Competitive and Cooperative Interactions Mediate RNA Transfer from Herpesvirus Saimiri ORF57 to the Mammalian Export Adaptor ALYREF

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.
Author Summary Herpes viruses invade cells, hijacking cellular components to sustain their lifecycle and replicate. A critical step of infection is the export of viral mRNA from the nucleus to the cytoplasm, where the molecular machinery to produce proteins is located. To provide a link between their mRNA and cellular components of the mRNA export pathway, all herpesviruses use special adaptor proteins. These adaptor proteins specifically select viral mRNAs from the mixture present in the nucleus, and introduce them to cellular mRNA export factors, such as ALYREF. How these viral adaptors manage to trick ALYREF to accept foreign genetic material has not been understood on a molecular level. In this study we reveal how a typical viral adaptor protein ORF57 recognizes specific viral RNA motifs, and also how it binds to the cellular protein ALYREF. We uncover details of how ORF57 transfers the viral RNA to ALYREF, locking it in the cooperative ternary complex. We also describe the atomic-resolution structure of ORF57-ALYREF interaction interface. Together the data provides the first molecular insight of how viral mRNA is transferred between viral and cellular proteins, thus helping virus to hijack a cell.