Internal RNA 2'O-methylation in the HIV-1 genome counteracts ISG20 nuclease-mediated antiviral effect El Kazzi, P. Rabah, N. Chamontin, C. Poulain, L. Ferron, F Debart, F. Canard, B. /Missé, Dorothée Coutard, B. Nisole, S. Decroly, E. RNA 2'O-methylation is a 'self' epitranscriptomic modification allowing discrimination between host and pathogen. Indeed, human immunodeficiency virus 1 (HIV-1) induces 2'O-methylation of its genome by recruiting the cellular FTSJ3 methyltransferase, thereby impairing detection by RIG-like receptors. Here, we show that RNA 2'O-methylations interfere with the antiviral activity of interferon-stimulated gene 20-kDa protein (ISG20). Biochemical experiments showed that ISG20-mediated degradation of 2'O-methylated RNA pauses two nucleotides upstream of and at the methylated residue. Structure-function analysis indicated that this inhibition is due to steric clash between ISG20 R53 and D90 residues and the 2'O-methylated nucleotide. We confirmed that hypomethylated HIV-1 genomes produced in FTSJ3-KO cells were more prone to in vitro degradation by ISG20 than those produced in cells expressing FTSJ3. Finally, we found that reverse-transcription of hypomethylated HIV-1 was impaired in T cells by interferon-induced ISG20, demonstrating the direct antagonist effect of 2'O-methylation on ISG20-mediated antiviral activity. 2023 text https://www.documentation.ird.fr/hor/fdi:010086598 fdi:010086598 El Kazzi P., Rabah N., Chamontin C., Poulain L., Ferron F, Debart F., Canard B., Missé Dorothée, Coutard B., Nisole S., Decroly E.. Internal RNA 2'O-methylation in the HIV-1 genome counteracts ISG20 nuclease-mediated antiviral effect. 2023, 51 (6), 2501-2515 EN