In a recent study published in bioRxiv*, researchers observed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in the loss of the N6-methyladenosine (m6A) modification in ribonucleic acids cellular (RNA).
Study: Global loss of cellular m6A RNA methylation after infection with different SARS-CoV-2 variants. Image credit: MattLphotography/Shutterstock
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m6A, a predominant internal RNA modification, regulates several biological processes, including mRNA translation, cell differentiation, and stress granule (SG) formation. This modification has also been observed in viral genomic RNA. In addition, the m6A modification is introduced by host proteins into the SARS-CoV-2 genome; where it favors viral replication and limits immune responses.
Depletion of the cytoplasmic m6A reporter, catalytic subunit m6A-methyltransferase 3 (METTL3) suppresses SARS-CoV-2 replication. Despite recent advances in the understanding of m6A in the SARS-CoV-2 genome, how it affects the host m6A profile during infection remains to be elucidated.
The study and conclusions
In the present study, the researchers evaluated the effects of SARS-CoV-2 infection on m6A RNA modification in different cell types. Vero cells were infected with SARS-CoV-2 variant B.1, B.1.1.7 (Alpha) or B.1.351 (Beta).
RNA was isolated and assessed for alterations in gene expression by RNA sequencing (RNA-seq). Expression levels per gene were similar for all three variants, with viral reads accounting for 1.2% to 2.8% of total reads. Analysis of differentially expressed genes (DEGs) in Vero cells identified 998 up-regulated genes and 950 down-regulated genes.
Gene expression patterns were variant specific and more similar between alpha and beta variants. Pathway analysis showed enrichment of altered gene cilia assembly, RNA catabolism, and protein localization pathways. The researchers then focused on pathways associated with RNA catabolism enriched in dysregulated genes and visualized their interactions using network analysis.
The authors observed frequent deregulation of m6A-associated genes in Vero cells during infection, although the expression of the main m6A writers was not significantly changed. RNA isolated from infected and uninfected Vero cells was then supplemented with spiked bacterial RNA for m6A RNA immunoprecipitation and sequencing. The team observed a loss of m6A peaks in cellular RNA after infection, which was more profound with the B.1 and Alpha variants than with the Beta variant.
In addition, the authors observed that METTL3 was partially localized to the cytoplasm, which normally localizes to the nucleus, and cotranscriptionally introduces the m6A modification. This cytoplasmic localization was more prominent with the B.1 and Alpha variants than with the Beta variant. However, the localization of METTL14 in the nucleus was not affected during infection.
The team speculated whether partial cytoplasmic localization of METTL3 might compromise the formation of the functional METTL3/METTL14 complex. To this end, a proximity ligation assay (PLA) performed to detect the complex revealed a significant decrease in the METTL3/METTL14 signal in infected cells.
In addition, m6A-enriched regions were detected at various locations in the positive (genomics) and negative (replication intermediates) strands of SARS-CoV-2 RNA, with an average of 10 m6A modifications per viral genome. The researchers observed the regulation of exportin 1 (XPO1), a nuclear export protein, and its interactions with METTL3 during SARS-CoV-2 infection.
Cells were then treated with an XPO1 inhibitor (Selinexor) during infection. Selinexor treatment restored METTL3 localization to the nucleus; furthermore, the METTL3/METTL14 PLA signal was robust in infected cells. This suggested that a change in METTL3 localization during infection disrupted the formation of the METTL3/METTL14 complex.
Interestingly, Selinexor treatment also reduced SARS-CoV-2 infection without affecting cell viability. Since SARS-CoV-2 infection compromises SG formation, the research team noted that Selinexor-mediated restoration of METTL3 localization promoted SG formation in infected cells. Selinexor treatment also restored the expression of the four down-regulated genes that interact with an SG protein and an m6A reader.
The researchers assessed the localization of METTL3 after SARS-CoV-2 infection in BEAS-2B cells, a bronchial epithelial cell line. These infected cells showed peak positivity and partial cytoplasmic localization of METTL3. Likewise, this partial cytoplasmic localization was also observed in primary human bronchial epithelial cells in monolayer culture.
Human bronchial epithelium (HPE) reconstituted in air-liquid interface (ALI) cultures was then used as a model of infection. High viral replication was observed in HBE at four and seven days post-infection (dpi). SARS-CoV-2 reads accounted for 0.48% and 0.47% of total reads at 4 and 7 dpi, respectively. As in Vero cells, m6A peaks were markedly reduced in HBE after infection.
m6A peaks were detected in both SARS-CoV-2 RNA strands at 4 dpi, but only in the positive strand at 7 dpi. Genes associated with host immune responses to viruses and interferon signaling pathways were upregulated in HBE after infection, while genes related to cilium organization were upregulated in the drop These findings were also recapitulated in ALI cultures of human nasal epithelium (HNE).
Conclusions
Overall, the present study illustrated that SARS-CoV-2 infection leads to a global loss of m6A methylation in cellular RNAs, while viral RNA remained modified with m6A. m6A-modified transcripts were preferentially up-regulated after infection. Infection resulted in partial localization of METTL3 to the cytoplasm, compromising the formation of the METTL3/METTL14 complex in the nucleus.
Global loss of m6A was also observed in human airway epithelial cells, implying that loss of m6A was a characteristic of cells infected with SARS-CoV-2. Inhibition of nuclear export protein (XPO1) restored METTL3 localization and SG formation and increased mRNA expression. The authors postulate that rescue of METTL3 localization could be explored as a novel antiviral strategy for SARS-CoV-2 infection.
*Important news
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guide clinical practice/health-related behavior, or be treated as established information.