title: Competitive and cooperative interactions mediate RNA transfer from herpesvirus saimiri ORF57 to the mammalian export adaptor ALYREF.
abstract: The essential herpesvirus adaptor protein HVS ORF57, which has homologs in all other herpesviruses, promotes viral mRNA export by utilizing the cellular mRNA export machinery. ORF57 protein specifically recognizes viral mRNA transcripts, and binds to proteins of the cellular transcription-export (TREX) complex, in particular ALYREF. This interaction introduces viral mRNA to the NXF1 pathway, subsequently directing it to the nuclear pore for export to the cytoplasm. Here we have used a range of techniques to reveal the sites for direct contact between RNA and ORF57 in the absence and presence of ALYREF. A binding site within ORF57 was characterized which recognizes specific viral mRNA motifs. When ALYREF is present, part of this ORF57 RNA binding site, composed of an α-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the α-helix, but contact with RNA is still maintained by a flanking region. At the same time, the flexible N-terminal domain of ALYREF comes into contact with the viral RNA, which becomes engaged in an extensive network of synergistic interactions with both ALYREF and ORF57. Transfer of RNA to ALYREF in the ternary complex, and involvement of individual ORF57 residues in RNA recognition, were confirmed by UV cross-linking and mutagenesis. The atomic-resolution structure of the ORF57-ALYREF interface was determined, which noticeably differed from the homologous ICP27-ALYREF structure. Together, the data provides the first site-specific description of how viral mRNA is locked by a herpes viral adaptor protein in complex with cellular ALYREF, giving herpesvirus access to the cellular mRNA export machinery. The NMR strategy used may be more generally applicable to the study of fuzzy protein-protein-RNA complexes which involve flexible polypeptide regions.
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Richard B
authors.initials: RB
authors.lastname: Tunnicliffe
authors.affiliation: RNA Biology Laboratory, Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Guillaume M
authors.initials: GM
authors.lastname: Hautbergue
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, United Kingdom.
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Wilson
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Priti
authors.initials: P
authors.lastname: Kalra
authors.affiliation: Manchester Institute of Biotechnology and Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom.
authors.forename: Alexander P
authors.initials: AP
authors.lastname: Golovanov
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000502:physiology; D006566:Herpesviridae Infections / Q000378:metabolism*; D006567:Herpesvirus 2, Saimiriine / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity; D006790:Host-Parasite Interactions / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D034443:RNA Transport / Q000502:physiology; D012367:RNA, Viral / Q000032:analysis / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000378:metabolism*; D014412:Tumor Virus Infections / Q000378:metabolism*
chemical_list: C105242:ALYREF protein, human; D009687:Nuclear Proteins; C415247:ORF57 protein, Herpesvirus saimiri; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors