Abstract
Viral RNA genomes are modified by epitranscriptomic marks, including 2′-O-methylations that are added by cellular or viral methyltransferases. 2′-O-methylations modulate RNA structure, function, and discrimination between self and non-self RNA by innate immune sensors such as RIG-I like receptors. This is illustrated by HIV-1 that decorates its RNA genome through hijacking the cellular FTSJ3 2′-O-methyltransferase, thereby limiting immune sensing and interferon production. However, the impact of such RNA modification during viral genome replication is poorly understood. Here we show by performing endogenous reverse transcription on methylated or hypomethylated HIV-1 particles, that 2′-O-methylations negatively affect HIV-1 reverse transcriptase (RT) activity. Biochemical assays confirm that RNA 2′-O-methylation impedes RT activity, especially at low dNTP concentrations reflecting those in quiescent cells, by reducing nucleotide incorporation efficiency and impairing translocation. Mutagenesis highlights K70 as a critical amino-acid for the RT to bypass 2′-O-methylations. Hence, the observed antiviral effect due to viral RNA 2′-O-methylation antagonizes the FTSJ3-mediated proviral effects, suggesting the fine tuning of RNA methylation during viral replication.