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. 2019 Feb 28;47(4):1987-2001.
doi: 10.1093/nar/gky1181.

Structural Identification of Conserved RNA Binding Sites in Herpesvirus ORF57 Homologs: Implications for PAN RNA Recognition

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Free PMC article

Structural Identification of Conserved RNA Binding Sites in Herpesvirus ORF57 Homologs: Implications for PAN RNA Recognition

Richard B Tunnicliffe et al. Nucleic Acids Res. .
Free PMC article

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) transcribes a long noncoding polyadenylated nuclear (PAN) RNA, which promotes the latent to lytic transition by repressing host genes involved in antiviral responses as well as viral proteins that support the latent state. KSHV also expresses several early proteins including ORF57 (Mta), a member of the conserved multifunctional ICP27 protein family, which is essential for productive replication. ORF57/Mta interacts with PAN RNA via a region termed the Mta responsive element (MRE), stabilizing the transcript and supporting nuclear accumulation. Here, using a close homolog of KSHV ORF57 from herpesvirus saimiri (HVS), we determined the crystal structure of the globular domain in complex with a PAN RNA MRE, revealing a uracil specific binding site that is also conserved in KSHV. Using solution NMR, RNA binding was also mapped within the disordered N-terminal domain of KSHV ORF57, and showed specificity for an RNA fragment containing a GAAGRG motif previously known to bind a homologous region in HVS ORF57. Together these data located novel differential RNA recognition sites within neighboring domains of herpesvirus ORF57 homologs, and revealed high-resolution details of their interactions with PAN RNA, thus providing insight into interactions crucial to viral function.

Figures

Figure 1.
Figure 1.
Interaction mapping of RNA with KSHV ORF57 residues 68–178 by NMR. (A) Schematic of domain organization within the HVS and KSHV ORF57 proteins, and the boundaries of the constructs used throughout this study. N-terminal intrinsically disordered domain is shown as white bar with positions of nuclear localization sequences (NLS) marked, and C-terminal globular ICP27-homology domain (IHD) is shown as black bar. (B) 15N HSQC spectra of free protein (colored grey) overlaid with spectra of protein bound with RNA oligos, (i) 14merS oligo (colored red) or (ii) PAN17 oligo (colored purple). (C) Signal shifts and intensity perturbations induced by RNA oligos mapped onto the sequence of ksORF57 from analysis of spectra in panel A. Residues with signals broadened are marked by circles, and moderate and large signal shifts indicated by light and dark blocks, respectively. The positions of three NLS motifs are marked.
Figure 2.
Figure 2.
The structure of the C-terminal IHD from HVS ORF57. (A) Analysis of oligomeric state of IHD constructs hvsORF57Δ146 and ksORF57Δ153 by SEC-MALS indicated homo-dimerization. Size-exclusion chromatogram of purified proteins, dashed lines indicate refractive index with scale on left axis, solid lines represent molar mass quantified with scale on right axis. (B) X-ray crystal structure of the hvsORF57Δ146 homo-dimer. Chain A is colored blue through red from N- to C-termini with α-helices labelled and shown as cylinders, while chain B is colored grey. CHCC Zinc-finger locations are indicated by the spheres marked Zn2+ and the lower insert shows detail this feature. (C) Protein surface view colored by electrostatic potential in an orthogonal orientation to panel B, as indicated by eye symbol. A narrow channel through the protein passing under helix α6 is apparent. (D) Detail of helices α6 and α7 that form the homo-dimer bridge feature. Colored as panel A with sidechain atoms shown as sticks; selected residues are labeled. Red dash marks hydrogen bond between the Y311 sidechains.
Figure 3.
Figure 3.
PAN RNA MRE U-bend motif interaction with hvsORF57Δ146. (A) Structure of PAN17 interaction with hvsORF57Δ146: polyuridine RNA interacts with the bridge formed by helix α6 at the homo-dimer interface. RNA is colored orange and blue on backbone and bases respectively, protein chain A is colored rainbow blue through red from N- to C-termini, chain B is shaded grey. (B) Orthogonal view of panel A showing protein surface colored cyan and grey for chains A and B respectively. (C) Detail of protein-RNA recognition of uridine bases at the homo-dimer interface. RNA bases U45 and U46 plus protein sidechains that contact RNA are shown as sticks and labelled, water molecules shown as red spheres. Hydrogen bonds indicated by black dashes. The two uridine recognition sites formed by each protein chains: (i) Chain A interaction with U45, (ii) Chain B interaction with U46. Electron density assigned to RNA is shown as blue mesh scaled to 1.5σ. (D) The nine sites on PAN RNA which KSHV ORF57 interacts identified by Sztuba-Solinska et al. (53) contain polyuridine sequences which allow generation of a consensus binding sequence logo. The direct interaction with 43–49 site marked with asterisk is observed in the structure presented on the left.
Figure 4.
Figure 4.
Sequence alignment and summary of binding motifs of ORF57 homologs, in comparison with HSV1 ICP27. The alignment was generated by Clustal omega (65) for HVS ORF57, KSHV ORF57 and EBV EB2 sequences, whereas the C-terminal IHD sequence of HSV 1 ICP27 was aligned with HVS ORF57 to match position of structural elements. Secondary structure elements for HVS, KSHV and ICP27 are marked, with α-helices and β-sheets colored red and yellow, respectively. NLS are highlighted in light blue. The PxxP motif of ksORF57 implicated in ALYREF interactions is marked by a grey dashed box (30). Residues colored green are located in RNA binding sites in HVS and KSHV ORF57 and were identified by NMR or X-ray crystallography data, with conserved sites in γ-herpesviruses marked by dashed green boxes and labelled R1, R2 and R3. Conserved zinc coordinating residues are marked by black dashed boxes.
Figure 5.
Figure 5.
Comparison of the ICP27-homology domain structures. (A) Orthogonal views of a superposition of the X-ray crystal structures of hvsORF57Δ146 (colored blue and grey for chains A and B respectively) and HSV-1 ICP27Δ241 (colored green and cyan for chains A and B respectively). Dashed lines mark structural motifs shown in detail in panel (B). (C) Superposition of hvsORF57Δ146 (chains A and B colored blue and light blue respectively) with KSHV ORF57 homo-dimer coordinates, PDB: 5ZB3 (chains A and B colored green and light green respectively). The locations of ksORF57 motifs previously targeted by mutations are labelled: the isolated RGG sequences are marked with dashes and regions previously referred to as Leucine-zippers are marked by red dots. (D) The PAN17 RNA U-bend coordinates from the structure determined here (shown in stick representation) are docked onto the conserved binding surface of ksORF57 (colored green and grey for chains A and B respectively) (E) Detail of the docked PAN17 RNA poly-uridine sequence interaction with the ksORF57 coordinates, colored as in panel D. Generated by UCSF Chimera (73).

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