Structural Basis for the Recognition of Cellular mRNA Export Factor REF by Herpes Viral Proteins HSV-1 ICP27 and HVS ORF57

The herpesvirus proteins HSV-1 ICP27 and HVS ORF57 promote viral mRNA export by utilizing the cellular mRNA export machinery. This function is triggered by binding to proteins of the transcription-export (TREX) complex, in particular to REF/Aly which directs viral mRNA to the TAP/NFX1 pathway and, subsequently, to the nuclear pore for export to the cytoplasm. Here we have determined the structure of the REF-ICP27 interaction interface at atomic-resolution and provided a detailed comparison of the binding interfaces between ICP27, ORF57 and REF using solution-state NMR. Despite the absence of any obvious sequence similarity, both viral proteins bind on the same site of the folded RRM domain of REF, via short but specific recognition sites. The regions of ICP27 and ORF57 involved in binding by REF have been mapped as residues 104–112 and 103–120, respectively. We have identified the pattern of residues critical for REF/Aly recognition, common to both ICP27 and ORF57. The importance of the key amino acid residues within these binding sites was confirmed by site-directed mutagenesis. The functional significance of the ORF57-REF/Aly interaction was also probed using an ex vivo cytoplasmic viral mRNA accumulation assay and this revealed that mutants that reduce the protein-protein interaction dramatically decrease the ability of ORF57 to mediate the nuclear export of intronless viral mRNA. Together these data precisely map amino acid residues responsible for the direct interactions between viral adaptors and cellular REF/Aly and provide the first molecular details of how herpes viruses access the cellular mRNA export pathway

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 a-helix, binds preferentially to ALYREF. This competitively displaces viral RNA from the a-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

Typical effects of complex formation and RNA→protein ST on selected signals of ALYREF^1–155 and ORF57^8–120.

<p>The ¹H dimension slices through ¹H-¹⁵N-correlation spectra are displayed for three representative signals of each protein, on the left for ALYREF and on the right for ORF57. The residue assignments in the free form are labeled at the top, and same signals are shown below each other for different complexes, as indicated. First type of signal (ALYREF^M1 and ORF57^E24) is not significantly affected by any complex formation, or ST. Second type (ALYREF^A34 and ORF57^Y81) is not affected much by protein and marginally affected by RNA binding, but is altered or displays significant ST effect in the ternary complex (percentage drop in signal intensity is indicated in blue, and ST spectral traces shown in red). These are residues likely contributing to cooperative ternary complex formation, forming contacts with RNA. Third type of signals originates from the structured regions of proteins (ALYREF^A104 and ORF57^R111). ALYREF^A104 signal is not significantly affected in protein-protein complex, but shows significant increase in ST effect in the ternary complex, suggesting that ORF57 recruits RNA to the proximity of this residue. ORF57^R111 signal is broadened beyond detection in protein-protein complex, and remains broadened in the ternary complex. For this signal strong ST effect is observed when in complex with RNA. This residue is involved in initial viral RNA recognition, but then RNA is displaced from this site by ALYREF binding.</p

NMR calculation statistics for an ensemble of the 20 lowest energy structures of ALYREF fragment (ALYREF^54–155) bound to ORF57^103–120 (PDB code 2YKA).

<p>NMR calculation statistics for an ensemble of the 20 lowest energy structures of ALYREF fragment (ALYREF^54–155) bound to ORF57^103–120 (PDB code 2YKA).</p

Overview of RNA interaction sites mapped on ORF57^8–120 and ALYREF^1–155 using different approaches.

<p>Backbone amide chemical shift change (δCS) and saturation transfer (ST) data are shown for ORF57^8–120 (A) and ALYREF^1–155 (B) with different ligands added as labeled. Crosses indicate residues with signal broadened beyond detection. For δCS, large (>0.1) and moderate (>0.04) chemical shift changes of each protein relative to signals in its free state, are shown as solid and broken bars, respectively. For ST data, signal intensity ratios significantly different from the background mean values are represented by solid (>6 SD) and broken (>3 SD) bars, respectively. Labels indicate the state of the protein for each dataset, data shown for interactions with non-specific RNA (green), ORF57 specific RNA (red/orange) and ALYREF-ORF57-RNA complex (blue).</p

Probing ORF57-RNA binding by mutations and UV cross-linking, and ALYREF-ORF57-RNA remodeling assay.

<p>(<i>A</i>) Purified hexa-histidine tagged GB1 (negative control) or ORF57^8–120 WT and point mutants (as labeled) were incubated with end-labeled 14merS RNA oligonucleotide before the mixture was subjected to UV cross-link (+) or not (−). Similarly in the remodeling assay, WT ORF57^8–120 was incubated with end-labeled 7merS (<i>B</i>) or 14merS (<i>C</i>), before the mixture was added to purified GST-ALYREF immobilized onto glutathione coated beads. Purified complexes were eluted in native conditions and UV cross-linked (+) or not (−). All samples were finally analyzed on 15% SDS-PAGE stained with Coomassie blue and by PhosphoImaging.</p

Model of the passage of RNA between ORF57 and ALYREF.

<p>Local protein interactions with RNA are detected by moderate (orange) and large (red) saturation transfer effects (represented by ‘lightning bolts’) observed by ST-HSQC or ST-IDIS-TROSY and mapped onto respective regions. Broadened residues are colored light-yellow. Linked black circles represent a position for transiently bound RNA. ALYREF (green) binds RNA 14merS weakly via its RRM and N-terminal regions (A), whereas ORF57 (blue) binds 14merS tightly mainly via the R-b helix and also the aa81–92 region (B). Interaction of ORF57-RNA complex with ALYREF partially displaces the RNA from the R-b helix, while RNA maintains contact with ORF57 aa81–92 and also forms new contacts with ALYREF's aa22–48 and helix-2 of the RRM domain (C). The RNA contacts with ALYREF within the ALYREF-ORF57-RNA ternary complex are more abundant than for just ALYREF-RNA, and thus ORF57 enhances the interaction of viral RNA with ALYREF.</p

Fluorescent studies and simulations of the ternary complex formation between ORF57, O, fragment of viral RNA, R, and ALYREF, A.

<p>(<i>A</i>) Simultaneous non-linear fit of normalized fluorescence-derived parameters <i>Δλ^N_bcm</i> (blue shift of emission signal) and <i>ΔI^N</i> (fluorescence quenching) to the non-redundant three-equation model using DynaFit software, to obtain <i>K_d</i> for the ternary complex. The experimental values of thus determined <i>K_d^OA+R</i>, as well as <i>K_d</i>'s for other complexes measured earlier, are summarized on two illustrative thermodynamic cycles for the ternary complex assembly presented on panels <i>(B)</i> and <i>(C)</i>. Simulations (using COPASI software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003907#ppat.1003907-Hoops1" target="_blank">[67]</a>) for these two possible cycles illustrate an increase in the concentration of ternary ORF57-RNA-ALYREF complex when ORF57 is added to 10 µM equimolar mixture of ALYREF and RNA, assuming the simplest four-state equilibrium model (<i>B</i>), or six-state model which additionally takes into account weak nonspecific ALYREF-RNA binding (<i>C</i>). The arrow marks a point where all the components of ternary complex are present in equimolar amounts. The presence of equimolar ORF57 significantly increases the concentration of RNA in complex with ALYREF (i.e., [OAR] vs [AR]_[O] = 0). The baseline concentrations [AR]_[O] = 0 are indicated on the panel (<i>C</i>) on the left, assuming two different conservative estimates for values of <i>K</i>_d for nonspecific ALYREF-RNA binding.</p