AbstractRNA 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.