Dr Andrei A Krasilin
email: ikrasilin@mail.ioffe.ru

Ekaterina K Khrapova

Dr Alexandre Nominé

Dr Jaafar Ghanbaja

Thierry Belmonte

Victor V Gusarov

Dr A Nominé

Prof. T Belmonte

Cation Redistribution along the Spiral of Ni-Doped Phyllosilicate Nanoscrolls: Energy Modelling and STEM/EDS Study

Keywords: Crystal growth, Doping, Electron microscopy, Hydrothermal synthesis, Phyllosilicates

Here, we study the stress-induced self-organization of Mg 2+ and Ni 2+ cations in the crystal structure of multiwalled (Mg1-x,Nix)3Si2O5(OH)4 phyllosilicate nanoscrolls. The phyllosilicate layer strives to compensate size and surface energy difference between the metal oxide and silica sheets by curling. But as soon as the layer grows, the scrolling mechanism becomes a spent force. An energy model proposes secondary compensation of strain: two cations distribute along the nanoscroll spiral in accordance with preferable radii of curvature. To reveal this, we study synthetic (Mg1-x,Nix)3Si2O5(OH)4 nanoscrolls by the scanning transmission electron microscopy/energy-dispersive X-ray spectroscopy (STEM/EDS) technique. For a number of scrolls, we have found indeed a change of Ni concentration with increase in distance from the nanoscroll central axis. The concentration gradient, according to our estimates, can reach 50 at.% over 25 nm of the wall thickness.

Introduction

Some of 1:1 phyllosilicates and clays possess a very intriguing feature, which consists in spontaneous curling of crystalline layer with a formation of nanotubes and nanoscrolls. Three of the most known representatives are chrysotile Mg3Si2O5(OH)4, [1][2][3] halloysite Al2Si2O5(OH)4, [4][5][6] and imogolite Al2SiO3(OH)4. [7,8] Because of wide occurrence in nature, their tubular shape had been revealed decades before the carbon [9][10] and chalcogenide [11,12] nanotubes discoveries. Such hydrosilicate layers consist of brucite-like or gibbsite-like octahedral sheet bonded to a SiO2 tetrahedral sheet. In the case of imogolite, single SiO4 tetrahedra fit into voids of the same gibbsite-like sheet. Size difference between the sheets (or between the void and the tetrahedra) creates internal stress of the whole layer and initiates its curling.

Single-walled and multi-walled nanotubes and nanoscrolls formed by this process have a range of interesting properties and related applications. The inner channel grants large specific surface area (up to 400 m 2 /g) [5,13] as well as active sites for adsorption [14,15] and encapsulation of functional substance. [16,17] The outer and inner hydrosilicates surfaces saturated with OH groups can be modified by various organic compounds as well as be decorated by metallic or oxide phases. [18,19] High aspect ratio affects on particles aggregation: entangling of nanotubes and nanoscrolls is used for production of self-supported and composite membranes. [20,21] Individual nanoscrolls demonstrate good mechanical properties, [22,23] which are appropriate for reinforcement of polymer composites. Change of Mg by a delement like Ni enhances magnetic properties of the particles. [ 24,25] The delement can then be reduced from the silicate matrix by hydrogen in order to produce composite materials with catalytic activity. [26,27] Cations substitutions in the phyllosilicate layer change size and morphology of the resulting nanotubes and nanoscrolls. In the case of chrysotile to date there are series of reports on change of Mg and (or) Si by Fe, [24,28,29] Ni, [30][31][32] Ti, [33,34] and Ge. [35] Halloysite and imogolite were successfully doped by Ge [5,36] and Fe [37] as well.

In this paper we would like to drive an attention to some feature which can be observed during partial substitution by a guest cation. Thill et al. [36] have claimed that in the Al2(Six,Ge1-x)O3(OH)4 system single-walled and double-walled imogolite nanotubes were formed at x = 1 and x=0, correspondingly. If the value was 0 < x < 1 -i. e. both Si and Ge cations were present -triple-walled nanoscrolls were formed. Bloise et al. [30] have found that considerable amounts of particles with telescopic and conical morphology can be obtained by hydrothermal treatment of Ni-doped forsterite. [38] The same situation was observed in case of Fe-containing precursor. [28] Krasilin et al. have showed [39,40] that conical scrolls content has a maximum provided that both Mg and Ni are present in (Mg1-x,Nix)3Si2O5(OH)4 nanoscrolls with chrysotile structure. These features could owe to a sort of inhomogeneity in cations distribution between the particles as well as within the layer. Krasilin et al. have demonstrated [41] that Mg/Ni ratio changes in correlation with the number of layers (outer diameter) of the nanoscroll. Here we attempt to deepen the study of chemical composition of individual (Mg,Ni)3Si2O5(OH)4 multi-walled nanoscrolls using transmission electron microscopy in order to reveal a consistency in cations distribution along the phyllosilicate layer.

Results and Discussion

Energy Effect of Cation Redistribution

In the previous studies [32,40,42] we have developed an energy model of scrolling of a 1:1 phyllosilicate flat layer with chrysotile structure, which is based on the models proposed elsewhere. [36,43,44] Here we would like to pay attention to some chemical composition features that could exist in case several cations constitute the phyllosilicate octahedral sheet. First, let us consider the layer as a continuous media of two chemically bonded sheets (Figure 1) of MeO6 octahedra and SiO4 tetrahedra. These two sheets possess slightly different sizes bo and bt that result in an appearance of an internal stress in the layer. Its bending and scrolling are one of the effective relaxation paths.

Full compensation of the size difference between the sheets is achieved at the following radius of curvature: (1) where h is the layer thickness. This radius can be determined in a result of diffraction studies [46,47] or structure optimization. [48] For the case of Mg3Si2O5(OH)4 nanoscrolls these approaches yield r0,Mg value around 8.8 nm. Substitution of ions in the crystal structure, obviously, will affect bo, bt and r0 values. Figure 1 shows that the structure of the octahedral sheet is mostly equivalent to that of layered hydroxide, for example, Mg(OH)2. If the guest cation Me can form the layered hydroxide of similar structure, the r0,Me value can be recalculated on the basis of cell parameters of these hydroxides, assuming negligible bt and h change:

(2)

For the case of Ni3Si2O5(OH)4 nanoscrolls this equation yields r0,Ni = 15 nm. [32] The r0 value is important, but it is not the only factor that governs the scrolling process. To understand that it is sufficient to look at the morphology of Mg3Si2O5(OH)4 and Ni3Si2O5(OH)4 nanoscrolls: they never form single-walled nanotubes of 18 and 30 nm in diameter except very initial stages of growth. [3,49] On the contrary, they are multi-walled. This issue can be explained by the following energy model.

We consider an energy effect (J/mol) of the scrolling process consisting of three components: (3) where v is a quantity of substance; Es is a strain energy change; Σs is a surface energy change; Ua is an adhesion energy change. Each of the three energy components are differences between energy values of scrolled and flat phyllosilicate layers.

The adhesion energy differs from zero only if the number of layers n in the scroll is more than 1. The Ua value is proportional to the surface area of contact between two adjacent wraps. It can be calculated by integration over the Archimedean spiral: (4) where ua is a specific adhesion energy; L2 is the nanoscroll length; f = (h + t) / 2π is the spiral constant; t is the interlayer distance; rin is inner (starting) radius of the spiral; ϕ is an angle coordinate.

Surface energy of the scrolled state of the layer can be obtained by the analogical procedure taking into the account all the surfaces of the layer: (5) where σout, σin, σ1, and σ2 are specific surface energies of outer surface, inner surface, ends and edges of the scroll; L1,out, L1,in are lengths of coils formed by outer and inner surfaces; L1 is coil length at middle of the layer. For the flat layer these lengths are equal.

Assuming small bending, strain energy of the scroll is proportional to squared difference of curvatures: (6) where Ds is bending stiffness, and r(ϕ) is current radius of curvature. According to the general equation of the Archimedean spiral: (7) The flat layer has zero curvature, and the equation ( 6) simplifies: (8) These basic equations allow us to determine preferable size parameters -length, outer and inner diameter -of cylindrical nanoscroll of certain mass, which includes only one type of cation in the octahedral sheet (for example, Mg 2+ or Ni 2+ , see Figure 1). If the sheet contains two or more cations, all the parameters in the equation should be estimated (as a first approximation) on the basis of an interpolation procedure.

Two cations can be randomly or nonrandomly distributed in the layer. The latter means that parameters like Ds, σi, ua, and r0 will become coordinate-dependent. Further on we will concentrate on the analysis of the strain energy contribution ( 6) and ( 8) only for the case of substitution of Mg 2+ by Ni 2+ assuming negligible surface and adhesion energy change. This approximation is justified by close σi values of the corresponding oxides. [32] The flat layer, as it seems, does not get any energy preference from nonrandom distribution (redistribution) of the cations (8). Nevertheless, it could affect steadiness of the scrolling process and lead to formation of noncylindrical scrolls because of different r0 values in different places of the layer. In case of cylindrical scroll, redistribution along cylinder length L2 does not cause strain decrease (6). On the contrary, redistribution of Mg 2+ and Ni 2+ along the length L1 could cause minimization of the squared difference under the integral: (9) To achieve this, the r0(ϕ) value should closely follow current radius of curvature r(ϕ). Because r(ϕ) is a linear function of ϕ (7), the form of r0(ϕ) should be close to straight line as well.

We try to reveal this dependence by electron microscopy study of Ni concentration changes in single (Mg,Ni)3Si2O5(OH)4 nanoscrolls.

PXRD and SEM/EDS

Figure 2 shows PXRD patterns of initial composition and its hydrothermal treatment product. Co-precipitation reaction in presence of amorphous SiO2 yields poorly crystalline phase with layered structure. Its hydrothermal treatment leads to formation of a phase with a set of reflections corresponding to those of #10-381 Mg3Si2O5(OH)4 or #49-1859 Ni3Si2O5(OH)4. An isostructural compound which would include both Mg 2+ and Ni 2+ ions is absent in the database. Hydrothermal treatment product possesses smaller interlayer period in comparison with that of the initial composition, probably because of removal of excessive amount of water molecules between the layers during crystal growth. PXRD does not reveal any other crystalline phases in the samples.

Table 1 demonstrates element composition of the hydrothermal treatment product measured by conventional SEM/EDS. Average Ni concentration CNi in the sample is a bit higher than the estimated one (50 at.%), as a consequence, the ratio R is higher than 1.5. Nevertheless, measured values coincide with the estimated ones within error.

TEM Study

Figures 3a-e represent different nanoscroll morphologies that are formed under hydrothermal conditions.

First group consists of aggregates of small scrolls up to 30 nm in diameter and 100-200 nm in length, despite their tubular form may not be always recognizable. Second group includes separate cylindrical and conical scrolls with outer diameter in the 30-70 nm range and length reaches up to several microns. It seems that a kind of attachment mechanism [50] can play an important role in the formation of these particles. Figures 3b-e show some of the particles which probably exhibit this type of growth. Also, there are scrolls whose outer diameters overcome 100 nm. Often, they have telescopic morphology (Figure 3c). Their outer diameter values are not usual for the case of pure Mg-based and Ni-based phyllosilicate nanoscrolls, [32] and it can be related with simultaneous Mg 2+ and Ni 2+ presence in the system. Thill et al. [36] have reported diameter increase for the case of Ge-doped imogolite as well. The inner diameter of scrolls falls within the range 6-12 nm range. The inner diameter decreases with increase of the outer diameter value in consistency with the previous studies. [ 41] We measured an interlayer period by analyzing a grey level profile of the TEM image (Figure 4). No correlation between the period and the distance from centre was found. Weight average value of the period was 0.72 nm, and it fluctuated in the range 0.68 to 0.78 nm. Mean d-spacing of the (002) X-ray peak was 0.73 nm.

For the case of Mg3Si2O5(OH)4 nanotubes Foresti et al. [1] have reported 0.734 nm d-spacing, and White et al. [51] have reported around 0.7 nm for Ni3Si2O5(OH)4 nanotubes. Alvarez-Ramírez et al. [52] also have noted that the d-spacing of Ni3Si2O5(OH)4 nanotubes varies in the range 0.75 to 0.85 nm depending on the number of layers. The observed variation can be related with different degree of layers hydration, which should increase d-spacing value. Taking into the account this feature, it is hard to reveal a probable effect of d-spacing change due to Mg 2+ and Ni 2+ redistribution, which should occur because of their different ionic radii.

STEM/EDS Study

As a first attempt we determined Ni concentration in one nanoscroll with step variation of outer diameter (Figure 5). Except two areas, change of the CNi value stands in correlation with the outer diameter, although the its spread is only 3 at.%. Note all CNi values in each of the areas is lower than that measured by conventional SEM/EDS (Table 1). On the other hand, we should not omit some systematic shift between the values obtained by the two techniques. We have showed [21] that Ni concentration in small particles is almost equal to the initial concentration set at co-precipitation stage. For bigger scrolls there was a shift of concentration which depended on their number of layers, or wall thickness. Here, we can observe this feature even within the single scroll.

Figure 6 shows the results of Ni concentration measurement by a line scan procedure. One can see an almost double increase of apparent Ni concentration with distancing from the nanoscroll central axis. The value drops down beyond the nanoscroll outer radius. Mean value of apparent concentration is 53 at.%. The reconstruction procedure (see Methods section) demonstrates that first layers of the nanoscroll contain around 10 at.% of Ni. The concentration grows linearly at subsequent layers (distance from centre). Line scan EDS mode brings considerable damage to the nanoscroll, so it is more reliable to collect and process EDS maps. Figure 7 shows the results of Ni concentration profile measurement carried out by this approach. Average value of apparent concentration is 39 at.%. The CNi value, as it was in the previous case, decreases with decrease in the distance from the nanoscroll centre, but here a behavior of the reconstructed CNi profile is more complex. This may be related to specific conditions of the nanoscroll growth or with close presence of other particles, which could distort the EDS results.

The last case showed in Figure 8 is a result of EDS mapping procedure carried out at higher magnification. We divided the nanoscroll map into series of 2 nm slices parallel to the nanoscroll axis (see Figure SI5 for details). Average apparent Ni concentration is 44 at.%. The reconstructed concentration profile shows that, again, the CNi value gradually increases in the direction from inner layers to outer ones.

Redistribution Mechanism

We have demonstrated several cases of Ni concentration change with change of the layer curvature, which is equivalent to increase in distance from the nanoscroll centre. It can be revealed by different modes of STEM/EDS analysis, including measurement of average concentration in the "tube in tube" particle (Figure 5), line scan perpendicular to the nanoscroll (Figure 6), and processing of EDS maps (Figure 7 and8).

We suppose that the effect of Mg 2+ and Ni 2+ ions redistribution can take place during the nanoscroll growth (Figure 9a). The scroll that has been just formed accounts only several layers in its structure with a radius of curvature close to r0 for given Ni concentration (around 50 at.%). Under hydrothermal conditions nanoscroll begins to grow by virtue of species sedimentation on the outer and on the inner surface, and by stacking of adjacent scrolls (Figure 9a). At this stage, the redistribution can occur: layers with r(ϕ) < r0,Mg are more favorable for Mg-rich species, and layers with r(ϕ) > r0,Ni are more suitable for Ni-rich species. Layers with r0,Mg < r(ϕ) < r0,Ni are situated in the intermediate zone.

Assuming r0(ϕ) can be converted to CNi(ϕ) on the basis of the additivity rule, maximum Ni concentration change with increase in distance from centre should be from 0 at.% at 8.8 nm up to 100 at.% at 15 nm (dashed line on Figure 9b). 678) deviate from the theoretically proposed line. First and foremost, real nanoscrolls have inner and outer radii, different from the ideal situation (r0,Mg as the inner radius, and r0,Ni as the outer one). As a consequence, Ni concentration profile can spread between the actual radii, and the slope decreases. Second reason of slope decrease is that the energy model does not take into account kinetic hindrances which could accompany the nanoscroll growth process. Third, other energy components (see equations ( 3)-( 5)) can play a part in the redistribution process sophisticating the CNi(ϕ) behavior. Besides possible contribution of surface energy (see equation ( 5)) a kind of configuration entropy could act against cations redistribution.

Solid lines obtained by approximation of reconstructed CNi values (see Figures

Conclusions

To summarize, here we demonstrate both theoretical and experimental basis for (Mg1x,Nix)3Si2O5(OH)4 phyllosilicate nanoscroll to redistribute Mg 2+ and Ni 2+ cations along the spiral in order to minimize an excessive internal stress. The stress originates from size difference between octahedral and tetrahedral sheets of the layer. In this particular example, Mg 2+ having larger ionic radius (720 pm) concentrates in the inner layers of the scroll, whereas Ni 2+ (690 pm) prefers the outer layers of smaller curvature. This redistribution can occur during the nanoscroll recrystallization process under hydrothermal conditions by selective sedimentation of Ni-rich and Mg-rich species on the outer and on the inner surfaces of the scroll, correspondingly. According to the energy model, concentration change should be close to linear function of the radius of curvature (of angle). Despite the need of further theoretical development, conclusions driven here should be valid also for a more general case of cations substitutions in octahedral and/or tetrahedral sheets of curved hydrosilicates like chrysotile, halloysite, and imogolite.

Experimental Section (Mg1-x,Nix)3Si2O5(OH)4 Nanoscroll Synthesis

Hydrosilicate nanoscrolls with estimated x = 0.5 and (Mg + Ni) / Si = 1.5 molar ratio were synthesized by a hydrothermal technique. On the first step, initial composition was prepared via reverse coprecipitation of Mg(OH)2 and Ni(OH)2 from 400 ml of 1 M MgCl2 and NiCl2 water solutions in the presence of amorphous SiO2 (aerosil A-300) by adding 400 ml of 2 M NaOH water solution. The precipitate was washed several times with distilled water until neutral pH and negative results of the reaction: (10) Then the precipitate was dried at 90°C in air and ground in an agate mortar. Approximately 0.6 g of the powder was put in a stainless-steel vessel of total 400 ml capacity, and 250 ml of 0.5 M NaOH water solution was added as a hydrothermal medium. Hydrothermal treatment was carried out at 350°C and 14 MPa during 12 hours at constant stirring. After the treatment, the product was washed several times with distilled water and then dried at 90°C on air.

Sample Characterization

Phase composition of samples after the co-precipitation and hydrothermal treatment was studied by the X-ray powder diffractometer (PXRD) Rigaku Smartlab SE (CuKα radiation λ= 0.15406 nm). A PDF-2 database was used for peak indexing. Scanning electron microscope (SEM) FEI Quanta 200 equipped with EDAX energy dispersive X-ray spectrometer (EDS) was used for general morphology and chemical composition analysis.

Nanoscroll structure and chemical composition features were investigated by a transmission electron microscope JEOL -ARM 200F Cold FEG operating at 200kV and equipped with a spherical aberration (Cs) probe corrector (point resolution 0.19 nm in TEM mode and 0.078 nm in STEM mode) together with Gatan Digital Micrograph and JEOL Analysis Station softwares.

Chemical Composition Measurements by STEM/EDS

We obtained Ni concentration profile by performing an EDS line scan perpendicular to the nanoscroll axis, and by collecting and processing of EDS intensity maps. First approach is simpler but is more invasive for the nanoscroll than the second one. Phyllosilicate nanoscroll got burn through at each point of the line scan. EDS mapping technique also caused considerable damage to initial structure of the scroll after certain number of frames being collected, so we limited the number of frames used for further processing. EDS intensity maps were processed involving two approaches. First, intensity map was converted to EDS quantity map using a built-in JEOL algorithm. Then Ni concentration (at.%) normalized on sum of Mg and Ni was extracted from the desired profile perpendicular to the nanoscroll axis. In the other case, EDS intensity maps were divided into series of rectangular areas with long edges parallel to the nanoscroll axis (see Figure SI5), and Ni concentration was calculated by processing the average X-ray spectra of each area. The data obtained by these two methods was then represented versus distance from the nanoscroll central axis (distance from centre). The data points situated on equal distance to left and to the right from the nanoscroll central axis were averaged.

The Ni concentration profile obtained by this way should be further corrected, because X-ray signal comes from different layers of the nanoscroll (Figure SI1). It is assumed that apparent Ni concentration C * at k layer situated at distance from centre Rk is a weighted average of true concentrations Ci in current and all the outer layers of effective thickness wi on the way of the electron beam: (11) Effective thickness of k layer has a maximum: (12) which then drops to a constant thickness equal to 2p. Here R is the outer radius of the scroll. The value of thickness p does not have to be equal to the real phyllosilicate thickness h. It is determined by current magnification or number of points in line scan mode.

Assuming apparent concentration at the first (outer) layer is equal to true concentration it is possible to recalculate, going to the centre step by step (Figure SI1), Ck (k = 2..n) values using the equation inspired by the Abel inversion procedure: (13) where Ci are true Ni concentrations of the layers which are outer in relation to the current one. This method of reconstruction has a crucial drawback related with an accumulation of error while going from the outer layers to the inner ones:

Figure 1 .

 1 Figure 1. Crystal structure of Ni-doped chrysotile based the data of Foresti et al.[1] Visualization was made using VESTA 3 software.[45] Bending of the layer is caused by size difference between octahedral and tetrahedral sheets. Preferable radius of curvature is influenced by ionic radii of constituting cations.

Figure 2 .

 2 Figure 2. PXRD patterns of initial composition (1) and its hydrothermal treatment product (2). Vertical lines represent chrysotile phase (card #10-381, PDF-2 database). Inset shows first reflection (002) of hydrosilicate phase with a maximum at 0.73 nm.

Figure 3 .

 3 Figure 3. a) TEM image of (Mg,Ni)3Si2O5(OH)4 nanoscrolls. b-e) Magnified fragments of the TEM image. The arrows denote particles, which probably have grown involving an attachment mechanism.

Figure 4 .

 4 Figure 4. a) TEM image of the nanoscroll wall. Area of inspection is shown by rectangle. b) A grey value profile of the area averaged parallel to the layers. Dots denote change of the interlayer period with distancing from the nanoscroll centre. c) Distribution of the interlayer period with a maximum at 0.72 nm.

Figure 5 .

 5 Figure 5. a) STEM image of telescopic particle and its EDS mapping (composed of two frames). The numbers denote areas, at which Ni concentration was averaged. b) Ni concentration at different areas of the nanoscroll in comparison with its outer diameter. The inset shows magnified STEM image of the area #4.

Figure 6 .

 6 Figure 6. a) Change of apparent (solid circles) and reconstructed (hollow squares) Ni concentration with increase of distance from the nanoscroll centre. Inset shows STEM image of the nanoscroll and position of the line scan (large STEM image is available on Figure SI2). Mean value of apparent concentration is 53 at.% b) X-ray spectra in points situated near the nanoscroll (1), on the outer surface (2), and near the inner channel (3). Insets show MgK and NiK lines.

Figure 7 .

 7 Figure 7. a) STEM image of the nanoscroll. Apparent Ni concentration was calculated along the line perpendicular to the nanoscroll axis, and spectra were averaged along the wide edge of selected rectangular area (large scale image is shown on Figure SI3). Mean value of apparent concentration is 39 at.% b) Change of apparent (solid circles) and reconstructed (hollow squares) Ni concentration with increase of distance from the nanoscroll centre.

Figure 8 .

 8 Figure 8. a) STEM image and EDS maps of the nanoscroll (for details on how spectra were obtained please see Figure SI5). Average apparent Ni concentration is 44 at.% b) Change of apparent (solid circles) and reconstructed (hollow squares) Ni concentration with increase of distance from the nanoscroll centre.

Figure 9 .

 9 Figure 9. a) Possible mechanism of Mg2+ and Ni2+ cations redistribution as a result of the nanoscroll growth process. b) Results of linear regression of the data represented on Figure 6a (line 1), Figure 7b (line 2), and Figure 8b (line 3) in comparison with maximal gradient (dashed line), predicted by the equation (9).

Acknowledgements

The research was supported by the Russian Science Foundation grant 16-13-10252. The RXRD studies were performed using the equipment of the Engineering Centre of Saint-Petersburg State Technological Institute (Technical University). AK is grateful to Programme Metchnikov for mobility support by the Embassy of France in Russia and Campus France. AN, TB and JG are grateful to Programme Hubert Curien -Kolmogorov for mobility support through NSPH project. AN and TB benefited from the support of the project CEENEMA ANR-15-CE05-0005-01 of the French National Research Agency (ANR).


Camila Pereira-Montecinos

Daniela Toro-Ascuy

Sebastián Cecilia Rojas-Fuentes

Sebastián Riquelme-Barrios

Bárbara Rojas-Araya

Francisco García-De-Gracia

Paulina Aguilera-Cortés

Catarina Ananías-Sáez

Grégoire De Bisschop

Jonás Chaniderman

; Grégoire De Bisschop

Mónica L Acevedo

Bruno Sargueil

Fernando Valiente-Echeverría

Ricardo Soto- Rifo
email: rsotorifo@uchile.cl

Ricardo Soto

An epitranscriptomic switch at the 5´-UTR controls genome selection during HIV-1 genomic RNA packaging

Keywords: N 6 -methyladenosine, HIV-1 genomic RNA, RNA Packaging, 5´-UTR, Gag

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ABSTRACT

During retroviral replication, the full-length RNA serves both as mRNA and genomic RNA (gRNA). While the simple retrovirus MLV segregates its full-length RNA into two functional populations, the HIV-1 full-length RNA was proposed to exist as a single population used indistinctly for protein synthesis or packaging. However, the mechanisms by which the HIV-1 Gag protein selects the two RNA molecules that will be packaged into nascent virions remain poorly understood. Here, we demonstrate that HIV-1 full-length RNA packaging is regulated through an epitranscriptomic switch requiring demethylation of two conserved adenosine residues present within the 5´-UTR. As such, while m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A deposition by METTL3/METTL14 onto the full-length RNA was associated with increased Gag synthesis and reduced packaging, FTO-mediated demethylation was required for the incorporation of the full-length RNA into viral particles. Interestingly, HIV-1 Gag associates with the RNA demethylase FTO in the nucleus and drives full-length RNA demethylation. Finally, the specific inhibition of the FTO RNA demethylase activity suppressed HIV-1 full-length RNA packaging. Together, our data propose a novel epitranscriptomic mechanism allowing the selection of the fulllength RNA molecules that will be used as viral genomes.

INTRODUCTION

Retroviral full-length RNA plays two key functions in the cytoplasm of infected cells. First, it is used as the mRNA template for the synthesis of Gag and Gag-Pol precursors and, second, it serves as the genomic RNA (gRNA) packaged into newly produced viral particles [START_REF] Karn | Transcriptional and posttranscriptional regulation of HIV-1 gene expression[END_REF][START_REF] Butsch | Destiny of unspliced retroviral RNA: ribosome and/or virion?[END_REF][START_REF] Mailler | The Life-Cycle of the HIV-1 Gag-RNA Complex[END_REF] . In contrast to the simple retrovirus murine leukemia virus (MLV), which was shown to segregate its full-length RNA into two functionally different populations serving as template for translation (mRNA) or packaging (gRNA), the HIV-1 and HIV-2 full-length RNA were proposed to exist as a single population acting indistinctly as mRNA and gRNA [START_REF] Levin | Deficiency of 60 to 70S RNA in murine leukemia virus particles assembled in cells treated with actinomycin D[END_REF][START_REF] Levin | Synthesis of murine leukemia virus proteins associated with virions assembled in actinomycin D-treated cells: evidence for persistence of viral messenger RNA[END_REF][START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] . However and despite several years of efforts, there is still an important gap in our knowledge regarding the molecular mechanisms behind the selection of the full-length RNA molecules that will be incorporated into assembling viral particles.

The 5´-untranslated region (5´-UTR) present within the HIV-1 full-length RNA is the most conserved region of the viral genome and contains several high order structural motifs involved in different steps of the viral replication cycle from transcription, reverse transcription, splicing, translation to dimerization and packaging [START_REF] Mailler | The Life-Cycle of the HIV-1 Gag-RNA Complex[END_REF][START_REF] Berkhout | Structure and function of the human immunodeficiency virus leader RNA[END_REF][START_REF] Kuzembayeva | Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles[END_REF] . Since the full-length RNA serves both as mRNA and gRNA, translation and packaging are expected to be mutually exclusive events [START_REF] Butsch | Destiny of unspliced retroviral RNA: ribosome and/or virion?[END_REF] . The Gag protein recognizes cis-acting RNA elements present at the 5´-UTR and the beginning of the Gag coding region and drives the selective incorporation of two copies of the gRNA into assembling viral particles. Indeed, there is accumulating evidence showing that dimerization and packaging of the HIV-1 full-length RNA are two tightly interconnected processes dependent on the Gag protein [START_REF] Laughrea | Mutations in the kissing-loop hairpin of human immunodeficiency virus type 1 reduce viral infectivity as well as genomic RNA packaging and dimerization[END_REF][START_REF] Chen | HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein[END_REF][START_REF] Clever | Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation[END_REF] . Structural and mutational analyses proposed that a conformational switch within the 5´-UTR regulates the transition from translation to dimerization and packaging in vitro [START_REF] Huthoff | Two alternating structures of the HIV-1 leader RNA[END_REF][START_REF] Berkhout | In vitro evidence that the untranslated leader of the HIV-1 genome is an RNA checkpoint that regulates multiple functions through conformational changes[END_REF][START_REF] Abbink | A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon[END_REF][START_REF] Keane | RNA structure. Structure of the HIV-1 RNA packaging signal[END_REF] . In such models, the 5´-UTR alternates in conformations that occlude the dimerization initiation signal (DIS) or the Gag start codon thus, favoring translation or dimerization and packaging, respectively [START_REF] Mailler | The Life-Cycle of the HIV-1 Gag-RNA Complex[END_REF][START_REF] Kuzembayeva | Life of psi: how full-length HIV-1 RNAs become packaged genomes in the viral particles[END_REF] . However, chemical probing performed in cells and purified viral particles showed that a single structure, in which DIS is accessible for dimerization and packaging, predominates in these biological states [START_REF] Paillart | First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions[END_REF][START_REF] Wilkinson | High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states[END_REF] . Moreover, the packaging prone structure does not interfere with full-length RNA translation suggesting that other factors rather than structural rearrangements are involved in the regulation of the cytoplasmic sorting of the HIV-1 full-length RNA [START_REF] Abbink | The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation[END_REF][START_REF] Boeras | The basal translation rate of authentic HIV-1 RNA is regulated by 5'UTR nt-pairings at junction of R and U5[END_REF] .

It was recently reported that the HIV-1 full-length RNA contains N [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] -methyladenosine (m 6 A) residues located at the 5´-and the 3´-UTR as well as at internal positions such as the Rev response element (RRE) [START_REF] Lichinchi | Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells[END_REF][START_REF] Kennedy | Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression[END_REF][START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF] . Methylation of adenosines at the RRE and the 3´-UTR was shown to promote Gag synthesis by favoring nuclear export and/or the intracellular accumulation of viral transcripts at late stages of the replication cycle [START_REF] Lichinchi | Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells[END_REF][START_REF] Kennedy | Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression[END_REF][START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF][START_REF] Lu | N(6)-methyladenosine-binding proteins suppress HIV-1 infectivity and viral production[END_REF] . However, it was also reported that the presence of m 6 A could also induce the degradation of the incoming gRNA early during infection [START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF][START_REF] Lu | N(6)-methyladenosine-binding proteins suppress HIV-1 infectivity and viral production[END_REF] . These controversial data prompted us to study whether m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A could serve as a mark that defines the functions of the HIV-1 full-length RNA as template for translation or packaging during viral replication.

Here, we show that methylation of two adenosine residues within the 5´-UTR by the METTL3/METLL14 complex inhibits full-length RNA packaging. m 6 A-seq analysis revealed that the full-length RNA present in purified viral particles lacks m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A at the 5´-UTR suggesting the existence of two populations that differ in their m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A patterns. Further bioinformatic analyses identified two highly conserved nucleotides A 198 and A 242 within the 5´-UTR as the key residues involved in the m 6 A-mediated regulation of gRNA packaging. We also observed that the full-length RNA is a substrate for the RNA demethylase FTO, which together with Gag drives RNA demethylation to promote packaging. Finally, the pharmacological targeting of FTO activity resulted in impaired full-length RNA metabolism and a strong inhibition of packaging. Together, our data reveal a novel mechanism by which Gag selects the molecules of gRNA that will be used for packaging, which is regulated by an epitranscriptomic switch within the 5´-UTR.

RESULTS

The presence of m 6 A alters the in vitro folding and dimerization of the HIV-1 full-length

RNA 5´-UTR

Since previous studies suggesting that a conformational switch of the 5´-UTR could regulate the ability of the HIV-1 full-length RNA to function as mRNA or gRNA have not considered the presence of RNA modifications such as m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A, we first sought to determine the impact of adenosine methylation on the folding of the 5´-UTR. As a first approach to study the impact of m 6 A on RNA structure, we generated an in vitro-transcribed 5'-UTR containing unmodified adenosines or a 5´-UTR in which all adenosines were replaced by m 6 A. Both m 6 A-5'-UTR and A-5'-UTR were submitted to 1M7 SHAPE analysis in parallel as described in Methods. Reactivity towards SHAPE reagents reveals the ribose flexibility, and as a consequence, the pairing status of each nucleotide. As such, higher reactivity of a given nucleotide means a higher probability to be in a single strand conformation. Comparative analysis of the SHAPE reactivity profiles indicates that the presence of m 6 A significantly alters the folding of the 5´-UTR (Fig. 1a and Supplementary Fig. 1a). The first interesting observation from our SHAPE data is that we do not only observe a reactivity modification for As or Us. Nucleotides in white are of equivalent reactivity. Sites where the reactivity could not be determined are depicted in grey. The histogram shows the reactivity difference between 0% m 6 A 5'-UTR and 100% m 6 A 5'-UTR (lower). Blue and red bars highlight nucleotides with a significantly lower or higher reactivity in the 100% m6A RNA than in the 0% m6A RNA (pvalue < 0.05 and reactivity difference > 0.4), respectively. Significative differences that are below the threshold of 0. Moreover, although all adenosines were methylated in the m 6 A-5'-UTR RNA only local reactivity changes, often clustered, were observed suggesting that the presence of m 6 A influences the folding of specific domains or motifs within the 5´-UTR. The presence of m 6 A is predicted and has been observed to destabilize A-U pairings embedded in helical regions. This is mostly in agreement with the destabilizations (increase in reactivity) that are often observed for As, Us or in nucleotides in close proximity with proposed A-U base pairs. In particular, this could explain the reactivity enhancement within the poly-A stem, which is likely to be globally destabilized by m 6 A. However, none of the other nucleotides for which we observe a reactivity increase is involved or are close to A-U pairings and most of them are not even predicted to be base paired in most of the published models. In contrast, m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A has been shown to stabilize A-U pairings when preceded in 5' by a bulged nucleotide. This does not offer a rationale for any of the reactivity drop we observe which are mostly predicted to be in single strand regions thus, suggesting that m 6 A modulates a higher order structure and/or tertiary pairings yet to be identified. This prompted us to monitor dimerization of the 5'-UTR and m 6 A-5´-UTR in vitro. We observed that the presence of m 6 A reduces but not abolish the efficiency of dimerization (Fig. 1b). The structure of the HIV-1 full-length RNA dimer is still poorly defined and a matter of debate, thus the dimerization deficiency observed does not provide a straightforward explanation for all the alterations of the SHAPE profile but could reveal unknown rearrangements.

Together, these data indicate that the presence of m 6 A may play an important role in the folding and dimerization of the 5´-UTR and prompted us to study this feature in a cellular context.

The presence of m 6 A within the full-length RNA favors Gag synthesis but interferes with packaging

In order to study the role of m 6 A on the cytoplasmic fate of the full-length RNA during viral replication, we first determined the effects of METTL3/14 overexpression on Gag and fulllength RNA levels obtained from cell extracts and purified viral particles (see scheme in Supplementary Fig. 2a). m 6 A-RIP analysis from METTL3/14 overexpressing cells showed an increase in the m 6 A/A ratio of the full-length RNA compared to the control indicating that the viral transcript is hypermethylated under these experimental conditions (Supplementary Fig. 2b). Consistent with the positive role of m 6 A on Gag synthesis previously described [START_REF] Lichinchi | Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells[END_REF][START_REF] Kennedy | Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression[END_REF][START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF] , we observed that increased methylation of the full-length RNA by METTL3/14 overexpression results in increased levels of Gag and its processing products with minor effects on the intracellular levels of the full-length RNA (Fig. 2a). Quantification of viral particles produced from the same cells revealed a slight increase in Gag levels (as judged by anti-CAp24 ELISA) from METTL3/14 overexpressing cells, which could be attributed to the increased Gag synthesis observed (Fig. 2b). Then, we quantified the full-length RNA from equal amounts of viral particles and observed that viral particles produced under METTL3/14 overexpression contain around 3-fold less packaged gRNA indicating that hypermethylation of the full-length RNA impedes its packaging into nascent particles (Fig. 2c). We were not able to observe a similar effect of murine METTL3/14 overexpression on the simple retrovirus MLV, which was shown two segregate their full-length RNA into two specialized populations for translation and packaging further suggesting that MLV and HIV-1 might evolved diverse mechanisms for the cytoplasmic sorting of their full-length RNA (Supplementary Fig. 2c).

Since results presented above indicate that m 6 A deposition by METTL3/14 affects the cytoplasmic fate of the full-length RNA, we wanted to investigate where within the cell the m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A writer complex modifies the viral RNA. For this, we analyzed the interaction between the full-length RNA and METTL3 by in situ hybridization coupled to the proximity ligation assay (ISH-PLA) [START_REF] Toro-Ascuy | A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA[END_REF] . Confocal microscopy analyses revealed a predominant interaction within the nucleus, which suggests that the full-length RNA must be methylated in the nucleus and reach the cytoplasm in a methylated form (Fig. 2d and Supplementary Fig. 2d).

Together, these data suggest that nuclear methylation of the HIV-1 full-length RNA by METTL3/14 favors its use as mRNA for Gag synthesis but interferes with its incorporation into viral particles.

Methylation of A 198 and A 242 within the 5´-UTR interferes with HIV-1 full-length RNA packaging

From data presented above, it seems that the presence of m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A interferes with the function of the HIV-1 full-length RNA as gRNA. Thus, to gain further insights into this regulation, we employed the m 6 A-seq strategy to determine the m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A patterns of the intracellular and viral particle-associated HIV-1 full-length RNA. In agreement with previous data reported for the NL4.3 and LAI.2 strains in T-lymphocytes and HEK293T cells [START_REF] Lichinchi | Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells[END_REF][START_REF] Kennedy | Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression[END_REF][START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF] , we identified m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A peaks mainly at the 5´-UTR and a cluster of peaks at the 3´ end of the intracellular full-length RNA (Fig. 3a, see intracellular full-length RNA). Interestingly, we observed that the full-length RNA from purified viral particles maintains the m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A peak at the 3´-UTR but lacks the m 6 A peak at the 5´-UTR. This observation together with data from Fig. 2c suggests that the presence of m 6 A at the 5´-UTR interferes with the incorporation of the full-length RNA into viral particles (Fig. 3a, see packaged full-length RNA). Of note, this difference in the methylation patterns between intracellular and packaged RNA was not observed in the host 7SL RNA, which is also packaged at high levels into HIV-1 particles [START_REF] Onafuwa-Nuga | 7SL RNA, but not the 54-kd signal recognition particle protein, is an abundant component of both infectious HIV-1 and minimal virus-like particles[END_REF] , indicating a very specific effect of m 6 A on full-length RNA packaging (Supplementary Fig. 3a). These data strongly suggest that full-length RNA molecules lacking m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A at the 5´-UTR are primarily selected by Gag as gRNA to be incorporated into viral particles.

A bioinformatic prediction of the potentially methylated residues within the 5-UTR of the NL4.3 strain identified A 198 and A 242 in a very favorable methylation context (Supplementary Fig. 3b). Both residues are contained within the m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A peak we have identified within the 5´-UTR of the intracellular full-length RNA (Supplementary Fig. 3c) and were also identified in previous m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A-seq data obtained from T-lymphocytes and HEK293T cells [START_REF] Lichinchi | Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells[END_REF][START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF] . Thus, we deleted A 198 , A 242 or both from the NL4.3 provirus in order to determine the role of these adenosine residues on the m 6 A-mediated regulation of full-length RNA packaging. We observed that ΔA 198 and ΔA 242 single mutant proviruses were slightly resistant to the effects of METTL3/14 overexpression on full-length RNA packaging (Supplementary Fig. 3d).

However, the ΔA 198 /ΔA 242 double mutant provirus was refractory to the positive effects of METTL3/14 overexpression on Gag synthesis and the negative effects on full-length RNA packaging observed with the wild type provirus indicating that A 198 and A 242 are key residues involved in this m 6 A-mediated regulation (Fig. 3b and Supplementary Fig. 3e). Next, we sought to investigate the role of A 198 and A 242 on HIV-1 full-length RNA metabolism. A comparison between wild type and the ΔA 198 /A 242 provirus showed that the double mutant virus accumulates more intracellular Gag but releases significantly less viral particles (Fig. 3c and Supplementary Fig. 3f). Moreover, quantification of the full-length RNA from equal amounts of viral particles revealed a defect in packaging in the ΔA 198 /A 242 double mutant provirus thus, confirming the critical relevance of these two adenosine residues for the incorporation of the HIV-1 full-length RNA into viral particles (Fig. 3c). An analysis of 890 sequences from the HIV database (www.hiv.lanl.gov) indicate that A 198 and A 242 are highly conserved within the 5´-UTR of isolates suggesting that this epitranscriptomic regulation must be a common feature of different HIV-1 subtypes including the highly prevalent subtypes C and B as well as circulating recombinant forms (Fig. 3d and Supplementary Fig.

Demethylation by a Gag-FTO complex favors HIV-1 full-length RNA packaging

Then, we sought to determine whether this m 6 A-mediated regulation of full-length RNA packaging was a dynamic process. This was important considering that the reversible nature of adenosine methylation in cellular mRNA has been challenged [START_REF] Darnell | pre-mRNA processing includes N6 methylation of adenosine residues that are retained in mRNA exons and the fallacy of "RNA epigenetics[END_REF] . For this, we overexpressed the RNA demethylase FTO and the analysis of the m 6 A/A ratio of the fulllength RNA in control and FTO overexpressing cells revealed that the viral RNA is indeed a substrate for this m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A eraser (Supplementary Fig. 4a). Consistent with a positive role of m 6 A on Gag synthesis, we observed that FTO-induced demethylation of the full-length RNA results in a reduction of Gag levels despite a slight increase in intracellular full-length RNA levels (Fig. 4a). We also observed minimal changes in the CAp24 levels from purified viral particles produced under RNA demethylation conditions (Fig. 4b). However and in agreement with a negative role of m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A on full-length RNA packaging, we observed that viral particles produced from FTO overexpressing cells contain around 3-fold more packaged gRNA compared to the control (Fig. 4c). It should be mentioned that we were not able to observe similar results with the RNA demethylase ALKBH5 suggesting that full-length RNA demethylation by FTO is important for packaging (Supplementary Fig. 4b).

Considering that m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A demethylation favors full-length RNA packaging, we wanted to know where within the cell the viral RNA became demethylated by FTO. For this, we analyzed the interaction of the between the full-length RNA and FTO in cells by ISH-PLA but despite several attempts, we were not able to detect a direct interaction regardless all the components were correctly expressed within the cells (Supplementary Fig. 4c). This observation suggests that either there is no a massive interaction between the full-length RNA and FTO or that such interactions occurs very transiently (or at very low rates) being below the detection limit of our ISH-PLA strategy.

The lack of a detectable interaction between the full-length RNA and FTO prompted us to investigate whether Gag could interact with FTO and drive full-length RNA demethylation. In order to test this possibility, we employed the proximity ligation assay (PLA) and observed that Gag and FTO indeed form complexes in cells (Fig. 4d). Interestingly, quantification of the dots per cell localizing with the nuclear staining as well as 3D reconstitutions of representative images indicate that Gag and FTO mostly associates within the nucleus (Figs. 4e and4f). Indeed, we observed that FTO overexpression increases the nuclear localization of Gag (Supplementary Fig. 4d). extracts were used to perform RNA extraction followed by an immunoprecipitation using an anti-m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A antibody (m 6 A-RIP as described in Methods). The full-length RNA from the input ("A" fraction) and from the immunoprecipitated material ("m 6 A" fraction) was quantified by RT-qPCR. The m 6 A/A ratio was normalized to pNL4.3 wild type (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (*P<0.05, t-test).

From these results, it was tempting to speculate that the full-length RNA is methylated within the nucleus by METLL3/14 and Gag interacts with FTO in the nucleus to drive demethylation of the full-length RNA molecules that will be incorporated into assembling viral particles.

Thus, we analyzed the m 6 A content of the full-length RNA in the presence or absence of Gag by using the wild type NL4.3 provirus and a mutant provirus containing premature stops codons that abolish Gag synthesis (GagStop provirus). Compared to the wild type full-length RNA, the level of m 6 A increases when Gag is absent and is restored or even decreased when the Gag protein was expressed in trans indicating that Gag regulates the methylation status of the full-length RNA (Fig. 4g).

Together, these results strongly indicate that the FTO-mediated demethylation is required for full-length RNA packaging in a process supported by the Gag precursor.

Inhibition of FTO demethylase activity impacts full-length RNA metabolism and blocks packaging

We finally sought to determine whether this epitranscriptomic regulation of the HIV-1 fulllength RNA packaging was a potential therapeutic target for pharmacological intervention.

For this, we took advantage of the ester form of meclofenamic acid (MA2), which was shown to specifically interfere with FTO-mediated m 6 A demethylation [START_REF] Huang | Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5[END_REF] . Therefore, we analyzed Gag and the full-length RNA in cells treated with DMSO (as a control) or MA2 and observed a reduction in Gag synthesis and the intracellular levels of the full-length RNA indicating that FTO-mediated demethylation is required for proper metabolism of the full-length RNA within the cell (Fig. 5a). Consistent with a perturbed intracellular full-length RNA metabolism, we observed a decrease in the viral particles released from MA2-treated cells (Fig. 5b).

Strikingly, quantification of the packaged full-length RNA from equal amounts of viral particles indicates that inhibition of FTO activity by MA2 almost abolished packaging (Fig. 5c). Taking together, these results confirm that FTO-mediated demethylation is critical for HIV-1 full-length RNA packaging and this process is a potential target for the design of novel antiretroviral drugs.

DISCUSSION

Assembly of human immunodeficiency virus type-1 particles is a highly regulated process in which the major structural polyprotein Gag together with other viral and cellular components are recruited to the plasma membrane for the release of the viral progeny. The assembly process occurs in multiple steps driven by the different functional domains that compose the Gag precursor. As such, while the nucleopcapsid (NC) domain specifically recruits two copies of the full-length RNA, the matrix (MA) domain allows targeting of the complex to specific plasma membrane micro-domains and the capsid (CA) domain drives Gag multimerization at such sites. Packaging of two copies of full-length RNA by the NC domain of Gag is highly specific and occurs selectively over thousands of cellular and viral RNA species. This selectivity was proposed to be possible by the presence of cis-acting RNA signatures spanning the 5´-UTR and the beginning of the Gag coding region. However, the full-length RNA also serves as mRNA for the synthesis of Gag and Gag-Pol precursors and thus, translation and packaging are expected to be two mutually exclusive processes. Although the adoption of a branched multiple hairpin (BMH) conformation of the 5´-UTR was initially proposed to favor dimerization and packaging over translation [START_REF] Huthoff | Two alternating structures of the HIV-1 leader RNA[END_REF][START_REF] Berkhout | In vitro evidence that the untranslated leader of the HIV-1 genome is an RNA checkpoint that regulates multiple functions through conformational changes[END_REF][START_REF] Abbink | A novel long distance base-pairing interaction in human immunodeficiency virus type 1 RNA occludes the Gag start codon[END_REF] , it was later demonstrated that translation of the full-length RNA is under positive selection and thus, not regulated by a conformational switch of the 5´-UTR [START_REF] Abbink | The HIV-1 leader RNA conformational switch regulates RNA dimerization but does not regulate mRNA translation[END_REF][START_REF] Boeras | The basal translation rate of authentic HIV-1 RNA is regulated by 5'UTR nt-pairings at junction of R and U5[END_REF] . Additional structural studies carried out in cells and virions also argued against structural rearrangements as drivers of the transition between translation and packaging of the HIV-1 full-length RNA [START_REF] Paillart | First Snapshots of the HIV-1 RNA Structure in Infected Cells and in Virions[END_REF][START_REF] Wilkinson | High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states[END_REF][START_REF] Watts | Architecture and secondary structure of an entire HIV-1 RNA genome[END_REF] . Therefore, the mechanism by which Gag selects "packageable" from "translatable" full-length RNA molecules still remains as one of the long-lasting questions in Retrovirology. In this work, we showed that demethylation of two highly conserved adenosine residues within the 5´-UTR is critical for packaging of the HIV-1 full-length RNA. Interestingly, we observed that Gag associates with the RNA demethylase FTO in the nucleus and promotes demethylation of the full-length RNA, suggesting that Gag may drive FTO-mediated demethylation of those RNA molecules that will be used as gRNA to be incorporated into assembling viral particles (Fig. 6). This differential epitranscriptomic regulation exerted on the full-length RNA depending on its functions (mRNA or gRNA) may also help to explain the controversies reported in the literature [START_REF] Riquelme-Barrios | Emerging Roles of N(6)-Methyladenosine on HIV-1 RNA Metabolism and Viral Replication[END_REF] . As such, while the presence of m 6 A favors Gag synthesis through YTHDF proteins acting on the full-length RNA molecules destined to serve as mRNA [START_REF] Kennedy | Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression[END_REF] , the same cytoplasmic m 6 A readers may recognize specific features and drive degradation of the incoming viral RNA early upon infection (i.e., when the full-length RNA acts as gRNA) [START_REF] Tirumuru | N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression[END_REF] .

Figure 6 Working model for the epitranscriptomic regulation of HIV-1 full-length RNA packaging. The HIV-1 full-length RNA is methylated by METTL3/14 complex in the nucleus (for simplicity, only the presence of m 6 A on the 5´-UTR is shown). However, the structural protein Gag interacts with the m 6 A eraser FTO and drives demethylation of adenosines residues present at the 5´-UTR in a process required for full-length RNA packaging. Further studies are required to elucidate the mechanism by which the binding of YTHDF proteins to m 6 A residues negatively impact full-length RNA metabolism in the absence of translation and whether methylation of the 5´-UTR is involved. In addition, the molecular mechanism by which the presence of m 6 A at the 5´-UTR interferes with full-length RNA packaging also deserves further investigation. One of the most plausible explanations is that recognition of m 6 A residues at the 5´-UTR by reader proteins interferes with Gag binding and/or full-length RNA dimerization. However, it is also possible that the presence of m 6 A itself directly repeals the recruitment of Gag or alters the optimal RNA conformation of the dimerization and/or packaging signal. Indeed, our in vitro structural and dimerization analyses support this last idea as they suggest that the presence of m 6 A affects the folding and dimerization of the 5´-UTR. Of note, although neither A 198 nor A 242 showed a significant reactivity alteration this does not exclude that their modification may have influenced the reactivity of close nucleotides such as G 240 and G 241 . To date no studies have monitored the effect of m 6 A on such a complex structure and here we clearly show that this modification can have not only local but more global effects on RNA folding. Indeed, we observed that the introduction of m 6 A alters dimerization of the 5´-UTR, which could at least partly explain why methylated full-length RNA are not recovered within viral particles. Nevertheless, our experiments using the ΔA 198 /ΔA 242 provirus strongly indicate that these two adenosine residues play a major role in packaging. While A 198 is located within the region complementary to the tRNA Lys3 at the primer binding site (PBS), A 242 is located in the AGGA bulge at the base of the SL1 region and corresponds to a Gag-binding domain previously

shown by chemical probing in vitro and in virio as well as by CLIP-seq studies [START_REF] Wilkinson | High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states[END_REF][START_REF] El-Wahab | Specific recognition of the HIV-1 genomic RNA by the Gag precursor[END_REF][START_REF] Kutluay | Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis[END_REF] .

Further studies are required to fully understand the role of these residues in full-length RNA packaging.

In contrast to the simple retrovirus MLV, which segregates its full-length RNA into two separate populations for translation and packaging, the full-length RNA of the human lentivirus HIV-1 was proposed to exist as a single population that can indistinctly serve as mRNA or gRNA. While the presence of two specialized full-length RNA populations supports the lack of an epitranscriptomic regulation of MLV packaging, our data obtained with HIV-1 suggest that its full-length RNA may also exist as two populations with different m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A patterns. From these two populations, those molecules lacking m 6 A at the 5´-UTR will be preferentially selected by Gag for packaging (Fig. 6). In this regard, the HCV core protein was also shown to bind preferentially to RNA molecules lacking m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A most probably to avoid YTHDF proteins-mediated degradation upon viral entry [START_REF] Gokhale | N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection[END_REF] . Whether the packaging of hypomethylated RNA genomes is a conserved mechanism evolved by RNA viruses to avoid early degradation by cytoplasmic m 6 A readers must be determined.

Although the reversibility of adenosine methylation as well as the main target of the RNA demethylase FTO have been recently challenged [START_REF] Darnell | pre-mRNA processing includes N6 methylation of adenosine residues that are retained in mRNA exons and the fallacy of "RNA epigenetics[END_REF][START_REF] Mauer | m(6) Am , and the hypothesis of reversible epitranscriptomic mRNA modifications[END_REF] , we showed that the HIV-1 full-length RNA is a substrate for FTO and this RNA demethylase regulates the incorporation of the viral genome into released viral particles. Interestingly, treatment of HIV-1 producer cells with the specific FTO inhibitor meclofenamic acid resulted in an impairment of full-length RNA metabolism with a potent effect on packaging, confirming the critical role of the demethylase activity of FTO during viral replication. In addition to meclofenamic acid, two small molecules developed by structure-based rational design were recently described as specific inhibitors of FTO m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A demethylase activity with the potential to be used as a treatment for adult myeloid leukemia [START_REF] Huang | Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia[END_REF] . Therefore, this novel epitranscriptomic mechanism regulating packaging of the HIV-1 full-length RNA could also be exploited as a target for pharmacological intervention.

METHODS

Selective 2´-hydroxyl acylation analyzed by primer extension (SHAPE):

The HIV-1 fulllength RNA 5`-UTR was in vitro transcribed using the T7 RNA polymerase as described [START_REF] De Bisschop | HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity[END_REF] .

For m 6 A RNAs, ATP was substituted by N

Figure 1 :Figure 1 :

 11 Figure 1:The presence of m[START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A alters the in vitro folding and dimerization of the HIV-1 full-length RNA 5´-UTR. a, SHAPE reactivity differences between in vitro transcribed 5´-UTR RNA containing 0% m 6 A or 100% m[START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A plotted on the secondary structure model of the HIV-1 full-length RNA 5'-UTR (upper panel). Nucleotides in red are significantly more reactive with m 6 A whereas those in blues are significantly less reactive (p-value < 0.05 and reactivity difference > 0.4). Nucleotides in white are of equivalent reactivity. Sites where the reactivity could not be determined are depicted in grey. The histogram shows the reactivity difference between 0% m 6 A 5'-UTR and 100% m 6 A 5'-UTR (lower). Blue and red bars highlight nucleotides with a significantly lower or higher reactivity in the 100% m6A RNA than in the 0% m6A RNA (p-value < 0.05 and reactivity difference > 0.4), respectively. Significative differences that are below the threshold of 0.4 are indicated in black. b, Fraction of dimers determined by electromobility shift assay for different concentrations of in vitro transcribed 5'-UTR (1 µM, 0.5 µM, 0.25 µM, 127 nM, 65 nM, 33 nM, 18 nM, 2 nM) harboring either 0% of m 6 A (grey) or 100% of m 6 A (red) with the dissociation constants obtained from the data points (see Methods for details).

Figure 2 :Figure 2 :

 22 Figure 2: The presence of m 6 A within the full-length RNA favors Gag synthesis but interferes with packaging. HEK293T cells were transfected with pNL4.3 and pCMV-VSVg together with pCDNA-Flag-METTL3 and pCDNA-Flag-METTL14 or pCDNA-d2EGFP as a control. a, At 24 hpt cells extracts were used to detect Gag, Flag-METTL3 and Flag-METTL14 by Western blot. GAPDH was used as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). Intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (*P<0.05, t-test). b, Supernatants from cell cultures in (a) were filtered and viral particles were purified by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA. The level of CAp24 was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (n.s; nonsignificant, t-test). c, Viral particles purified from (b) were used to perform RNA extraction and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (***P<0.001, t-test). d, HeLa cells were transfected with pNL4.3, pCMV-VSVg and pCDNA-Flag-METTL3. At 24 hpt, the interaction between the full-length RNA and the Flag-tagged METTL3 was analyzed by ISH-PLA as described in the Methods section. Red dots indicate the interactions between the full-length RNA and the METTL3. Scale bar 10 µm. A quantification of the red dots in the nucleus (co-localizing with the DAPI staining) and the cytoplasm of 14 cells is presented on the right (****P<0.0001, Mann-Whitney test).

  3g). Taking together, these results indicate that the HIV-1 full-length RNA may exist as two different populations that differ at least in the m[START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A residues present within the 5´-UTR. Only the full-length RNA molecules lacking m 6 A at positions 198 and 242 might be recognized by Gag for packaging.

Figure 3 :

 3 Figure 3: Methylation of A 198 and A 242 within the 5´-UTR interferes with HIV-1 full-length RNA packaging. a, HEK293T cells were transfected with pNL4.3 and pCMV-VSVg. Intracellular polyA RNA or viral particle-associated RNA was extracted at 24 hpt, fragmented and used for m 6 A-seq as described in Methods. Peak calling results for the intracellular (left) and packaged (right) full-length RNA is shown. b, HEK293T cells were transfected with pNL4.3 wild type or pNL4.3

Figure 4 :

 4 Figure 4: Demethylation by a Gag-FTO complex favors HIV-1 full-length RNA packaging. HEK293T cells were transfected with pNL4.3 and pCMV-VSVg together with pCDNA-3XFlag-FTO or pCDNA-3XFlag-d2EGFP as a control. a, At 24 hpt cells extracts were used to detect Gag and 3XFlag-FTO by Western blot. GAPDH was used as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). Intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the

Figure 5 :

 5 Figure 5: Inhibition of FTO demethylase activity impacts full-length RNA metabolism and blocks packaging. HEK293T cells were transfected with pNL4.3 and pCMV-VSVg and were treated with MA2 or DMSO as a control. a, At 24 hpt cells extracts were used to detect Gag and GAPDH as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). The intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (***P<0,001, t-test). b, At 24 hpt the supernatant was filtered and viral particles were purified by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA, normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (n.s; non-significant, t-test). c, Purified viral particles from (b) were used to perform an RNA extraction and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (****P<0.0001, t-test).

Figure 5 :

 5 Figure 5: Inhibition of FTO demethylase activity impacts full-length RNA metabolism and blocks packaging. HEK293T cells were transfected with pNL4.3 and pCMV-VSVg and were treated with MA2 or DMSO as a control. a, At 24 hpt cells extracts were used to detect Gag and GAPDH as a loading control (left panel). In parallel, cells extracts were used to perform RNA extraction and the full-length RNA was quantified by RT-qPCR (right panel). The intracellular full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (***P<0,001, t-test). b, At 24 hpt the supernatant was filtered and viral particles were purified by ultracentrifugation. The level of CAp24 was quantified by an anti-CAp24 ELISA, normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (n.s; non-significant, t-test). c, Purified viral particles from (b) were used to perform an RNA extraction and the packaged full-length RNA from CAp24 equivalents was quantified by RT-qPCR. Packaged full-length RNA was normalized to the control (arbitrary set to 100%) and presented as the mean +/-SD of three independent experiments (****P<0.0001, t-test).

Figure 6

 6 Figure 6 Working model for the epitranscriptomic regulation of HIV-1 full-length RNA packaging. The HIV-1 full-length RNA is methylated by METTL3/14 complex in the nucleus (for simplicity, only the presence of m 6 A on the 5´-UTR is shown). However, the structural protein Gag interacts with the m 6 A eraser FTO and drives demethylation of adenosines residues present at the 5´-UTR in a process required for full-length RNA packaging.

  0 (Fujifilm). The fraction of dimer was calculated as the ratio "dimer -background" over "dimer + monomer -2*background". Data were fitted to Fraction dimer = Bmax*[RNA]/(Kd+[RNA]) using Prism 5.02 (GraphPad Software).Cell culture, DNA transfection and viral particle purification: HEK293T and HeLa cells were maintained in DMEM (Life technologies) supplemented with 10% FBS (Hyclone) and antibiotics (Hyclone) at 37ºC and 5% CO 2 atmosphere. Cells growing in 6-well plates (2.5x10 5 cells/well) were transfected using linear PEI ∼25000 Da (Polyscience) as described previously[START_REF] Toro-Ascuy | A Rev-CBP80-eIF4AI complex drives Gag synthesis from the HIV-1 unspliced mRNA[END_REF] . Cells were transfected using a ratio µg DNA/µl PEI of 1/15 and the DNA/PEI mix was incubated for 20 min at room temperature before adding to the cells. For experiments involving the FTO inhibitor, the culture medium was replaced by medium containing dimethyl sulfoxide (DMSO) as a control or 80 µM of an ethyl ester form of meclofenamic acid diluted in DMSO (MA2) prior DNA transfection. For viral particle purification, the supernatant was collected and filtered by passing through a 0.22 µm filter and then ultracentrifugated at 25.000 rpm for 2 hours at 4ºC in a 20% sucrose cushion (prepared previously and stored at 4 ºC). Purified viral particles were resuspended in 100 µl of PBS and stored in aliquots at -80 ºC to then perform anti-CAp24 ELISA (HIV-1) or Western blot (MLV) or RNA extraction. Cells were also collected to perform Western blot and RNA extraction as described in Supplementary Methods.

m 6 A

 6 -seq: Poly(A) RNA was purified from 100 µg of total RNA extracted from HEK293T cells previously transfected with pNL4.3 and pCMV-VSVg. Briefly, total RNA in 500 µl of water was incubated at 65ºC for 10 minutes and incubated with 3 µl of oligo dT-Biotin (dT-B; IDT Technologies) (50 pmol/µl) and 13 µl of SSC Buffer 20X (Santa Cruz Biotechnology) and allowed to cool at room temperature. 600 µg of Dynabeads ™ Streptavidin (60 µl; Thermo Fisher) were washed three times with Buffer SSC 0.5X and resuspended in 100 µl of Buffer SSC 0.5X. Then, the RNA/oligo dT-B mix was incubated with the streptavidin beads at room temperature for 10 minutes in head-over-tail rotation. RNA-beads were washed four times with 300 µl Buffer SSC 0.1X and bound RNA was eluted twice with 100 µl of water. The RNA was precipitated with 10 mM MgCl 2 , 20 µg glycogen (Thermo Fisher) and 2.5 volume of ETOH 100% overnight at -20 ºC and then washed with ETOH 70%. Poly(A) RNA as well as RNA obtained from purified viral particle were fragmented using Fragmentation Reagent (Thermo Fisher). For this, 2 µg of RNA in 9 µl of water was incubated with 1 µl of Fragmentation Buffer 10X for 15 minutes at 70 ºC, then 1 µl of Stop solution was added and incubated on ice. The RNA fragmented was precipitated overnight as described above.Fragmented RNA diluted in 380 µl was heated at 70 ºC for 5 minutes, placed on ice for 3 minutes. The denatured RNA was mixed with 1 µl of rRNasin® (Promega), 5 µl VRC, 100 µl of IP Buffer 5X (50 mM Tris-Hcl pH7.4, 750 mM NaCl and 0.5% NP-40) and 5 µl of an antim[START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A antibody (0,5 mg/mL; Synaptic System #202003) and incubated for 2 hours at 4 ºC with head-over-tail rotation. At the same time, 600 µg of Dynabeads ™ Protein A magnetic beads (20 µl; Thermo Fisher) were washed in 1 ml of IP Buffer 1X with 1 µl of VRC and were incubated with 500 µl of Buffer IP 1X with 0.5 mg/ml of BSA for 2 hours at 4 ºC with headover-tail rotation. Then, beads were washed with 500 µl of IP Buffer 1X and added to the RNA/anti-m 6 A antibody mix. The RNA-beads mix was incubated for 2 hours at 4ºC in headover-tail rotation. After incubation, the RNA-beads mix was washed twice with 500 µl IP Buffer 1X. Bound RNA was eluted with 100 µl of Elution Buffer (5mM Tris-HCl, 1mM EDTA and 0.05% SDS) and 1 µl of Proteinase K (New England BioLabs) and incubated for 1.5 hour at 50ºC. RNA was extracted from supernatant using TRIzol ™ (ThermoFisher). The RNA recovered was precipitated with 10 mM MgCl 2 , 20 µg glycogen (Thermo Fisher) and 2.5 volumes of ETOH 100% overnight at -20 ºC and then washed with ETOH 70%. Equal amounts of RNA from input and immunoprecipitation were used for RT-qPCR. cDNA libraries preparations and RNAseq was performed at Genoma Mayor. All the samples were

Packaged 0 20 40 60 80 100 120 pNL4.3 wt pNL4.3 ∆198/242 CAp24 level (% of control) * pNL4.3 wt pNL4.3 ΔA 198 /ΔA 242 0 20 40 60 80 100 120 pNL4.3 wt pNL4.3 ∆198/242 Packaged full-length RNA (% of control) pNL4.3 wt ΔA 198 /ΔA 242 pNL4.3 ΔA 198 /ΔA 242

					Intracellular
																Particle
	120.000										120.000
	110.000										110.000
	100.000										100.000
	90.000										90.000
	80.000										80.000
	60.000 70.000									FPKM	60.000 70.000
	50.000										50.000
	40.000										40.000
	30.000										30.000
	20.000										20.000
											10.000
											0
	0	1.000	2.000	3.000	4.000	5.000	6.000	7.000	8.000	9.000	0	1.000	2.000	3.000	4.000	5.000	6.000	7.000	8.000	9.000
				Nucleotide position in HIV-1 genome				0		2000	4000 Nucleotide position in HIV-1 genome 6000		8000		9256
												gag				vif		env		nef
											5´UTR			pol		vpr		rev			3´UTR
																		tat
																	vpu
	b										0	gag	2000		4000	vif	6000	env	8000	nef	9256
											5´UTR			pol		vpr		rev			3´UTR
																		tat
																	vpu

  [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] -methyl-ATP (Jena Bioscience). RNA was quantified by measurement of the OD 256 using a BioSpec-nano (Shimadzu). RNA integrity was assessed by agarose gel electrophoresis. SHAPE probing was conducted essentially as in[START_REF] Wilkinson | High-throughput SHAPE analysis reveals structures in HIV-1 genomic RNA strongly conserved across distinct biological states[END_REF] with minor modifications. Briefly, 6 pmol of in vitro transcribed RNA containing 100% m[START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A or 0% m 6 A were diluted in 24 µl of water, denatured at 80ºC for 2 minutes and ice cooled. After addition of 3 µl of 10X Folding Buffer (HEPES Ph7.5 400 nM, KCl 1M, MgCl 2 50 mM), samples were incubated for 10 minutes at room temperature and then 10 minutes at 37ºC. Then, the RNA solution was added to 3 µl of 20 mM 1M7 (2mM final) (AEchem Electropherograms were analyzed using QuSHAPE[START_REF] Karabiber | QuShape: rapid, accurate, and best-practices quantification of nucleic acid probing information, resolved by capillary electrophoresis[END_REF] . Raw data were processed by excluding the 2% of the highest values and normalizing the remaining values by the mean of the next 8% highest values[START_REF] Low | SHAPE-directed RNA secondary structure prediction[END_REF] . The experiments were performed three times and reproducibility was assessed by calculating the standard error of the mean.Secondary structure was drawn using VaRNA[START_REF] Darty | VARNA: Interactive drawing and editing of the RNA secondary structure[END_REF] . Samples were run for 1 hour at 100V on 4% native acrylamide mini-gels containing 34 mM Tris, 54 mM HEPES, 0.1 mM EDTA and 2.5 mM MgCl 2 . Dried gels were quantified using a BAS-5000 phosphorimager and MultiGauge 3.

	Dimerization assay: In vitro transcribed 5`-UTR was serially diluted in 10 mM Tris pH 7, 10
	mM NaCl, 140 mM KCl to obtain a final concentration ranging from 0 to 1 µM. 20 fmol (2
	nM final) of radiolabeled RNA was added. Samples were denatured at 95ºC for 3 minutes and
	then ice cooled. After addition of 1 mM MgCl 2 , RNA was allowed to fold for 30 minutes at
	37°C. Samples were subsequently chilled on ice, mixed with a 5X native loading buffer
	(glycerol 20%, xylene cyanol 0.1%, bromophenol blue 0.1%) and loaded on a native 4%
	acrylamide gel.
	Scientific Corporation) or to 3 µl of DMSO (control) and incubated for 6 minutes at 37ºC.
	RNA was subsequently precipitated in presence of 1 µl of 20 mg/ml of glycogen, 3 µl of 5M
	sodium acetate, 100 µl of ETOH for 1 hour at -20ºC and then washed with ETOH 70% and
	resuspended in water. For primer extension, RNA was treated with 1 µl of DMSO and
	denatured for 3 minutes at 95ºC and cooled at 4ºC. Samples were mixed with 3 µl of 2µM
	fluorescent primer (D4-5'-TTTCTTTCCCCCTGGCCTT for the probed/control samples and
	D2-5'-TTTCTTTCCCCCTGGCCTT for the sequencing reaction, Sigma) and incubated for 5
	minutes at 65ºC, 10 minutes at 35ºC and then 1 minute on ice. 5 µl of Reverse Transcription
	Mix (10 µl of 10 mM dNTPs and 40 µl of 5X MMLV RT Buffer Promega), 1 µl of 10 mM
	ddTTP for the sequencing reaction and 1 µl of MMLV Reverse Transcriptase RNAse H
	minus (Promega) were finally added and the reverse transcription was performed at 35ºC for
	2 minutes, 42ºC for 30 minutes and 55ºC for 5 minutes. Samples were then ice cooled and
	precipitated with ETOH for 2 hours. Pellets were resuspended in 40 µl of Sample Loading

Solution (Beckman Coulter). Reverse transcription products were resolved on a CEQ-8000 sequencer (Beckman Coulter).

FUNDING

This work was funded through the FONDECYT Program (Nº 1160176 and 1190156 to RSR and 1180798 to FVE). CPM, FGdeG, SRB, BRA and PAC are recipients of a National Doctoral fellowship from CONICYT. Research at BS laboratory was funded by CNRS, Université Paris Descartes and a grant from Fondation pour la Recherche Médicale (FRM #DBI20141231337). Exchanges between RSR and BS laboratories were funded by a LIA grant from CNRS. GdB is a recipient of a fellowship from the French Ministry for "Enseignement Superieur et Recherche".

DATA AVAILABILITY

m [START_REF] Dorman | Comparison of viral genomic RNA sorting mechanisms in human immunodeficiency virus type 1 (HIV-1), HIV-2, and Moloney murine leukemia virus[END_REF] A-seq data were deposited at the GEO upon accession number GSE130687.

FV-E and BS contributed to data analysis and manuscript writing. RS-R contributed to the concept and design, data analysis and manuscript writing. All the authors read and approved the final version of the manuscript.

COMPETING FINANCIAL INTERESTS

Authors declare no competing financial interests.