m(6)A 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+).