Figure 1. HIV-1 Rev protein has a conserved ULM resembling motif. (A)

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Figure 1. HIV-1 Rev protein has a conserved ULM resembling motif. (A) Figure 1. HIV-1 Rev protein has a conserved ULM resembling motif. (A). Schematic representation of ULM–UHM interactions ... Figure 1. HIV-1 Rev protein has a conserved ULM resembling motif. (A). Schematic representation of ULM–UHM interactions required for constitutive and alternative splicing. Pro – proline-rich region; Zn – zinc-finger domain; BPS – branch point sequence; Py – polypyrimidine; RS – arginine–serine-rich domain. (B). Domain organization of full-length Rev. (C). Alignment of ULM sequences of HIV-1 Rev, SF3b155 (ULM5), SF1 and U2AF65. Conserved residues are marked: blue – basic residues preceding the conserved tryptophan; green – conserved tryptophan; red – acidic and N/Q-type residues following the conserved tryptophan. (D). Alignment of the ULM motif in selected HIV-1, HIV-2 and SIV strains. With the exception of single HIV-2 and SIV strains, the tryptophan is strictly conserved in all aligned isolates (a total of 2000 HIV-1, HIV-2 and SIV sequences was used for the alignment). Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, gkz185, https://doi.org/10.1093/nar/gkz185 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2. Rev ULM binds U2AF65 and SPF45 UHMs Figure 2. Rev ULM binds U2AF65 and SPF45 UHMs. (A) 1H,15N-HSQC spectra of free 250 μM UHMs ... Figure 2. Rev ULM binds U2AF65 and SPF45 UHMs. (A) <sup>1</sup>H,<sup>15</sup>N-HSQC spectra of free 250 μM UHMs (black) titrated with increasing amounts of Rev ULM 38–50 (blue 1:0.3; red 1:0.6; green 1:1.3). Arrows in the close-up views indicate the direction of chemical shift perturbations. (B and C, left panels) Surface representation of U2AF65 and SPF45 UHMs in complex with SF1 and SF3b155 ULMs (PDB: 4FXW and 2PEH), respectively. The chemical shift perturbations induced by Rev ULM are mapped on the surface of the structures. Color gradients (white to green for U2AF65 and white to magenta for SPF45) indicate the extent of chemical shift perturbations. (B and C) right panels. Isothermal titration calorimetry of U2AF65/SPF45 UHMs and Rev ULM (38–51). 900 μM Rev peptide was titrated to 90 μM UHMs. The graphs are representative of five independent ITC measurements. Control titration of Rev peptide to the buffer was subtracted from experimental runs before K<sub>D</sub> calculations. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, gkz185, https://doi.org/10.1093/nar/gkz185 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3. Crystal structure of SPF45 UHM bound to Rev ULM Figure 3. Crystal structure of SPF45 UHM bound to Rev ULM. (A) Cartoon representation of SPF45 UHM with ball-and-stick ... Figure 3. Crystal structure of SPF45 UHM bound to Rev ULM. (A) Cartoon representation of SPF45 UHM with ball-and-stick representation of the bound Rev ULM surrounded by experimental omit-electron density map contoured at 1.5σ. Please note, that a number of alternative conformations of amino acid side chains are present in the structural model. In order to simplify the graphical representation of the structure, the conformations presented in this figure corresponds to the electron density with higher occupancy. (B) Recognition of the critical Rev tryptophan (W45) by the hydrophobic cleft of the SPF45 UHM-fold. Interacting residues are shown as sticks and numbered. SPF45 residues of the RXF motif are underlined. (C) Network of intermolecular hydrogen bonds that stabilize the UHM–ULM interaction. Residues are shown as sticks and numbered. (D) Overlay of SPF45, U2AF65, U2AF35 and CAPERα UHMs bound to ULM peptides (PDB: 6HIP, 2PEH, 4FXW, 1JMT, 4OZ1) Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, gkz185, https://doi.org/10.1093/nar/gkz185 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4. Tryptophan at position 45 is required for proper biogenesis of HIV-1 mRNA species and viral production. ... Figure 4. Tryptophan at position 45 is required for proper biogenesis of HIV-1 mRNA species and viral production. (A) Left. Three different primer pairs were used to amplify alternatively spliced mRNAs produced during HIV-1 life cycle: fully spliced, single spliced and unspliced transcripts. Right. HeLafB cells expressing the Rev W45A mutant show increased levels of single spliced mRNAs compared to cells expressing Rev WT, as measured by RT-PCR. (B) Supernatant of HeLafB cells expressing the Rev W45A mutant show reduced p24 levels compared to cells expressing Rev WT, as measured by p24 ELISA. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, gkz185, https://doi.org/10.1093/nar/gkz185 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5. W45-dependent effect of Rev on the biogenesis of RRE-containing mRNA in HeLa cells. (A) Schematic ... Figure 5. W45-dependent effect of Rev on the biogenesis of RRE-containing mRNA in HeLa cells. (A) Schematic representation of pDM128. The intronic sequence is represented as a dashed line, SD4 – splice donor 4, SA7 – spliced acceptor 7. The location of qPCR amplicons detecting either spliced or unspliced mRNA is shown. (B, C) HeLa cells were transiently transfected with pDM128 and Rev WT or W45A (+ pRLSV40 to control for transfection efficiency and pBSIISK+ as plasmid DNA for dilution). Total RNA was isolated 48 hours after transfection, reverse-transcribed with an oligo(dT) primer. (B) The relative abundance of spliced to unspliced poly(A)+ RNA was assessed by qPCR (left graph). (C) In order to look at the levels of spliced and unspliced mRNAs separately instead of as a ratio, the same qPCR data were plotted comparing changes in the levels of spliced and unspliced mRNA species in cells transfected with RevW45A versus Rev WT (right graph). (D) HeLa cells were transiently transfected with GFP-tagged Rev WT or W45A and assayed 48 hours after transfection. Extracts from HeLa cells were incubated with or without RNase A and subjected to immunoprecipitation with α-GFP antibody. 1% of input and 50% of the immunoprecipitate was analyzed by Western blot, using α-GFP and α-U2AF65 antibodies. The experiments were performed three times, a representative gel is shown. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, gkz185, https://doi.org/10.1093/nar/gkz185 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6. Model presenting possible mechanisms of Rev functions in HIV-1 mRNA splicing inhibition. Rev binds to the RRE ... Figure 6. Model presenting possible mechanisms of Rev functions in HIV-1 mRNA splicing inhibition. Rev binds to the RRE in HIV-1 mRNA and interacts with the nuclear export factor CRM1 in order to transfer the transcript to the cytoplasm. Independently of its function in mRNA export, Rev can also inhibit the process of HIV-1 mRNA splicing by interfering with UHM–ULM interactions required for spliceosome assembly. We propose that Rev interactions with splicing factors may contribute to the protection of HIV-1 mRNA from splicing in order to promote rapid and efficient formation of CRM1-Rev nuclear export complex and subsequent transport of the mRNA to the cytoplasm. SS – splice site; -W – conserved tryptophan residue from the ULM motifs. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Nucleic Acids Res, gkz185, https://doi.org/10.1093/nar/gkz185 The content of this slide may be subject to copyright: please see the slide notes for details.