The risk of COVID-19 can be predicted by a nomogram based on m6A-related genes.

BACKGROUND: The expression of m6A-related genes and their significance in COVID-19 patients are still unknown. METHODS: The GSE177477 and GSE157103 datasets of the Gene Expression Omnibus were used to extract RNA-seq data. The expression of 26 m6A-related genes and immune cell infiltration in COVID-19 patients were analyzed. Finally, we built and validated a nomogram model to predict the risk of COVID-19 infection. RESULTS: There were significant differences in 11 m6A regulatory factors between patients with COVID-19 and healthy individuals. The classification of disease subtypes based on m6A-related gene levels can be distinguished. COVID-19 patients in GSE177477 were classified into two categories based on m6A-related genes. The patients in cluster A were all symptomatic, while those in cluster B were asymptomatic. A significant correlation was also found between immune cells and m6A-related genes. Finally, seven m6A-related disease-characteristic genes, HNRNPA2B1, ELAVL1, RBM15, RBM15B, YTHDC1, HNRNPC, and WTAP, were screened to construct a nomogram model for predicting risk. The calibration curve, decision curve analysis, and clinical impact curve analysis were used to show that the nomogram model was effective and had a high net efficacy for risk prediction. CONCLUSIONS: m6A-related genes were correlated with immune cells. The nomogram model effectively predicted COVID-19 risk. Moreover, m6A-related genes may be associated with the presence or absence of symptoms in COVID-19 patients.

Epitranscriptomic N6-Methyladenosine Profile of SARS-CoV-2-Infected Human Lung Epithelial Cells.

N6-methyladenosine (m6A) is a dynamic posttranscriptional RNA modification that plays an important role in determining transcript fate. The functional consequence of m6A deposition is dictated by a group of host proteins that specifically recognize and bind the m6A modification, leading to changes in RNA stability, transport, splicing, or translation. The cellular m6A methylome undergoes changes during certain pathogenic conditions such as viral infections. However, how m6A modification of host cell transcripts and noncoding RNAs change during severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection has not been reported. Here, we define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells compared to uninfected controls. We identified mRNA and long and small noncoding RNA species that are differentially m6A modified in response to SARS-CoV-2 infection. The most significantly differentially methylated transcript was the precursor of microRNA-4486 (miRNA-4486), which showed significant increases in abundance and percentage of methylated transcripts in infected cells. Pathway analyses revealed that differentially methylated transcripts were significantly associated with several cancer-related pathways, protein processing in the endoplasmic reticulum, cell death, and proliferation. Upstream regulators predicted to be associated with the proteins encoded by differentially methylated mRNAs include several proteins involved in the type-I interferon response, inflammation, and cytokine signaling. IMPORTANCE Posttranscriptional modification of viral and cellular RNA by N6-methyladenosine (m6A) plays an important role in regulating the replication of many viruses and the cellular immune response to infection. We therefore sought to define the epitranscriptomic m6A profile of human lung epithelial cells infected with SARS-CoV-2. Our analyses demonstrate the differential methylation of both coding and noncoding cellular RNAs in SARS-CoV-2-infected cells compared to uninfected controls. Pathway analyses revealed that several of these RNAs may be involved in the cellular response to infection, such as type-I interferon. Our study implicates m6A modification of infected-cell RNA as a mechanism of posttranscriptional gene regulation during SARS-CoV-2 infection.

WTAP Targets the METTL3 m6A-Methyltransferase Complex to Cytoplasmic Hepatitis C Virus RNA to Regulate Infection.

Modification of the hepatitis C virus (HCV) positive-strand RNA genome by N6-methyladenosine (m6A) regulates the viral life cycle. This life cycle takes place solely in the cytoplasm, while m6A addition on cellular mRNA takes place in the nucleus. Thus, the mechanisms by which m6A is deposited on the viral RNA have been unclear. In this work, we find that m6A modification of HCV RNA by the m6A-methyltransferase proteins methyltransferase-like 3 and 14 (METTL3 and METTL14) is regulated by Wilms' tumor 1-associating protein (WTAP). WTAP, a predominantly nuclear protein, is an essential member of the cellular mRNA m6A-methyltransferase complex and known to target METTL3 to mRNA. We found that HCV infection induces localization of WTAP to the cytoplasm. Importantly, we found that WTAP is required for both METTL3 interaction with HCV RNA and m6A modification across the viral RNA genome. Further, we found that WTAP, like METTL3 and METTL14, negatively regulates the production of infectious HCV virions, a process that we have previously shown is regulated by m6A. Excitingly, WTAP regulation of both HCV RNA m6A modification and virion production was independent of its ability to localize to the nucleus. Together, these results reveal that WTAP is critical for HCV RNA m6A modification by METTL3 and METTL14 in the cytoplasm. IMPORTANCE Positive-strand RNA viruses such as HCV represent a significant global health burden. Previous work has described that HCV RNA contains the RNA modification m6A and how this modification regulates viral infection. Yet, how this modification is targeted to HCV RNA has remained unclear due to the incompatibility of the nuclear cellular processes that drive m6A modification with the cytoplasmic HCV life cycle. In this study, we present evidence for how m6A modification is targeted to HCV RNA in the cytoplasm by a mechanism in which WTAP recruits the m6A-methyltransferase METTL3 to HCV RNA. This targeting strategy for m6A modification of cytoplasmic RNA viruses is likely relevant for other m6A-modified positive-strand RNA viruses with cytoplasmic life cycles such as enterovirus 71 and SARS-CoV-2 and provides an exciting new target for potential antiviral therapies.

m6A Demethylase ALKBH5 Restrains PEDV Infection by Regulating GAS6 Expression in Porcine Alveolar Macrophages.

Porcine epidemic diarrhea virus (PEDV) is a burdensome coronavirus for the global pig industry. Although its fecal-oral route has been well-recognized, increasing evidence suggests that PEDV can also spread through airborne routes, indicating that the infection may also occur in the respiratory tract. N6-methyladenosine (m6A) has been known to regulate viral replication and host immunity, yet its regulatory role and molecular mechanism regarding PEDV infection outside the gastrointestinal tract remain unexplored. In this study, we demonstrate that PEDV can infect porcine lung tissue and the 3D4/21 alveolar macrophage cell line, and the key m6A demethylase ALKBH5 is remarkably induced after PEDV infection. Interestingly, the disruption of ALKBH5 expression remarkably increases the infection's capacity for PEDV. Transcriptome profiling identified dozens of putative targets of ALKBH5, including GAS6, which is known to regulate virus infectivity. Further, MeRIP-qPCR and mRNA stability analyses suggest that ALKBH5 regulates the expression of GAS6 via an m6A-YTHDF2-dependent mechanism. Overall, our study demonstrates that PEDV can infect porcine lung tissue and 3D4/21 cells and reveals the crucial role of ALKBH5 in restraining PEDV infections, at least partly, by influencing GAS6 through an m6A-YTHDF2-dependent mechanism.

Computational Prediction of N6-methyladenosine (m6A) RNA Methylation in SARS-CoV-2 Viral Transcripts

SARS-CoV-2 caused atypical pneumonia (COVID-19) is an ongoing pandemic that seriously threat the global public health. Many people die from this disease with severe symptoms. The most prevalent m6A RNA modification may be involved in by assisting the virus escaping from the host cell immune system attack. We provided here the first computational prediction study of RNA methylation sites in SARS-CoV-2. Based on virus sequence information, we predict the potential virus m6A sites and hope to make anyhow contributions to this unprecedented situation. As a result, we found 27 most frequent m6A sequences (41 bp) in SARS-CoV-2, and two of them are quite near to the spike protein stop codon position. © 2021 ACM.

Methyltransferase-Like Protein 14 Attenuates Mitochondrial Antiviral Signaling Protein Expression to Negatively Regulate Antiviral Immunity via N6 -methyladenosine Modification.

Mitochondrial antiviral signaling (MAVS) protein is the core signaling adaptor in the RNA signaling pathway. Thus, appropriate regulation of MAVS expression is essential for antiviral immunity against RNA virus infection. However, the regulation of MAVS expression at the mRNA level especially at the post transcriptional level is not well-defined. Here, it is reported that the MAVS mRNA undergoes N6 -methyladenosine (m6 A) modification through methyltransferase-like protein 14 (METTL14), which leads to a fast turnover of MAVS mRNA. Knockdown or deficiency of METTL14 increases MAVS mRNA stability, and downstream phosphorylation of TBK1/IRF3 and interferon-ß production in response to RNA viruses. Compared to wild-type mice, heterozygotes Mettl14+/- mice better tolerate RNA virus infection. The authors' findings unveil a novel mechanism to regulate the stability of MAVS transcripts post-transcriptionally through m6 A modification.

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

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 ß-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 ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.

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

N6-methyladenosine (m6A) is the most frequent chemical modification in eukaryotic mRNA and is known to participate in a variety of physiological processes, including cancer progression and viral infection. The reversible and dynamic m6A modification is installed by m6A methyltransferase (writer) enzymes and erased by m6A demethylase (eraser) enzymes. m6A modification recognized by m6A binding proteins (readers) regulates RNA processing and metabolism, leading to downstream biological effects such as promotion of stability and translation or increased degradation. The m6A writers and erasers determine the abundance of m6A modifications and play decisive roles in its distribution and function. In this review, we focused on m6A writers and erasers and present an overview on their known functions and enzymatic molecular mechanisms, showing how they recognize substrates and install or remove m6A modifications. We also summarize the current applications of m6A writers and erasers for m6A detection and highlight the merits and drawbacks of these available methods. Lastly, we describe the biological functions of m6A in cancers and viral infection based on research of m6A writers and erasers and introduce new assays for m6A functionality via programmable m6A editing tools.

Methyltransferase-like 3 Modulates Severe Acute Respiratory Syndrome Coronavirus-2 RNA N6-Methyladenosine Modification and Replication.

The coronavirus disease 2019 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is an ongoing global public crisis. Although viral RNA modification has been reported based on the transcriptome architecture, the types and functions of RNA modification are still unknown. In this study, we evaluated the roles of RNA N6-methyladenosine (m6A) modification in SARS-CoV-2. Our methylated RNA immunoprecipitation sequencing (MeRIP-Seq) and Nanopore direct RNA sequencing (DRS) analysis showed that SARS-CoV-2 RNA contained m6A modification. Moreover, SARS-CoV-2 infection not only increased the expression of methyltransferase-like 3 (METTL3) but also altered its distribution. Modification of METTL3 expression by short hairpin RNA or plasmid transfection for knockdown or overexpression, respectively, affected viral replication. Furthermore, the viral key protein RdRp interacted with METTL3, and METTL3 was distributed in both the nucleus and cytoplasm in the presence of RdRp. RdRp appeared to modulate the sumoylation and ubiquitination of METTL3 via an unknown mechanism. Taken together, our findings demonstrated that the host m6A modification complex interacted with viral proteins to modulate SARS-CoV-2 replication. IMPORTANCE Internal chemical modifications of viral RNA play key roles in the regulation of viral replication and gene expression. Although potential internal modifications have been reported in SARS-CoV-2 RNA, the function of the SARS-CoV-2 N6-methyladenosine (m6A) modification in the viral life cycle is unclear. In the current study, we demonstrated that SARS-CoV-2 RNA underwent m6A modification by host m6A machinery. SARS-CoV-2 infection altered the expression pattern of methyltransferases and demethylases, while the expression level of methyltransferase-like 3 (METTL3) and fat mass and obesity-associated protein (FTO) was linked to the viral replication. Further study showed that METTL3 interacted with viral RNA polymerase RNA-dependent RNA polymerase (RdRp), which influenced not only the distribution but also the posttranslational modification of METTL3. Our study provided evidence that host m6A components interacted with viral proteins to modulate viral replication.

Post-Transcriptional Regulation of Viral RNA through Epitranscriptional Modification.

The field of mRNA modifications has been steadily growing in recent years as technologies have improved and the importance of these residues became clear. However, a subfield has also arisen, specifically focused on how these modifications affect viral RNA, with the possibility that viruses can also be used as a model to best determine the role that these modifications play on cellular mRNAs. First, virologists focused on the most abundant internal mRNA modification, m6A, mapping this modification and elucidating its effects on the RNA of a wide range of RNA and DNA viruses. Next, less common RNA modifications including m5C, Nm and ac4C were investigated and also found to be present on viral RNA. It now appears that viral RNA is littered with a multitude of RNA modifications. In biological systems that are under constant evolutionary pressure to out compete both the host as well as newly arising viral mutants, it poses an interesting question about what evolutionary benefit these modifications provide as it seems evident, at least to this author, that these modifications have been selected for. In this review, I discuss how RNA modifications are identified on viral RNA and the roles that have now been uncovered for these modifications in regard to viral replication. Finally, I propose some interesting avenues of research that may shed further light on the exact role that these modifications play in viral replication.

The networks of m6A-SARS-CoV-2 related genes and immune infiltration patterns in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with a poor prognosis. The current coronavirus disease 2019 (COVID-19) shares some similarities with IPF. SARS-CoV-2 related genes have been reported to be broadly regulated by N6-methyladenosine (m6A) RNA modification. Here, we identified the association between m6A methylation regulators, COVID-19 infection pathways, and immune responses in IPF. The characteristic gene expression networks and immune infiltration patterns of m6A-SARS-CoV-2 related genes in different tissues of IPF were revealed. We subsequently evaluated the influence of these related gene expression patterns and immune infiltration patterns on the prognosis/lung function of IPF patients. The IPF cohort was obtained from the Gene Expression Omnibus dataset. Pearson correlation analysis was performed to identify the correlations among genes or cells. The CIBERSORT algorithm was used to assess the infiltration of 22 types of immune cells. The least absolute shrinkage and selection operator (LASSO) and proportional hazards model (Cox model) were used to develop the prognosis prediction model. Our research is pivotal for further understanding of the cellular and genetic links between IPF and SARS-CoV-2 infection in the context of the COVID-19 pandemic, which may contribute to providing new ideas for prognosis assessment and treatment of both diseases.

COVID-19: A methyl-group assault?

The socio-economic implications of COVID-19 are devastating. Considerable morbidity is attributed to 'long-COVID' - an increasingly recognized complication of infection. Its diverse symptoms are reminiscent of vitamin B12 deficiency, a condition in which methylation status is compromised. We suggest why SARS-CoV-2 infection likely leads to increased methyl-group requirements and other disturbances of one-carbon metabolism. We propose these might explain the varied symptoms of long-COVID. Our suggested mechanismmight also apply to similar conditions such as myalgic encephalomyelitis/chronic fatigue syndrome. The hypothesis is evaluable by detailed determination of vitamin B12and folate status, including serum formate as well as homocysteine and methylmalonic acid, and correlation with viral and host RNA methylation and symptomatology. If confirmed, methyl-group support should prove beneficial in such individuals.