How is the host’s m6A mRNA profile affected during SARS-CoV-2 infection?

By Tarun Sai LomteDec 15 2022Reviewed by Danielle Ellis, B.Sc.

In a recent study posted to bioRxiv*, researchers observed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in loss of N6-methyladenosine (m⁶A) modification in cellular ribonucleic acids (RNAs).

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources

Background

m⁶A, a prevalent 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. Moreover, the m⁶A modification is introduced by host proteins in the SARS-CoV-2 genome; wherein it promotes viral replication and limits immune responses.

Depleting cytoplasmic m⁶A writer, m⁶A-methyltransferase 3 catalytic subunit (METTL3) suppresses SARS-CoV-2 replication. Despite the recent advances in understanding m⁶A in the SARS-CoV-2 genome, how it affects the host’s m⁶A profile during infection remains to be elucidated.

The study and findings

In the present study, researchers assessed the effects of SARS-CoV-2 infection on m⁶A RNA modification in different cell types. Vero cells were infected with SARS-CoV-2 B.1, B.1.1.7 (Alpha), or B.1.351 (Beta) variant.

RNA was isolated and evaluated for gene expression alterations using RNA sequencing (RNA-seq). The per-gene expression levels were similar for the three variants, and viral reads accounted for 1.2% to 2.8% of total reads. Analysis of differentially expressed genes (DEGs) in Vero cells identified 998 upregulated and 950 downregulated genes.

Gene expression patterns were variant-specific and more similar between Alpha and Beta variants. Pathway analysis showed enrichment of altered genes cilium assembly, RNA catabolism, and protein localization pathways. Next, the researchers focused on RNA catabolism-associated pathways enriched in deregulated genes and visualized their interactions using network analysis.

The authors noted frequent deregulation of m⁶A-associated genes in Vero cells during infection, albeit the expression of main m⁶A writers was not significantly changed. Next, isolated RNA from infected and non-infected Vero cells was supplemented with spike-in 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 B.1 and Alpha variants than with the Beta variant.

Furthermore, the authors observed that METTL3 was partially localized to the cytoplasm, which typically localizes in the nucleus and introduces the m⁶A modification co-transcriptionally. This cytoplasmic localization was more prominent with B.1 and Alpha variants than with the Beta variant. However, METTL14 localization in the nucleus was mostly unaffected during infection.

The team speculated whether the partial cytoplasmic localization of METTL3 could 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 in several locations in the positive (genomic) and negative (replication intermediates) strands of SARS-CoV-2 RNA, with an average of 10 m⁶A modifications per viral genome. The researchers observed the upregulation of exportin 1 (XPO1), a nuclear export protein, and its interactions with METTL3 during SARS-CoV-2 infection.

Next, cells were treated with an XPO1 inhibitor (Selinexor) during infection. Selinexor treatment restored the localization of METTL3 to the nucleus; moreover, the METTL3/METTL14 PLA signal was robust in infected cells. This suggested that a change in the localization of METTL3 during infection perturbed the formation of the METTL3/METTL14 complex.

Intriguingly, Selinexor treatment also reduced SARS-CoV-2 infection without affecting cell viability. Given that 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 downregulated genes that interact with an SG protein and m⁶A reader.

The researchers evaluated METTL3 localization after SARS-CoV-2 infection in BEAS-2B cells, a bronchial epithelial cell line. These infected cells exhibited spike positivity and partial cytoplasmic localization of METTL3. Likewise, this partial cytoplasmic localization was also observed in primary human bronchial epithelial cells in a monolayer culture.

Next, the reconstructed human bronchial epithelium (HPE) in air-liquid interface (ALI) cultures was used as the infection model. 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. Like in Vero cells, m⁶A peaks were markedly reduced in HBE post-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 showed upregulation in HBE upon infection, whereas cilium organization-related genes were downregulated. These findings were also recapitulated in ALI cultures of human nasal epithelia (HNE).

Conclusions

Overall, the present study illustrated that SARS-CoV-2 infection leads to a global loss of m⁶A methylation in cellular RNAs, whereas viral RNA remained m⁶A-modified. m⁶A-modified transcripts were preferentially downregulated after infection. The infection caused partial localization of METTL3 to the cytoplasm, compromising the formation of the METTL3/METTL14 complex in the nucleus.

The global loss of m⁶A was also observed in human airway epithelial cells, implying that m⁶A loss was a characteristic of cells infected with SARS-CoV-2. The nuclear export protein (XPO1) inhibition restored METTL3 localization and SG formation and increased mRNA expression. The authors posit that rescuing METTL3 localization could be explored as a novel antiviral strategy for SARS-CoV-2 infection.

This news article was a review of a preliminary scientific report that had not undergone peer-review at the time of publication. Since its initial publication, the scientific report has now been peer reviewed and accepted for publication in a Scientific Journal. Links to the preliminary and peer-reviewed reports are available in the Sources section at the bottom of this article. View Sources