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. 2018 Jun 19;9(3):e01112-18.
doi: 10.1128/mBio.01112-18.

The ICP27 Homology Domain of the Human Cytomegalovirus Protein UL69 Adopts a Dimer-of-Dimers Structure

Richard B Tunnicliffe  1   2 , Richard F Collins  3 , Hilda D Ruiz Nivia  4 , Rozanne M Sandri-Goldin  5 , Alexander P Golovanov  6   2
Affiliations

The ICP27 Homology Domain of the Human Cytomegalovirus Protein UL69 Adopts a Dimer-of-Dimers Structure

Richard B Tunnicliffe et al. mBio. .

Abstract

The UL69 protein from human cytomegalovirus (HCMV) is a multifunctional regulatory protein and a member of the ICP27 protein family conserved throughout herpesviruses. UL69 plays many roles during productive infection, including the regulation of viral gene expression, nuclear export of intronless viral RNAs, and control of host cell cycle progression. Throughout the ICP27 protein family, an ability to self-associate is correlated with the functions of these proteins in transactivating certain viral genes. Here, we determined the domain boundaries of a globular ICP27 homology domain of UL69, which mediates self-association, and characterized the oligomeric state of the isolated domain. Size exclusion chromatography coupled with multiangle light scattering (SEC-MALS) revealed that residues 200 to 540 form a stable homo-tetramer, whereas a shorter region comprising residues 248 to 536 forms a homo-dimer. Structural analysis of the UL69 tetramer by transmission electron microscopy (TEM) revealed a dimer-of-dimers three-dimensional envelope with bridge features likely from a region of the protein unique to betaherpesviruses. The data provide a structural template for tetramerization and improve our understanding of the structural diversity and features necessary for self-association within UL69 and the ICP27 family.IMPORTANCE Human cytomegalovirus (HCMV) infection is widespread in the human population but typically remains dormant in an asymptomatic latent state. HCMV causes disease in neonates and adults with suppressed or impaired immune function, as the virus is activated into a lytic state. All species of herpesvirus express a protein from the ICP27 family which functions as a posttranscriptional activator in the lytic state. In HCMV, this protein is called UL69. The region of sequence conservation in the ICP27 family is a folded domain that mediates protein interactions, including self-association and functions in transactivation. All members thus far analyzed homo-dimerize, with the exception of UL69, which forms higher-order oligomers. Here, we use biochemical and structural data to reveal that UL69 forms stable tetramers composed of a dimer of dimers and determine a region essential for cross-dimer stabilization.

Keywords: ICP27 homology domain; human cytomegalovirus; structural analysis; tetramerization; transmission electron microscopy.

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Figures

FIG 1
FIG 1
HCMV UL69 self-associates via a conserved ICP27 homology domain and forms a tetramer. (A) Location of the ICP27 homology domain (IHD) (orange) with the prototype from HSV-1 and the homologues from HHV-6A, HHV-6B, and HCMV. Prediction within HCMV UL69 of ordered/structured regions is indicated by green blocks, and the secondary structure is shown by red cylinders for α-helices and yellow blocks for β-sheets (29). The domain boundaries for constructs used here are indicated along with the molecular weight based on the primary sequence (30). (B) Primary sequence alignment of IHDs from UL69 homologues within the betaherpesvirus subfamily, produced with ESPrint (35). (c) SEC-MALS analysis of the molecular weights of purified UL69 constructs indicates that tetramer formation within UL69long is destabilized to a dimer by truncation of the region from aa 200 to 247 in UL69short.
FIG 2
FIG 2
3D structure of the UL69 tetramer determined by 3D reconstruction of negative-stain EM images. (A) A selection of 25 projection-class averages were calculated using reference-free multivariate statistical analysis (MSA). Box size = 33 nm. (B) Surface-rendered views of the final 3D reconstruction of the UL69long envelope orientated to match the bottom row initial class averages of panel A. (C) Surface-rendered views of the UL69 long structure. Five-nanometer scale bars are shown. (D) Segregation of the UL69long structure into two lobe volumes related by C2 symmetry and colored green and gray. Proposed bridge features are highlighted by dashed circles. (E) Each lobe of UL69 was fitted to the ICP27 IHD dimer coordinates. The surface of UL69long is shown as a mesh, and ICP27 (PDB accession number 4YXP) is shown as a ribbon trace, with one chain of the homo-dimer colored gray and the second chain in rainbow colors (blue to red, from the N terminus to the C terminus). The N termini of ICP27 are further highlighted by magenta circles, highlighting their proximity in space to each other and to the bridge regions.
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