, growth of HCoV-OC43 in the recommended human colon cancer (HRT-18) cells does not produce obvious cytopathic effect (CPE) and its titration in these cells requires expensive antibody-based detection. Consequently, multiple quantification approaches for HCoV-OC43 using alternative cell lines exist, which complicates comparison of research results. Hence, we investigated the basic growth parameters of HCoV-OC43 infection in three of these cell lines (HRT-18, human lung fibroblasts (MRC-5) and African green monkey kidney (Vero E6) cells) including the differential development of cytopathic effect (CPE) and explored reducing the cost, time and complexity of antibody-based detection assay. Multi-step growth curves were conducted in each cell type in triplicate at a multiplicity of infection of 0.1 with daily sampling for seven days. Samples were quantified by tissue culture infectious dose50(TCID50)/mL or plaque assay (cell line dependent) and additionally analyzed on the Sartorius Virus Counter 3100 (VC), which uses flow virometry to count the total number of intact virus particles in a sample. We improved the reproducibility of a previously described antibody-based detection based TCID50 assay by identifying commercial sources for antibodies, decreasing antibody concentrations and simplifying the detection process. The growth curves demonstrated that HCoV-O43 grown in MRC-5 cells reached a peak titer of ˜107 plaque forming units/mL at two days post infection (dpi). In contrast, HCoV-OC43 grown on HRT-18 cells required six days to reach a peak titer of ˜106.5 TCID50/mL. HCoV-OC43 produced CPE in Vero E6 cells but these growth curve samples failed to produce CPE in a plaque assay after four days. Analysis of the VC data in combination with plaque and TCID50 assays together revealed that the defective:infectious virion ratio of MRC-5 propagated HCoV-OC43 was less than 3:1 for 1-6 dpi while HCoV-OC43 propagated in HRT-18 cells varied from 41:1 at 1 dpi, to 329:4 at 4 dpi to 94:1 at 7 dpi. These results should enable better comparison of extant HCoV-OC43 study results and prompt further standardization efforts.

Dataset Information

Targeting the m⁶A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication.

Publications

Targeting the m<sup>6</sup>A RNA modification pathway blocks SARS-CoV-2 and HCoV-OC43 replication.

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