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

N6-methyladenosine (m6A) is a ubiquitous RNA post-transcriptional modification found in coding as well as non-coding RNAs. m6A has also been found in viral RNAs where it is proposed to modulate host-pathogen interactions. Two m6A 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 m6A sites have not been observed in other studies mapping m6A 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 m6A 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, m6A is unlikely to substantially alter the conformational properties of the RNA. Our results add to a growing view that the impact of m6A on RNA depends on sequence context and Mg2+.

A number of studies have reported m 6 A in the HIV-1 RNA genome [18,20,21]. One study examining HIV-1 infected human T cells [20] reported two m 6 A sites (A68 and A62, Fig 1) in the Rev response element (RRE) stem IIB. RRE is a~350 nt cis-acting RNA element that is recognized by viral Rev protein to promote the export of unspliced or partially spliced viral RNA to express the structural proteins required for viral replication [22][23][24]. The two m 6 A sites were found in stem IIB (RREIIB), which is the primary binding site for Rev [23,[25][26][27]. Knocking down the methyltransferase complex (METTL3/METTL14) was shown to suppress Rev-RRE mediated RNA export and viral replication, and point substitution mutation of one a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 of the two highly conserved adenines (A68) strongly suppressed viral replication (> 90%) [20]. It was proposed [20] that the methyl group of m 6 A68 may interact with Rev protein to stabilize Rev-RRE binding, and/or that m 6 A68 may alter the conformational properties of stem IIB to facilitate Rev recognition. Another different study employing distinct cell lines and mapping methods did not observe these m 6 A sites on RREIIB suggesting that m 6 A can enhance HIV-1 replication and mRNA expression through recruitment of the m 6 A reader proteins YTHdomain containing family (YTHDF) [21]. A third study found m 6 A in RRE but did not identify the specific site [18]. The study proposed that the YTHDF proteins inhibit HIV-1 replication and infection by blocking viral reverse transcription.
Here, we asked whether methylation of RREIIB with m 6 A leads to changes in its conformational and Rev arginine rich motif (ARM) binding properties. We were driven to test this hypothesis because our recent studies showed that the internal loop region of RREIIB near the A68 bulge is highly flexible, and can adopt conformations with alternative secondary structures ( Fig 1A) that have different export activities [28,29]. By redistributing this dynamic ensemble of RREIIB, m 6 A could potentially impact the Rev-RRE interaction and RNA export. Prior studies have shown that m 6 A can reshape RNA-protein and RNA-RNA interactions by modulating RNA structure [30]. For example, a single m 6 A was shown to destabilize RNA duplexes by 0.5-1.7 kcal/mol [31,32] thus enhancing the binding affinity of proteins to their single-stranded RNA targets [33]. The modification destabilizes A-U base pairs because hydrogen bonding requires that the N 6 -methyl group adopt the unfavorable anti conformation [34,35]. m 6 A has also been shown to disrupt the non-canonical sheared G-A base pairs to block the assembly of the box C/D snoRNP complexes [36].
We find that modification of the A68 bulge has little effect on the stability, structure, dynamics, as well as the Rev-ARM binding properties of RREIIB. The results indicate that if RREIIB is m 6 A modified, it is unlikely to substantially change the conformational properties of RRE although we cannot rule out that small changes in the conformational properties could modulate Rev-RRE binding in vivo. The results also add to a growing view that the impact of m 6 A on RNA structure depends on sequence context and Mg 2+ [31,32,36,37]. For example, while m 6 A has been shown to destabilize canonical duplexes [31,32], it can stabilize junctional A-U base pairs in a Mg 2+ and secondary structure dependent manner [37] as well as in contexts in which the m 6 A is in a dangling end [32].
All RNA samples were purified using 20% (w/v) denaturing polyacrylamide (29:1) gel within 8M urea, 20 mM Tris Borate and 1 mM ethylene-diaminetetraacetate (EDTA) TBE buffer followed by Elutrap electro-elution system (Whatmann, GE healthcare) with 40 mM Tris Acetate and 1 mM EDTA (TAE) buffer then ethanol precipitation. The RNA pellets were dissolved in water and annealed by heating at 95˚C for 10 mins then rapidly cooling on ice. After measuring the concentration, the RNA samples were buffer-exchanged into NMR buffer (15 mM sodium phosphate, 25 mM NaCl, 0.1 mM EDTA, with or without 3 mM MgCl 2 at pH = 6.4) three times using 3kDa Amicon Ultra centrifugal filters (EMD Millipore). The final RNA concentrations were~1.3 mM and~0.4 mM for RREIIB and RREII, respectively.

UV melting experiments
Thermal melting experiments were performed on m 6 A modified and unmodified RREIIB using a PerkinElmer Lambda 25 UV/VIS spectrometer equipped with an RTP 6 Peltier Temperature Programmer and a PCB 1500 Water Peltier System. All RNA samples were buffer exchanged into NMR buffer. RNA samples (concentration~1 mM) were then diluted (with NMR buffer) to 3 μM prior to UV melting measurement, which were performed in triplicate (or more) using a sample volume of 400 μL in a Teflon-stoppered 1 cm path length quartz cell. Data are presented as the mean ± standard deviation of at least three independent measurements. Uncertainty in the calculated thermodynamic parameters were determined by error propagation as previously described [48].
The absorbance at 260 nm was monitored while the temperature was varied between 15 and 95˚C at a rate of 1C/min. Thermodynamic parameters were obtained by fitting the UV melting curves using nonlinear model fitting in Mathematica 10.0 (Wolfram Research) as previously described [37].
ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi ffi where A is the measured fluorescence polarization; A free is the polarization without Rev-Fl binding; A bound is the polarization with saturated Rev-Fl binding; R T is the total RNA concentration; L T is the total Rev-Fl concentration; K d is the dissociation constant. The uncertainty in (A) was deduced based on the standard deviation over triplicate measurements.

m 6 A68 does not alter the thermal stability of RREIIB
We first used optical melting experiments to examine whether methylation of A68 impacts the thermal stability of RREIIB. All experiments were performed in the presence of 3 mM Mg 2+ unless stated otherwise. This is important given the impact of Mg 2+ on RNA folding and dynamics [41] and also given recent studies showing that the impact of m 6 A on RNA structural dynamics and stability can depend on Mg 2+ [37]. For these experiments, we used a stem IIB construct (Fig 1B) containing the wild-type sequence that was recently shown to recapitulate the conformation of stem IIB in the larger three-way junction context [28]. Prior X-ray [42,43], NMR [44,45], SAXS [46] as well as chemical probing data on larger fragments of the HIV genome (~350 nt) [47] indicate that RREII stem IIB adopts the predominant secondary structure shown in Fig 1A. In the dominant RREIIB ground state (GS) conformation, A68 adopts an unpaired bulged conformation. When located in bulged nucleotides, m 6 A has previously been shown to slightly destabilize RNA hairpins by 0.4-0.7 kcal/mol [37] most likely due to the disruption of stacking interactions in the flipped-in conformation. Based on the UV melting data, m 6 A68 had a negligible effect on RREIIB stability (Fig 1B and 1C). Interestingly, a small degree of destabilization (by~0.4 kcal/mol) was observed in the absence of Mg 2+ , most likely because A68 adopts a partially flipped in conformation in the absence of Mg 2+ , and such a conformation could be more susceptible to destabilization by m 6 A [28]. Thus, the m 6 A effect is overridden by Mg 2+ . Indeed, Mg 2+ stabilizes RREIIB by~2 kcal/mol while m 6 A only destabilizes it by~0.4 kcal/mol (Fig 1C).

m 6 A68 minimally affects structural and dynamic properties of the RREIIB ground state
We used NMR to examine whether methylation of A68 affects the conformation of RREIIB. A single resonance was observed for the N 6 -methyl group in 1D 1 H NMR spectra of RREIIB m6A68 , consistent with a single dominant conformation for m 6 A68 (Fig 2A). The 1D 1 H imino spectrum  of RREIIB m6A68 is virtually identical to its unmodified counterpart, indicating that the methylation does not alter the RREIIB secondary structure (Fig 2B). Very good agreement was also observed between the 2D [ 13 C, 1 H] HSQC spectra of modified and unmodified RREIIB with only few residues in the internal loop region (A68, U66, G50, G46) showing minor chemical shift perturbations ( Fig 2C). These results indicate that m 6 A68 modification does not substantially affect the structure of RREIIB GS.
Prior studies showed that m 6 A can impact RNA conformation in a Mg 2+ dependent manner [37]. Interestingly, the modification had a larger impact on NMR spectra of RREIIB recorded in the absence of Mg 2+ . Multiple resonances are observed for the N 6 -methyl group, indicating the co-existence of multiple conformations (Fig 2A). The methylation also induced larger perturbations at A52-U66 base pair near the m 6 A68 bulge (Fig 2B). In 2D [ 1 H, 13 C] HSQC spectra, the modification induced severe line broadening [49] for resonances belonging to residues G47, G48, G50, A68, C69, G71 and U72 in the internal loop, consistent with enhanced dynamics at the micro-to-millisecond timescales, where only G50 resonance shows broadening in the presence of Mg 2+ (Fig 2C and 2D). It is likely that the m 6 A induced line broadening in the absence of Mg 2+ is due to a changes in ES kinetics or populations. The UV melting data showing that m 6 A68 destabilizes RREIIB GS in the absence of Mg 2+ suggests that the enhanced ES dynamics could arise from destabilization of the GS in the absence of Mg 2+ . Similar NMR results were obtained for a double m 6 A modified RREIIB (RREIIB m6A62m6A68 ) (S1 Fig).

m 6 A68 minimally affects the structural and dynamic properties of the excited states in the more native RREII three-way junction
We recently showed that RREII transiently adopts two low-abundance alternative secondary structures ('excited states', ES) referred to as ES1 and ES2 (Fig 3A). A68 remains as a bulge in ES1 while forms a G50-A68 mismatch in ES2. The combined m 6 A effects to A68 bulge in ES1 and to the specific G(anti)-A(anti) mismatch in ES2 remain unknown. We therefore examined whether methylation of A68 impacts the dynamics between the GS and ESs in more native RREII three-way junction. This conformational exchange can in principle be measured quantitatively with the use of NMR R 1ρ relaxation dispersion (RD) data [28,[50][51][52]. However, in practice, severe line-broadening and resonance overlap in NMR spectra of the more native three-way junction (RREII) in the presence of Mg 2+ complicates measurements [28]. We therefore turned to an alternative approach which we recently developed which uses site-specific stable isotope labeling to directly observe the low-populated ES that form under slow exchange kinetics [28]. With this approach, we were previously able to directly observe imino resonances belonging to ES1 and ES2 in RREII, which slows down the exchange kinetics relative to RREIIB (Fig 3A) [28]. In particular, by site-specifically labeling 15 N3-U72, we observed the G48-U72 mismatch, which uniquely forms in both ES1 and ES2, based on the characteristic imino chemical shift of G-U mismatches (Fig 3A) [28].
We used the above strategy to examine how methylation of A68 impacts the GS-ES exchange in RREII. Samples of m 6 A68 modified and unmodified RREII were chemically synthesized with site-labeled 15 N3-U72 ( 15 N3-U72-RREII m6A68 and 15 N3-U72-RREII, respectively). The 1D 1 H imino spectra of modified and unmodified RREII were very similar ( Fig  3B) indicating that the modification minimally impacts the GS secondary structure even in the more native three-way junction context. The 2D [ 1 H, 15 N] HSQC spectrum of RREII m6A68 includes a resonance characteristic of the ES G48-U72 mismatch, and it shows excellent overlap with the corresponding resonance observed in unmodified RREII (Fig 3C). No other resonances were observed indicating that m 6 A does not lead to stabilization of alternative m 6 A minimally impacts the structure, dynamics, and Rev ARM binding properties of HIV-1 RRE stem IIB conformations in which U72 is base paired. Similar results were obtained in the absence of Mg 2+ (S2 Fig). These results indicate that m 6 A68 does not significantly impact structural properties of the GS or the populations of the ESs in the native three-way junction both in the presence and absence of Mg 2+ . Note that we cannot entirely rule out that m 6 A destabilizes ES2 or ES1 and that the observed G48-U72 mismatch reflects either ES1 or ES2, respectively.

m 6 A68 has a negligible effect on Rev-RRM binding to RREIIB
We used a fluorescence polarization binding assay to examine whether methylation of A68 impacts binding of fluorescein labeled Rev-ARM peptide (Rev-Fl) to RREIIB (Fig 4) [28,40]. Unmodified RREIIB binds to Rev-ARM peptide with apparent K d = 30.6 ± 5.8 nM, in agreement with prior studies [44,53]. The binding affinity decreased two-fold (K d = 62.2 ± 23.8 nM) for the modified RREIIB m6A68 , indicating that the binding affinity for m 6 A modified was only slightly weakened relative to unmodified RREIIB especially when considering the uncertainty.

Discussions
Methylation of RREIIB can promote Rev-RRE interaction by changing the RREIIB conformation so as to favorably bind to Rev or by promoting Rev-RRE binding through direct interaction involving the methyl group. Our results argue against a significant impact on RREIIB structure, as might be expected given placement of m 6 A68 in the bulge. In addition, the modification slightly weakened binding of the Rev-ARM, and this is consistent with prior NMR [54] and X-ray crystallography [55] studies showing that A68 bulge interacts with Rev protein through non-specific Van Der Waals contacts, and that deleting A68 has minor effects on Rev or Rev-ARM binding affinity to RRE stem IIB [26,56]. Our data cannot rule out that the exposed and accessible m 6 A enhances binding in the context of full-length Rev and RRE or promotes RNA export through the recruitment of other host export factors, and that this in turn gives rise to the reported~2-3 fold decrease in HIV viral RNA pull down using Rev when knocking down METTL3/METTL14 responsible for producing m 6 A [20]. It is also possible that the structural and dynamic properties of m 6 A modified RREIIB differ in vivo relative to in vitro. A recent study [29] showed that the key dynamic properties of unmodified RREIIB are similar in vitro and in cells. Further studies are needed to address these possibilities.
The modification had a greater effect on the stability and conformation of RREIIB in the absence of Mg 2+ . We previously showed that Mg 2+ redistributes the RREIIB ensemble by stabilizing the GS and ES1 relative to ES2 [28]. Our results indicate that Mg 2+ has a larger effect on the relative stability of these different conformations as compared to the methylation. This