m6A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB

N 6 -methyladenosine (m 6 A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m 6 A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m 6 A sites have been reported in the HIV-1 Rev response element (RRE) stem IIB, one of which was shown to enhance binding to the viral protein Rev and viral RNA export. However, because these m 6 A sites have not been observed in other studies mapping m 6 A in HIV-1 RNA, their significance remains to be firmly established. Here, using optical melting experiments, NMR spectroscopy, and in vitro binding assays, we show that m 6 A minimally impacts the stability, structure, and dynamics of RRE stem IIB as well as its binding affinity to the Rev arginine-rich-motif (ARM) in vitro . Our results indicate that if present in stem IIB, m 6 A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m 6 A on RNA depends on sequence context and Mg 2+ .