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

ABSTRACT 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.

1) Could the study provide a comparison of differentially expressed coding and non-coding RNAs between their study and the published SARS-CoV-2 studies with respect to m6A-modified RNA species (such as reference 14).
2) Can authors report whether miRNA-4486 upregulation has been reported for other CoVs (or other viruses in general)?
Reviewer #2 (Comments for the Author): Here, Phillips et al. describe changes to post-transcriptional modification of host RNA in cultured lung cells triggered by infection with the virus sudden acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) that causes COVID-19. The focus is N6methyladenosine (m6A) modifications that are commonly triggered by viral infection and may affect RNA stability or utilization relevant to mRNA translation or other regulatory processes. The study is straightforward-the authors isolate RNA from uninfected and infected A549 cells at 24 hours post-infection prior to m6A-immunoprecipitation for enrichment followed by commercial microarray to measure changes to relative m6A-modified transcript abundance. Broad changes to the host m6A "epitranscriptome" are identified. Most are moderate but with one particular hit standing out-m6A-modified miRNA-4486, enriched >100-fold relative to uninfected cells. Pathway analyses indicate several genes, e.g., MUSK, STAT3, and EGFR targets as having potential for future investigation. The paper is very cleanly written and the techniques and analytical tools are appropriate. The dataset seems robust and will be of use to the field, fitting the scope of the journal. The major limitation, however, is that only a single cell line (A549) is studied. Validation of these changes, in particular the compelling miRNA results, would seem warranted in at least one additional cell system. Major comments: 1. As noted above, the major limitation to the study is that it only features a single infected cell type at a single time point postinfection. Specifically, it seems important to confirm or deny that miRNA-4486 and other hits are common targets of SARS-CoV-2 infection.
Minor comments: 1. The authors' abstract claim that their data "...suggest that m6A modification of cellular RNA is an important mechanism regulating host gene expression during SARS-CoV-2 infection..." is not justified herein. Changes are observed and reported consistent with the scope of this journal. Mechanism and/or relevance should be either addressed experimentally or this type of language removed.
3. Figure 2A. Something about this scheme confused me-I think it is that the shifted positions of transcript pools A and B implied some sort of interaction between the two pools.
4. Figure 2B. The 4486 dot was not distinguishable in my version (all appear red to me, but could be that I am partially colorblind.). I would also recommend highlighting the other notable hits and what they are so that is less necessary to access the supplementary data. 5. Table 2. Please better explain the criteria for selection of genes being shown here and the ordering-seems more useful for readers to go straight to the spreadsheets where they can sort the data, table feels arbitrary (i.e., what is its utility?).

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Reviewer #1:
Phillips et al present data to define the epitranscriptomic m6A profile of SARS-CoV-2-infected human lung epithelial cells and draw comparison to uninfected control cells. The study identified mRNA, LncRNA and sncRNA species that are differentially m6A-modified in response to SARS-CoV-2 infection. Interestingly, expression of m6A-modified miRNA-4486 was significantly upregulated in SARS-CoV-2-infected cells. This is a very intriguing finding to report even though the study does not provide functional consequence of miRNA-4486 upregulation. However, a reasonable discussion is provided.

Some important suggestions for authors to consider:
1) Could the study provide a comparison of differentially expressed coding and non-coding RNAs between their study and the published SARS-CoV-2 studies with respect to m6A-modified RNA species (such as reference 14).
Author response: We agree that a comparison of differential m 6 A-modification across studies would be beneficial for our broader understanding of how m 6 A modification of host cell transcripts change in response to infection. Liu et al. (cited reference 14) reported "gain and loss" of m 6 A peaks from sequencing results derived from m 6 A-modified mRNA in SARS-CoV-2infected cells relative to mock controls. However, there are important differences in experimental design and methodology that make meaningful comparison of our results difficult: A) Liu et al. used Huh7 (hepatocyte-derived carcinoma cells) infected at an MOI of 0.05 for 120 hours. We used A549-hACE2 (lung-derived carcinoma cells) infected at an MOI of 1.0 for 24 hours. B) Liu et al. performed meRIP-seq to identify m 6 A-modified sites in cellular RNA. This method requires the fragmentation of RNA prior to m 6 A-IP. As a result, transcripts may have both gain and loss of peaks on the same RNA, depending on the exact site of modification. In contrast, the epitranscriptomic array utilized in our study does not involve RNA fragmentation. Therefore, our analyses will reveal a net overall increase or decrease in m 6 A modification, without information regarding site-specificity. While there is some overlap in the two data sets, we chose to focus our discussion on our results.

2)
Can authors report whether miRNA-4486 upregulation has been reported for other CoVs (or other viruses in general)?
Author response: To our knowledge, miR-4486 upregulation has not previously been implicated in the context of viral infection. We have added a comment reflecting such to the discussion (lines 261-263).

3) Does SARS-CoV-2 infection upregulate miRNA-4486 expression in primary airway epithelial cells (HBEs/HAEs) of human, mice, or NHP origin?
Author response: Like the reviewer, we are interested to know whether m 6 A modification of miRNA-4486 is upregulated in SARS-CoV-2-infected primary HBE or HAE culture. While these experiments are outside the scope of our current study, we have added language to the text (lines 304-308) to indicate that this will be an important area of future investigation.
Author response: We appreciate the reviewer raising this important point. We have clarified the results in the text (lines 149-152) and with additional data (Fig. 3C-D).

Reviewer #2:
Here, Phillips et al. describe changes to post-transcriptional modification of host RNA in cultured lung cells triggered by infection with the virus sudden acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) that causes COVID-19. The focus is N6-methyladenosine (m6A) modifications that are commonly triggered by viral infection and may affect RNA stability or utilization relevant to mRNA translation or other regulatory processes. The study is straightforward-the authors isolate RNA from uninfected and infected A549 cells at 24 hours post-infection prior to m6A-immunoprecipitation for enrichment followed by commercial microarray to measure changes to relative m6A-modified transcript abundance. Broad changes to the host m6A "epitranscriptome" are identified. Most are moderate but with one particular hit standing out-m6A-modified miRNA-4486, enriched >100-fold relative to uninfected cells. Pathway analyses indicate several genes, e.g., MUSK, STAT3, and EGFR targets as having potential for future investigation. The paper is very cleanly written and the techniques and analytical tools are appropriate. The dataset seems robust and will be of use to the field, fitting the scope of the journal. The major limitation, however, is that only a single cell line (A549) is studied. Validation of these changes, in particular the compelling miRNA results, would seem warranted in at least one additional cell system. Major comments: 1. As noted above, the major limitation to the study is that it only features a single infected cell type at a single time point post-infection. Specifically, it seems important to confirm or deny that miRNA-4486 and other hits are common targets of SARS-CoV-2 infection.
Author response: We agree that our study would be strengthened by comparing results from other cell types or times post-infection. Due to the cost associated with the epitranscriptomic array, we chose to specifically focus on a lung cell line, which we feel is more biologically relevant to SARS-CoV-2 infection than other commonly used cell types in the field, such as Vero or Huh7. We strategically chose 24 hours post-infection because our data (not shown) shows that this represents a spreading infection, and therefore should capture modified RNA representing each temporal phase of the viral life cycle. We have added text (lines 304-311) to make clear that this is a limitation of our study.