Figure 1.
Identification of regions for the interactions between REF and ICP27/ORF57.
(A) Subdivision of ICP27, ORF57 and REF2-I in fragments. (B) GST-REF pulls down ICP271–138 (NtS) as efficiently as full-length ICP27, but not ICP271–103 (Nt) and ICP27139–512 (Ct), indicating that residues 104–138 of ICP27 are involved in REF binding. (C) Fusion of the ICP27103–138 peptide to GST allows a specific pull down of REF, REF1–155 (NM) and REF54–155 (Δ53) and a very weak pull down of the REF1–70 (N); no interaction is detected with the control Ras protein. (D) Same pull down assay as panel C but using GST fusion of ORF57 aa 8–120 in place of ICP27.
Figure 2.
Identifying interaction sites within REF, ORF57 and ICP27 constructs via different NMR parameters.
The detailed data is presented in SI. Purple solid and broken bars mark residues with large and moderate reduction of mobility upon binding, respectively, as evidenced by the increase in 15N{1H} NOE. Orange stars mark residues with amide signals broadened beyond detection in the complex. Large and moderate chemical shift changes (δCS) of amide signals for each ligand as labelled are shown as solid and broken bars, respectively. Red bars mark residues forming direct inter-molecular NOE contacts in REF-ICP27 complex. (A) Changes in REF54–155 induced by addition of ICP27103–138 and ORF578–120. Position of secondary structure elements of REF (β-sheets, α-helices and loops) is shown in relation to its sequence. ICP27 and ORF57 bind on the same site on the REF RRM domain, with α-helices 1 and 2 and adjacent loop regions. (B) Changes in ORF578–120 upon addition of REF54–155. The main REF-interaction site is mapped to residues 103–120. (C) Changes in ICP27103–138 induced by addition of REF54–155. The REF-binding site is mapped to residues 104-112.
Figure 3.
Mapping of the ICP27- and ORF57-induced signal shifts onto the structure of REF RRM domain.
Backbone amide weighted chemical shift changes (δCS) are emphasized by colour. δCS >0.3 are red, 0.15–0.299 orange, 0.05–0.149 yellow, prolines are blue and regions unaffected are green. (A) Regions affected by addition of ICP27103–138. True ICP27 chain position determined based on NOE data is also shown for reference, with corresponded chemical shift changes due to REF binding similarly mapped. (B) Similar regions are affected by ORF578–120 and (C) synthetic peptide ICP27103–103. (D) Synthetic mutant peptide ICP27103–103 W105A binding affects the same RRM regions, however the value of shifts at the top of α-helix 1 is reduced, highlighting the binding site for W105 (encircled on panel C). The position and numbering of N- and C- terminal residues are indicated.
Figure 4.
Structure of the REF - ICP27 complex.
(A) Ribbon representation showing ICP27 coloured blue, and REF RRM coloured green, red and yellow for looped, α-helical and β-sheet regions, respectively. Positions of N- and C-termini of polypeptide chains are labelled. (B) Overlay of 20 lowest energy structures with backbone shown in the same orientation, and rotated 180°. The best-fit superposition is made using heavy backbone atoms of structurally defined regions aa 74–152 of REF and 102–112 of ICP27. Colour-coding is the same as on panel A. (C) Overlay of the RRM domains of free REF2-I (red, PDB code 2F3J), free murine Aly (purple, PDB code 1NO8) and ICP27-bound REF2-I determined here (green), demonstrating the shift in α-helix 1 position. (D) Representation of REF – ICP27 complex in the same orientation with partially transparent surface. (E) Schematic of the REF and ICP27 binding site. ICP27 residues are coloured blue and REF in black; the hydrophobic and electrostatic interactions are indicated by dashes coloured green and red, respectively. (F) Electrostatic surface with negative and positive charge coloured red and blue, respectively. Bottom row shows for comparison the known structures of RRM domains (green) with bound peptide ligands (orange). (G) REF54–155 in complex with ICP27103–138, determined here. (H) UHM domain of human SPF45 in complex with SF3B155-ULM5 (PDB code 2peh, [44]). (I) U2AF35 in complex with U2AF65 (PDB code 1jmt, [42]).
Table 1.
NMR calculation statistics for an ensemble of the 20 lowest energy structures of REF54–155 bound to ICP27103–138.
Figure 5.
Modelling of complex between human Aly and HSV-ICP27.
(A) Sequence alignment of RRM domains of murine REF2-I (mREF2-I: CAB76384) used in this study, murine Aly (mALY: AAC53117) and human Aly (hALY: AAD09608). Position of secondary structure elements (β-sheets, α-helices and loops) is shown in relation to the REF2-I. Resides in mALY and mREF2-I that differ from hALY are highlighted in light red. (B) A cartoon of the structure of REF-ICP27 with the position of the 7 amino acid differences indicated using red space-fill spheres, this orientation shown is the same as used in Fig. 3, whereas in (C) an alternative orientation is used for clarity. Only one amino acid difference is part of the ICP27 interaction site, namely Val138 of mREF2-I, which is a Phe in hALY. (D) A model of hsALY (blue) overlaid with the experimental structure of mREF2-I (green), with ICP27 also shown (orange). Residues that differ between the RRM domains of mREF2-I and hsALY are red sticks in the mREF2-I form and cyan sticks in hsALY. The sidechain of M145 altered in the modelling procedure is indicated by sticks. Also the ICP27 sidechain of L108 is indicated (orange) which is positioned within the hydrophobic pocket of REF. (E) Detailed view of the sidechains of Met 145, and Val138 and Phe138 in the modelled hsALY (blue) and experimental REF2-I (green) structures, plus L108 of ICP27 is shown (orange).
Table 2.
Dissociation constants for the interaction of REF54–155 with viral protein fragments.
Figure 6.
Functional importance of the identified REF binding site for the interaction of ORF57 with human Aly.
(A) Co-immunoprecipitation assays were performed to compare the binding of wild type ORF57-GFP and ORF57 mutants. Site-directed mutations were made within the REF binding site of ORF57 and within its vicinity using Quik-Change II (Stratagene), according to the manufacturer's instructions. 293T cells were transfected with GFP-tagged ORF57 and mutant proteins and after 24 hours cell lysates were incubated with Protein A agarose and a polyclonal GFP-specific antibody and precipitated proteins were analysed by Western blotting with Aly-specific antibody and monoclonal antibodies to GFP as a loading control. (B) The relative binding affinities in repeated experiments were analysed quantitatively by densitometry. Point mutations affecting recognition triad residues 108, 111 and 112 and double arginines 119,120 all significantly reduce ORF57-Aly interaction. Mutations of similar type outside the interaction site have not affected the interaction between the two proteins.
Figure 7.
Functional importance of triad residues 105, 107 and 108 for the interaction of ICP27 with human Aly.
(A) Co-immunoprecipitation assays were performed on 293T cells transfected with GFP-tagged ICP27 and its mutants. Mutations probed triad residues identified in the experimental structure formed between short protein fragments of ICP27 and REF. Cell lysates were incubated with Protein A agarose and a polyclonal GFP-specific antibody and precipitated proteins were analyzed by Western blotting with Aly-specific antibody and a monoclonal antibody specific to GFP as a loading control. (B) The relative binding affinities from 3 independent experiments were analysed quantitatively by densitometry. Point mutations of the REF recognition triad residues all significantly decrease binding between full-length ICP27 and endogenous Aly, confirming the functional significance of these residues identified by NMR.
Figure 8.
Perturbing the ORF57-REF/Aly interaction with point mutations decreases cytoplasmic accumulation of viral mRNA.
293T cells were transfected with the pORF47 reporter mRNA construct and the vectors containing wild type ORF57 or its mutants, and RNA was isolated from cytoplasmic fractions. The mutations probed residues from REF binding site, as well as from its vicinity. qRT-PCR was performed and data for mRNA reporter plus ORF57-transfected cells normalised against cells transfected with reporter in the presence of GFP. A ΔΔcT method was applied to determine the relative levels of reporter mRNA between samples. Point mutations of ORF57 residues implicated in direct interaction with REF, and reducing its binding, all caused significant decrease in cytoplasmic accumulation of viral mRNA.