FC0102
ORF57  -  RNA, ALYREF

Biological function
ORF57 is an essential herpesvirus adaptor protein, which 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 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.

Structural evidence
Signal perturbation mapping suggested a specific RNA binding site encompassing aa64–120 within ORF57^{8- 120}, whereas the ALYREF-binding region aa103–120 located within this site is not sufficient for the recognition of specific viral RNA. Saturation transfer mapping, in line with signal perturbation mapping showed that that ORF57^{8- 120} contacts the specific RNA motifs directly using primarily its regions aa107–120 and aa81–92, with additional contribution from residues within aa94–105 and aa64– 79. Addition of both specific and non-specific RNA oligos caused only small NMR signal shifts in the 10-40 AA acidic region of ORF57^8-120. Saturation transfer IDIS NMR experiments show that both ORF57 and ALYREF retained a high degree of flexibility, even for residues directly involved in interactions. Amide signals from flexible protein regions which become involved in direct contacts with RNA (as evidenced by RNA-protein saturation transfer), are only partially broadened in the complex suggesting that the interaction with RNA in these conditions was somewhat transient and did not lead to the formation of a rigid 3D structure.

Biochemical evidence
The ORF57 mutants Y81A+R82A, R88A+ significantly reduced the efficiency of cross-linking with RNA 14merS and R79A+V80A and R94A+I95A in the flanking region reduced the cytoplasmic accumulation of RNA. The experimental fluorescence equilibrium binding data reveals the overall cooperativity in the ternary complex formation when the components are present at or near stoichiometric amounts, and support a role of ORF57 as an adaptor introducing RNA to ALYREF. K_D of ORF57^8-120 and RNA is 7.5 μM, and K_D of RNA binding to the ORF57-ALYREF is 1.55±0.24 μM indicating cooperativity between ORF57 and ALYREF in RNA binding. The fluorescence data are also consistent with a local competitiveness of ORF57-ALYREF and ORF57-RNA interactions: this competitiveness becomes apparent only if RNA is in significant excess.

Structure/Mechanism
In its free form the N-terminal region aa8–120 of ORF57 is flexible and mainly unstructured, apart from the short α-helix aa108–118. It has been proposed that the positively charged region aa61–120 interacts transiently with the negatively charged aa12–28 part of the ORF57 polypeptide chain, keeping the molecule in a loosely "closed" conformation. When the specific viral mRNA motif binds, it is recognized by the extensive ORF57 region aa64–120, including the helical region and the flanking disordered segments. The aa64–120 fragment, which is rich with arginines, serines and aromatic residues, forms direct contacts with RNA, but without forming a stable 3D structure, and this RNA binding is expected to release the negatively-charged N-terminal part of ORF57. In the ternary complex ORF57 aa106–120 directly interacts with the ALYREF RRM, whereas flexible flanking regions of ORF57 (aa81–92) jointly with ALYREF keep hold of the viral RNA molecule.

Mechanism category
tethering

Significance
Transient interactions within fuzzy flanking regions contribute to the overall stability of the complex. ORF57 also serves to bridge the interaction between viral RNA and cellular ALYREF, cooperatively enhancing the formation of the ternary complex, without allosterically remodeling ALYREF.