N6-methyladenosine modification is not a general trait of viral RNA genomes

Abstract

Despite the nuclear localization of the m6A machinery, the genomes of multiple exclusively-cytoplasmic RNA viruses, such as chikungunya (CHIKV) and dengue (DENV), are reported to be extensively m6A-modified. However, these findings are mostly based on m6A-Seq, an antibody-dependent technique with a high rate of false positives. Here, we address the presence of m6A in CHIKV and DENV RNAs. For this, we combine m6A-Seq and the antibody-independent SELECT and nanopore direct RNA sequencing techniques with functional, molecular, and mutagenesis studies. Following this comprehensive analysis, we find no evidence of m6A modification in CHIKV or DENV transcripts. Furthermore, depletion of key components of the host m6A machinery does not affect CHIKV or DENV infection. Moreover, CHIKV or DENV infection has no effect on the m6A machinery’s localization. Our results challenge the prevailing notion that m6A modification is a general feature of cytoplasmic RNA viruses and underscore the importance of validating RNA modifications with orthogonal approaches.

Elucidation of the Epitranscriptomic RNA Modification Landscape of Chikungunya Virus

“…None of the detected modifications exhibited enrichment in CHIKV-infected cells compared to mock-infected cells; indeed, for some, modification levels were even lower (Figure 1). As shown before, consistent with m 6 A being the most common internal modification of cellular mRNAs, it was detected as the most abundant in both cell lines [12,17]. Inosine and 2 ′ -O-methyladenosine (Am) were amongst the most abundant RNA modifications in both mock-infected and CHIKV-infected cells, whereas pseudouridine (Ψ) was only detected in Huh7 cells (Figure 1a).…”

“…Solely when ADAR1 was overexpressed, a low level of AG editing was observed. In agreement with these observations, we should note that previous nanopore direct RNA sequencing (DRS) analyses comparing in vitro transcribed CHIKV RNA (unmodified) and CHIKV native RNA (extracted from HEK293T-infected cells) also did not reveal any differences in their RNA modification patterns [12] using the NanoConsensus software [39]. Notably, NanoConsensus has been found to be robust in the detection of many different RNA modification types in DRS datasets when comparing two conditions (e.g., control vs native); however, inosine was not explicitly included in the benchmarking of NanoConsensus [39].…”

“…Although MS represents a useful tool to trace such changes, putative RNA modifications must be validated by using independent research approaches since the contaminant presence of highly modified cellular RNAs, such as tRNA or rRNA, even at very low levels, can lead to misleading interpretations of the resulting LC-MS/MS data. This is also the case for transcriptome-wide mapping methods of RNA modifications, especially the antibody-dependent ones, for which false positives have been widely reported [11][12][13][14]. The importance of multidisciplinary approaches to confirm the absence of RNA modifications in viral RNA has recently been highlighted for N 6 -methyladenosine (m 6 A) and N 1 -methyladenosine (m 1 A) [12,15].…”

“…The importance of multidisciplinary approaches to confirm the absence of RNA modifications in viral RNA has recently been highlighted for N 6 -methyladenosine (m 6 A) and N 1 -methyladenosine (m 1 A) [12,15]. Moreover, extensive controls are required, for example, an in vitro transcribed (IVT) viral RNA of the complete genome, devoid of modifications, is a crucial negative control for transcriptome-wide mapping methods [12]. Therefore, any modification signal detected in a specific viral RNA position must be interrogated with an orthogonal approach and discarded if the same signal is observed in the IVT control.…”

“…This is also the case for transcriptome-wide mapping methods of RNA modifications, especially the antibody-dependent ones, for which false positives have been widely reported [ 11 , 12 , 13 , 14 ]. The importance of multidisciplinary approaches to confirm the absence of RNA modifications in viral RNA has recently been highlighted for N 6 -methyladenosine (m 6 A) and N 1 -methyladenosine (m 1 A) [ 12 , 15 ]. Moreover, extensive controls are required, for example, an in vitro transcribed (IVT) viral RNA of the complete genome, devoid of modifications, is a crucial negative control for transcriptome-wide mapping methods [ 12 ].…”