The HIV-1 protein Rev is essential for virus replication and ensures the expression of partially spliced and unspliced transcripts. We identified a ULM (UHM ligand motif) motif in the Arginine-Rich Motif (ARM) of the Rev protein. ULMs (UHM ligand motif) mediate protein interactions during spliceosome assembly by binding to UHM (U2AF homology motifs) domains. Using NMR, biophysical methods and crystallography we show that the Rev ULM binds to the UHMs of U2AF65 and SPF45. The highly conserved Trp45 in the Rev ULM is crucial for UHM binding in vitro, for Rev co-precipitation with U2AF65 in human cells and for proper processing of HIV transcripts. Thus, Rev-ULM interactions with UHM splicing factors contribute to the regulation of HIV-1 transcript processing, also at the splicing level. The Rev ULM is an example of viral mimicry of host short linear motifs that enables the virus to interfere with the host molecular machinery.

CEITEC Central European Institute of Technology Masaryk University Brno 62 500 Czech Republic

Center for Integrated Protein Science Munich Department Chemie TU München Garching 85748 Germany

EMBL Heidelberg Heidelberg 69 117 Germany

Institute of Structural Biology Helmholtz Zentrum München Neuherberg 85 764 Germany

Institute of Virology Helmholtz Zentrum München Neuherberg 85 764 Germany

Research Unit Cellular Signal Integration Helmholtz Zentrum München Neuherberg 85 764 Germany

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Hocine S., Singer R.H., Grunwald D.. RNA processing and export. Cold Spring Harb. Perspect. Biol. 2010; 2:a000752. PubMed PMC

Maniatis T., Tasic B.. Alternative pre-mRNA splicing and proteome expansion in metazoans. Nature. 2002; 418:236–243. PubMed

Nilsen T.W., Graveley B.R.. Expansion of the eukaryotic proteome by alternative splicing. Nature. 2010; 463:457–463. PubMed PMC

Wahl M.C., Will C.L., Luhrmann R.. The spliceosome: design principles of a dynamic RNP machine. Cell. 2009; 136:701–718. PubMed

Barash Y., Calarco J.A., Gao W., Pan Q., Wang X., Shai O., Blencowe B.J., Frey B.J.. Deciphering the splicing code. Nature. 2010; 465:53–59. PubMed

Stamm S., Ben-Ari S., Rafalska I., Tang Y., Zhang Z., Toiber D., Thanaraj T.A., Soreq H.. Function of alternative splicing. Gene. 2005; 344:1–20. PubMed

Hertel K.J. Combinatorial control of exon recognition. J. Biol. Chem. 2008; 283:1211–1215. PubMed

Maris C., Dominguez C., Allain F.H.. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 2005; 272:2118–2131. PubMed

Kielkopf C.L., Rodionova N.A., Green M.R., Burley S.K.. A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell. 2001; 106:595–605. PubMed

Selenko P., Gregorovic G., Sprangers R., Stier G., Rhani Z., Krämer A., Sattler M.. Structural basis for the molecular recognition between human splicing factors U2AF65 and SF1/mBBP. Mol. Cell. 2003; 11:965–976. PubMed

Kielkopf C.L., Lucke S., Green M.R.. U2AF homology motifs: protein recognition in the RRM world. Genes Dev. 2004; 18:1513–1526. PubMed PMC

Corsini L., Bonnal S., Basquin J., Hothorn M., Scheffzek K., Valcarcel J., Sattler M.. U2AF-homology motif interactions are required for alternative splicing regulation by SPF45. Nat. Struct. Mol. Biol. 2007; 14:620–629. PubMed

Thickman K.R., Swenson M.C., Kabogo J.M., Gryczynski Z., Kielkopf C.L.. Multiple U2AF65 binding sites within SF3b155: thermodynamic and spectroscopic characterization of protein–protein interactions among pre-mRNA splicing factors. J. Mol. Biol. 2006; 356:664–683. PubMed PMC

Corsini L., Hothorn M., Stier G., Rybin V., Scheffzek K., Gibson T.J., Sattler M.. Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60. J. Biol. Chem. 2009; 284:630–639. PubMed

de Chiara C., Menon R.P., Strom M., Gibson T.J., Pastore A.. Phosphorylation of S776 and 14-3-3 binding modulate ataxin-1 interaction with splicing factors. PLoS One. 2009; 4:e8372. PubMed PMC

Loerch S., Maucuer A., Manceau V., Green M.R., Kielkopf C.L.. Cancer-relevant splicing factor CAPERalpha engages the essential splicing factor SF3b155 in a specific ternary complex. J. Biol. Chem. 2014; 289:17325–17337. PubMed PMC

Jagtap P.K.A., Garg D., Kapp T.G., Will C.L., Demmer O., Luhrmann R., Kessler H., Sattler M.. Rational design of cyclic peptide inhibitors of U2AF Homology Motif (UHM) domains to modulate Pre-mRNA splicing. J. Med. Chem. 2016; 59:10190–10197. PubMed

Frankel A.D., Young J.A.. HIV-1: fifteen proteins and an RNA. Annu. Rev. Biochem. 1998; 67:1–25. PubMed

Karn J., Stoltzfus C.M.. Transcriptional and posttranscriptional regulation of HIV-1 gene expression. Cold Spring Harbor Perspect. Med. 2012; 2:a006916. PubMed PMC

Dlamini Z., Hull R.. Can the HIV-1 splicing machinery be targeted for drug discovery. HIV AIDS (Auckl.). 2017; 9:63–75. PubMed PMC

Pollard V.W., Malim M.H.. The HIV-1 Rev protein. Annu. Rev. Microbiol. 1998; 52:491–532. PubMed

Daugherty M.D., Liu B., Frankel A.D.. Structural basis for cooperative RNA binding and export complex assembly by HIV Rev. Nat. Struct. Mol. Biol. 2010; 17:1337–1342. PubMed PMC

DiMattia M.A., Watts N.R., Cheng N., Huang R., Heymann J.B., Grimes J.M., Wingfield P.T., Stuart D.I., Steven A.C.. The structure of HIV-1 rev filaments suggests a bilateral model for Rev-RRE assembly. Structure. 2016; 24:1068–1080. PubMed PMC

Bai Y., Tambe A., Zhou K., Doudna J.A.. RNA-guided assembly of Rev-RRE nuclear export complexes. eLife. 2014; 3:e03656. PubMed PMC

Sherpa C., Rausch J.W., Le Grice S.F., Hammarskjold M.L., Rekosh D.. The HIV-1 Rev response element (RRE) adopts alternative conformations that promote different rates of virus replication. Nucleic Acids Res. 2015; 43:4676–4686. PubMed PMC

Hammarskjold M.L., Heimer J., Hammarskjold B., Sangwan I., Albert L., Rekosh D.. Regulation of human immunodeficiency virus env expression by the rev gene product. J. Virol. 1989; 63:1959–1966. PubMed PMC

Malim M.H., Hauber J., Le S.Y., Maizel J.V., Cullen B.R.. The HIV-1 rev trans-activator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature. 1989; 338:254–257. PubMed

Felber B.K., Hadzopoulou-Cladaras M., Cladaras C., Copeland T., Pavlakis G.N.. rev protein of human immunodeficiency virus type 1 affects the stability and transport of the viral mRNA. Proc. Natl Acad. Sci. U.S.A. 1989; 86:1495–1499. PubMed PMC

Malim M.H., Cullen B.R.. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol. Cell Biol. 1993; 13:6180–6189. PubMed PMC

Kammler S., Otte M., Hauber I., Kjems J., Hauber J., Schaal H.. The strength of the HIV-1 3′ splice sites affects Rev function. Retrovirology. 2006; 3:89. PubMed PMC

Arrigo S.J., Chen I.S.. Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev. 1991; 5:808–819. PubMed

D’Agostino D.M., Felber B.K., Harrison J.E., Pavlakis G.N.. The Rev protein of human immunodeficiency virus type 1 promotes polysomal association and translation of gag/pol and vpu/env mRNAs. Mol. Cell Biol. 1992; 12:1375–1386. PubMed PMC

Groom H.C., Anderson E.C., Lever A.M.. Rev: beyond nuclear export. J. Gen. Virol. 2009; 90:1303–1318. PubMed

Kjems J., Frankel A.D., Sharp P.A.. Specific regulation of mRNA splicing in vitro by a peptide from HIV-1 Rev. Cell. 1991; 67:169–178. PubMed

Kjems J., Sharp P.A.. The basic domain of Rev from human immunodeficiency virus type 1 specifically blocks the entry of U4/U6.U5 small nuclear ribonucleoprotein in spliceosome assembly. J. Virol. 1993; 67:4769–4776. PubMed PMC

Tange T.O., Jensen T.H., Kjems J.. In vitro interaction between human immunodeficiency virus type 1 Rev protein and splicing factor ASF/SF2-associated protein, p32. J. Biol. Chem. 1996; 271:10066–10072. PubMed

Naji S., Ambrus G., Cimermancic P., Reyes J.R., Johnson J.R., Filbrandt R., Huber M.D., Vesely P., Krogan N.J., Yates J.R. 3rd et al. .. Host cell interactome of HIV-1 Rev includes RNA helicases involved in multiple facets of virus production. Mol. Cell. Proteomics: MCP. 2012; 11:M111 015313. PubMed PMC

Hadian K., Vincendeau M., Mausbacher N., Nagel D., Hauck S.M., Ueffing M., Loyter A., Werner T., Wolff H., Brack-Werner R.. Identification of a heterogeneous nuclear ribonucleoprotein-recognition region in the HIV Rev protein. J. Biol. Chem. 2009; 284:33384–33391. PubMed PMC

Delaglio F., Grzesiek S., Vuister G.W., Zhu G., Pfeifer J., Bax A.. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 1995; 6:277–293. PubMed

Ludwig E., Silberstein F.C., van Empel J., Erfle V., Neumann M., Brack-Werner R.. Diminished rev-mediated stimulation of human immunodeficiency virus type 1 protein synthesis is a hallmark of human astrocytes. J. Virol. 1999; 73:8279–8289. PubMed PMC

Vincendeau M., Kramer S., Hadian K., Rothenaigner I., Bell J., Hauck S.M., Bickel C., Nagel D., Kremmer E., Werner T. et al. .. Control of HIV replication in astrocytes by a family of highly conserved host proteins with a common Rev-interacting domain (Risp). AIDS. 2010; 24:2433–2442. PubMed

Davey N.E., Van Roey K., Weatheritt R.J., Toedt G., Uyar B., Altenberg B., Budd A., Diella F., Dinkel H., Gibson T.J.. Attributes of short linear motifs. Mol. Biosyst. 2012; 8:268–281. PubMed

Chemes L.B., de Prat-Gay G., Sanchez I.E.. Convergent evolution and mimicry of protein linear motifs in host-pathogen interactions. Curr. Opin. Struct. Biol. 2015; 32:91–101. PubMed

Hagai T., Azia A., Babu M.M., Andino R.. Use of host-like peptide motifs in viral proteins is a prevalent strategy in host-virus interactions. Cell Rep. 2014; 7:1729–1739. PubMed PMC

Dinkel H., Van Roey K., Michael S., Davey N.E., Weatheritt R.J., Born D., Speck T., Kruger D., Grebnev G., Kuban M. et al. .. The eukaryotic linear motif resource ELM: 10 years and counting. Nucleic Acids Res. 2014; 42:D259–D266. PubMed PMC

Kjems J., Calnan B.J., Frankel A.D., Sharp P.A.. Specific binding of a basic peptide from HIV-1 Rev. EMBO J. 1992; 11:1119–1129. PubMed PMC

Tan R., Chen L., Buettner J.A., Hudson D., Frankel A.D.. RNA recognition by an isolated alpha helix. Cell. 1993; 73:1031–1040. PubMed

Hadzopoulou-Cladaras M., Felber B.K., Cladaras C., Athanassopoulos A., Tse A., Pavlakis G.N.. The rev (trs/art) protein of human immunodeficiency virus type 1 affects viral mRNA and protein expression via a cis-acting sequence in the env region. J. Virol. 1989; 63:1265–1274. PubMed PMC

Hope T.J., Huang X.J., McDonald D., Parslow T.G.. Steroid-receptor fusion of the human immunodeficiency virus type 1 Rev transactivator: mapping cryptic functions of the arginine-rich motif. Proc. Natl Acad. Sci. U.S.A. 1990; 87:7787–7791. PubMed PMC

Wang W., Maucuer A., Gupta A., Manceau V., Thickman K.R., Bauer W.J., Kennedy S.D., Wedekind J.E., Green M.R., Kielkopf C.L.. Structure of phosphorylated SF1 bound to U2AF(6)(5) in an essential splicing factor complex. Structure. 2013; 21:197–208. PubMed PMC

Zhang Y., Madl T., Bagdiul I., Kern T., Kang H.-S., Zou P., Maeusbacher N., Sieber S.A., Kraemer A., Sattler M.. Structure, phosphorylation and U2AF65 binding of the N-terminal domain of splicing factor 1 during 3′-splice site recognition. Nucleic Acids Res. 2013; 41:1343–1354. PubMed PMC

Stepanyuk G.A., Serrano P., Peralta E., Farr C.L., Axelrod H.L., Geralt M., Das D., Chiu H.J., Jaroszewski L., Deacon A.M. et al. .. UHM–ULM interactions in the RBM39-U2AF65 splicing-factor complex. Acta Crystallogr. D Struct. Biol. 2016; 72:497–511. PubMed PMC

Davey N.E., Trave G., Gibson T.J.. How viruses hijack cell regulation. Trends Biochem. Sci. 2011; 36:159–169. PubMed

DiMattia M.A., Watts N.R., Stahl S.J., Rader C., Wingfield P.T., Stuart D.I., Steven A.C., Grimes J.M.. Implications of the HIV-1 Rev dimer structure at 3.2 A resolution for multimeric binding to the Rev response element. Proc. Natl. Acad. Sci. U.S.A. 2010; 107:5810–5814. PubMed PMC

Daugherty M.D., D’Orso I., Frankel A.D.. A solution to limited genomic capacity: using adaptable binding surfaces to assemble the functional HIV Rev oligomer on RNA. Mol. Cell. 2008; 31:824–834. PubMed PMC

Nagaraj N., Wisniewski J.R., Geiger T., Cox J., Kircher M., Kelso J., Paabo S., Mann M.. Deep proteome and transcriptome mapping of a human cancer cell line. Mol. Syst. Biol. 2011; 7:548. PubMed PMC

David C.J., Boyne A.R., Millhouse S.R., Manley J.L.. The RNA polymerase II C-terminal domain promotes splicing activation through recruitment of a U2AF65-Prp19 complex. Genes Dev. 2011; 25:972–983. PubMed PMC

Lallena M.J., Chalmers K.J., Llamazares S., Lamond A.I., Valcarcel J.. Splicing regulation at the second catalytic step by Sex-lethal involves 3′ splice site recognition by SPF45. Cell. 2002; 109:285–296. PubMed

Will C.L., Luhrmann R.. Spliceosome structure and function. Cold Spring Harb. Perspect. Biol. 2011; 3:a003707. PubMed PMC

Tazi J., Bakkour N., Stamm S.. Alternative splicing and disease. Biochim. Biophys. Acta. 2009; 1792:14–26. PubMed PMC