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. 2010 Oct;84(19):10026-37.
doi: 10.1128/JVI.00710-10. Epub 2010 Jul 21.

The glycoprotein B disintegrin-like domain binds beta 1 integrin to mediate cytomegalovirus entry

Adam L Feire  1 , René M Roy, Kate Manley, Teresa Compton
Affiliations

The glycoprotein B disintegrin-like domain binds beta 1 integrin to mediate cytomegalovirus entry

Adam L Feire et al. J Virol. 2010 Oct.

Abstract

Cellular integrins were identified as human cytomegalovirus (HCMV) entry receptors and signaling mediators in both fibroblasts and endothelial cells. The goal of these studies was to determine the mechanism by which HCMV binds to cellular integrins to mediate virus entry. HCMV envelope glycoprotein B (gB) has sequence similarity to the integrin-binding disintegrin-like domain found in the ADAM (a disintegrin and metalloprotease) family of proteins. To test the ability of this region to bind to cellular integrins, we generated a recombinant soluble version of the gB disintegrin-like domain (gB-DLD). The gB-DLD protein bound to human fibroblasts in a specific, dose-dependent and saturable manner that required the expression of an intact beta1 integrin ectodomain. Furthermore, a physical association between gB-DLD and beta1 integrin was demonstrated through in vitro pull-down assays. The function of this interaction was shown by the ability of cell-bound gB-DLD to efficiently block HCMV entry and the infectivity of multiple in vivo target cells. Additionally, rabbit polyclonal antibodies raised against gB-DLD neutralized HCMV infection. Mimicry of the ADAM family disintegrin-like domain by HCMV gB represents a novel mechanism for integrin engagement by a virus and reveals a unique therapeutic target for HCMV neutralization. The strong conservation of the DLD across beta- and gammaherpesviruses suggests that integrin recognition and utilization may be a more broadly conserved feature throughout the Herpesviridae.

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Figures

FIG. 1.
FIG. 1.
Relationships between glycoprotein B fragments used in this study. Schematic of HCMV envelope gB fragments. The amino-terminal gB disintegrin module is comprised of amino acids 57 to 146, while gB-651 represents a C-terminal gB fragment with no recognizable receptor-binding motifs.
FIG. 2.
FIG. 2.
Binding properties of the gB disintegrin-like domain. (A) gB-DLD binds permissive human fibroblasts with rapid kinetics. NHDF cells were incubated with a constant concentration (10 μg/ml) of gB-DLD at 4°C in 96-well plates after blocking with 1 mg/ml BSA. Cells were incubated for the indicated times, washed, fixed, and probed for the gB-DLD His6 tag by cell enzyme-linked immunosorbent assay (ELISA). (B) gB-DLD binds permissive human fibroblasts in a dose-dependent and saturable manner. Increasing concentrations of gB-DLD or gB-651 were added to NHDF cells for 60 min at 4°C, and cells were washed, fixed, and assayed for bound protein by cell ELISA. (C) Audioradiograph of 35S-labeled gB-DLD. E. coli cells were grown in the absence of sulfate prior to recombinant protein induction and the addition of 35S. A sample of the prep was separated by SDS-PAGE and exposed to film overnight. A prominent band at approximately 12 kDa corresponds to the predicted mass of gB-DLD. The specific activity of this preparation was 56,207 cpm/μg. (D) Homologous competition. A constant concentration of radiolabeled gB-DLD (1 μg/ml) was added to human fibroblasts in the presence of increasing concentrations of cold gB-DLD. Nonspecific binding was determined by the addition of a 100-fold molar excess and was determined to be 19%.
FIG. 3.
FIG. 3.
gB-DLD binding to cell surfaces is dependent on β1 integrin expression. (A) Increasing concentrations of gB-DLD were added to NHDF, GD25, GD25-β1, or GD25-β1YYFF cells, which rendered them unable to activate certain integrin-specific signal transduction cascades. Cells were then washed, fixed, and probed for the gB-DLD His6 tag by cell enzyme-linked immunosorbent assay. (B) A constant concentration of radiolabeled gB-DLD (1.0 μg/ml) was added to the indicated cell lines grown in DMEM (blue) or in sodium chlorate MEM (red) to inhibit heparan sulfate formation. The specific activity of the preparation was 56,207 cpm/μg.
FIG. 4.
FIG. 4.
gB-DLD physically associates with β1 integrin. (A) NHDFs were infected with adenovirus particles expressing full-length gB at an MOI of 20 PFU/cell. Protein expression was allowed to proceed for 72 h before the cells were washed and lysed. Complexes were immunoprecipitated, reduced, boiled, and separated by SDS-PAGE. Samples were then Western blotted as indicated. (B) NHDFs were lysed and incubated in the presence or absence of gB-DLD or gB-651 for 4 h, prior to the addition of Ni-NTA for 16 h. Beads were washed, resuspended in reducing sample buffer, boiled, and separated by SDS-PAGE. Samples were then separately blotted for either β1 integrin, β3 integrin, or His6 tag.
FIG. 5.
FIG. 5.
gB-DLD binding to host cells blocks HCMV infection at a postattachment step of virus entry. (A and B) Infectivity assays. gB-DLD or gB-651 was allowed to bind to permissive human fibroblasts, followed by washes and infection with HCMV-GFP or HSV-1(KOS)gL86 for 60 min. Cells were then citrate washed to remove unbound virus, and infection was allowed to proceed for 24 h followed by flow cytometry to assay for GFP-positive cells (HCMV) (A) or for 6 h followed by assaying for β-galactosidase activity (HSV-1) (B). (C) Entry assay. Human fibroblasts were incubated with increasing concentrations of gB-DLD or gB-651. Unbound protein was washed, and cells were infected with HCMV for 3 h. Virus was then washed off, cells were fixed, and an immunofluorescence assay was performed to visualize nuclear localization of HCMV tegument pp65. The percent pp65-positive nuclei per total 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei is shown for each panel. (D) HCMV-binding assay. Human fibroblasts were incubated with gB-DLD, gB-651, or heparin, followed by challenge with 3H-labeled HCMV at 4°C. Unbound virus was washed, cells were lysed, and scintillation counting was performed.
FIG. 6.
FIG. 6.
Polyclonal antibodies generated against gB-DLD neutralize HCMV infectivity. Rabbits were immunized with gB-DLD, and serum was collected and IgG purified. Either control rabbit IgG or gB-DLD rabbit IgG was incubated with HCMV-GFP prior to infection. After 60 min, unpenetrated HCMV was removed with citrate washes, and the infection was allowed to proceed for 24 h. To measure infectivity, cells were collected and flow cytometry was performed.
FIG. 7.
FIG. 7.
gB-DLD blocks the infectivity of endothelial cell-tropic HCMV. (A) HUVEC were incubated with increasing concentrations of gB-DLD or gB-651, washed, and infected with endothelial cell-tropic VHL/e. After 120 min, unpenetrated virus was removed with washes and infection was allowed to proceed for 24 h. Infection was assessed via immunofluorescence to HCMV immediate-early proteins and 4′,6-diamidino-2-phenylindole (DAPI) staining (total cells). (B) To quantify these data, numbers of immediate-early protein-positive nuclei per total DAPI-stained cells were counted and are represented as the percent infectivity. Data are shown relative to percent infectivity without any protein treatment.
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