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. 2016 Jun 6;1(8):16082.
doi: 10.1038/nmicrobiol.2016.82.

Platelet-derived growth factor-α receptor is the cellular receptor for human cytomegalovirus gHgLgO trimer

Anna Kabanova  1 , Jessica Marcandalli  1 , Tongqing Zhou  2 , Siro Bianchi  3 , Ulrich Baxa  4 , Yaroslav Tsybovsky  4 , Daniele Lilleri  5 , Chiara Silacci-Fregni  1 , Mathilde Foglierini  1 , Blanca Maria Fernandez-Rodriguez  1 , Aliaksandr Druz  2 , Baoshan Zhang  2 , Roger Geiger  1   6 , Massimiliano Pagani  7 , Federica Sallusto  1 , Peter D Kwong  2 , Davide Corti  3 , Antonio Lanzavecchia  1   6 , Laurent Perez  1
Affiliations

Platelet-derived growth factor-α receptor is the cellular receptor for human cytomegalovirus gHgLgO trimer

Anna Kabanova et al. Nat Microbiol. .

Abstract

Human cytomegalovirus encodes at least 25 membrane glycoproteins that are found in the viral envelope(1). While gB represents the fusion protein, two glycoprotein complexes control the tropism of the virus: the gHgLgO trimer is involved in the infection of fibroblasts, and the gHgLpUL128L pentamer is required for infection of endothelial, epithelial and myeloid cells(2-5). Two reports suggested that gB binds to ErbB1 and PDGFRα (refs 6,7); however, these results do not explain the tropism of the virus and were recently challenged(8,9). Here, we provide a 19 Å reconstruction for the gHgLgO trimer and show that it binds with high affinity through the gO subunit to PDGFRα, which is expressed on fibroblasts but not on epithelial cells. We also provide evidence that the trimer is essential for viral entry in both fibroblasts and epithelial cells. Furthermore, we identify the pentamer, which is essential for infection of epithelial cells, as a trigger for the ErbB pathway. These findings help explain the broad tropism of human cytomegalovirus and indicate that PDGFRα and the viral gO subunit could be targeted by novel anti-viral therapies.

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Conflict of interest statement

Competing interests. The authors declare no financial or commercial conflict of interests.

Figures

Figure 1
Figure 1. Evidence that trimer-PDGFR and pentamer-ErbB interactions determine cellular tropism for fibroblasts or epithelial cells
(a) Human epithelial cells (ARPE-19) or fibroblasts (MRC-9) were exposed to pentamer-sufficient virus (VR1814) or pentamer-deficient virus (AD169). After 10 minutes, the cells were lysed and the lysate was captured by antibodies specific for tyrosine kinase receptors and developed using an anti-phospho-tyrosine antibody. Coordinates of the Phospho-Receptor Tyrosine Kinase array are reported in Supplementary Fig. 1. (b) Soluble trimer and pentamer were used for immunoprecipitation of cell surface biotinylated proteins from MRC-9 or ARPE-19 cells followed by immunoblotting with streptavidin-HRP conjugate. (c) Pull down from ARPE-19 or MRC-9 cells with soluble trimer or soluble pentamer immobilized on streptactin beads. Precipitated material was submitted to western blot analysis with an anti-PDGFRα polyclonal antibody. (d) The indicated receptors were downregulated on ARPE-19 and MRC-9 by shRNA. Cells were then infected with VR1814 or AD169 viruses for 48 hours. Shown is the percentage of infected cells. (e) The indicated receptors were knocked out on HAP-1 cells by CRISPR/Cas9. Cells were infected with VR1814 or AD169 viruses and analyzed as above. (f) The indicated receptors were overexpressed in ARPE-19 or MRC-9 cells using lentiviral vectors. Cells were infected and analyzed as above. Data shown are mean ± s.d. of three independent experiments. Statistical significance was evaluated by Mann-Whitney U test *P < 0.05; ****P < 0.0001.
Figure 2
Figure 2. HCMV trimer is required for both fibroblasts and epithelial cells infection, while HCMV pentamer is required for epithelial cells only
(a–d) MRC-9 cells were exposed to VR1814 or AD169 viruses that had been pre-incubated with different concentrations of antibodies or soluble ligands, alone or in different combinations: neutralizing antibodies specific for gH (13H11) or UL128/130/131 (7I13) or gO (CVB234); blocking antibody to PDGFRα (αR1), soluble trimer, soluble pentamer or soluble PDGFRα. (e) ARPE-19 cells were exposed to VR1814 virus that had been pre-incubated with different concentrations of antibodies to pUL128, gH or gO, soluble trimer or soluble pentamer. (f) ARPE-19 cells were pre-incubated with ErbB ligands (EGF, HB-EGF or Nrg-1) and subsequently infected with VR1814. Shown is the percentage of infected cells relative to untreated controls. Data shown are mean ± s.d. of four independent experiments.
Figure 3
Figure 3. The gHgLgO trimer, but not the gHgL dimer, binds with high affinity to PDGFRα
(a,b) Protein-protein interaction determined by Surface Plasmon Resonance (SPR) analysis. PDGFRα–Fc was immobilized to a GLM sensor chip and various concentrations (12.5 to 100 nM) of soluble trimer (a) or soluble dimer (b) were injected. Response units (RU) were recorded using ProteOn. (c,d) Protein-protein interactions were determined by Biolayer interferometry (BLI). PDGFRα-Fc was loaded on Protein G tip, dipped into various concentrations (ranging from 0.3 to 25 nM) of trimer and response units recorded. (c). The immobilized PDGFRα/Trimer complex was dipped into a solution of neutralizing anti-gH (13H11 or 11B12) or anti-pUL128 (15D8) antibodies and response units recorded (d). SPR and BLI sensorgrams are representative examples of experiments repeated three times.
Figure 4
Figure 4. Visualization of the gHgLgO-PDGFRα complex
(a–c) Size exclusion chromatography profiles of gHgLgO alone (a) or in complex with soluble PDGFRα (b) or with PDGFRα and a Fab fragment of the anti-gH antibody 13H11 (c). The shifts of the elution time caused by formation of binary and ternary complexes were marked with green dashed lines. SDS-PAGE analysis under reducing conditions of individual protein and complexes containing gH/gL/gO, PDGFRα and 13H11 Fab in different combinations isolated by size-exclusion (d). (e–g) Negative-stain electron microscopy and reference free 2D class averages of gHgLgO and its complexes purified by size exclusion chromatography. One representative image from each purification step was zoomed in to show the shape of the molecules and addition of volume. Trimer alone (e) or with receptor PDGFRα bound to gO. Of note, for the gHgLgO-PDGFRα complex, PDGFRα is only well resolved in some averages (f) or with the 13H11 Fab bound to the twisted region of gH in the gHgLgO-PDGFRα complex (g). Three-dimensional map of gHgLgO at 19.3 Å of resolution.
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