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

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.This work was supported by funds from the Spanish Ministry of Science and Innovation (PID2022-136939OB-I00 funded by MICIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” and PID2019106959RB-I00/AEI/10.13039/50110001103 to J.D.), Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (PID2021-128193NB-100 to E.M.N.), the European Research Council (ERC-StG-2021 No. 101042103 to E.M.N.), an institutional “María de Maeztu” Programme for Units of Excellence in R&D (CEX2018-000792-M) and by the 2021 SGR 00176 grant from the Departament de Recerca i Universitats de la Generalitat de Catalunya. B.B.P. was the recipient of a Beatriu Pinós postdoctoral fellowship funded by the Secretary of Universities and Research (Generalitat de Catalunya) and by the Horizon 2020 programme of research and innovation of the European Union under the Marie Sklodowska-Curie grant agreement No. 801370. I.D.Y. and S.A.W. were supported by the Biotechnology and Biological Sciences Research Council, U.K. (BBSRC grant: BB/V00722X/1). A.D.T. and M.P.T. are supported by FPI Severo-Ochoa fellowships by the Spanish Ministry of Science and Innovation. We acknowledge the support of the MEIC to the EMBL partnership, Centro de Excelencia Severo Ochoa and CERCA Programme/Generalitat de Catalunya. We thank the Genomics Core Facility (UPF), in particular Núria Bonet Martín and Raquel Rasal Soteras for technical assistance with library preparation of m6A-Seq and plasmid DNA, and Marc Tormo for assisting with plasmid variant calling. The mass spectrometric analyses were performed in the CRG/UPF Proteomics Unit which is part of the Proteored, PRB3 and is supported by grant PT17/0019, of the PE I + D + i 2013–2016, funded by ISCIII and ERDF. We also thank Dr. Matthew Seddon for technical assistance for producing the data graphs. Figures 1a, c, d, 3a, 6a, b and 7c, d were created using Biorender for which we own a full licence

m⁶A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Our results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent–lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection