Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression.
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