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

Area of Science:

• Virology
• Molecular Biology
• Epigenetics

Background:

• The N6-methyladenosine (m6A) epitranscriptomic mark is crucial in regulating RNA function.
• While the m6A machinery is nuclear, m6A modification has been reported in cytoplasmic RNA viruses like CHIKV and DENV.
• Previous findings relied heavily on m6A-Seq, an antibody-dependent method prone to false positives.

Purpose of the Study:

• To rigorously investigate the presence of m6A modification in chikungunya virus (CHIKV) and dengue virus (DENV) RNA.
• To validate or refute the widespread m6A modification in exclusively cytoplasmic RNA viruses.
• To assess the functional relevance of the host m6A machinery in CHIKV and DENV infections.

Main Methods:

• Combined m6A-Seq with antibody-independent techniques like SELECT and nanopore direct RNA sequencing.
• Conducted functional, molecular, and mutagenesis studies.
• Assessed the impact of depleting host m6A machinery components on viral infection and localization.

Main Results:

• No evidence of m6A modification was detected in CHIKV or DENV transcripts using orthogonal validation methods.
• Depletion of key host m6A machinery components did not affect CHIKV or DENV infection levels.
• CHIKV and DENV infections did not alter the localization of the host m6A machinery.

Conclusions:

• The prevailing notion of widespread m6A modification in cytoplasmic RNA viruses is challenged.
• Orthogonal validation is critical for confirming RNA modifications, especially when using antibody-dependent methods.
• m6A modification is not a general feature of CHIKV and DENV RNA, and the host m6A machinery is not essential for their replication.