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. 2019 Jun 20;10(1):2699.
doi: 10.1038/s41467-019-10587-1.

CD46 facilitates entry and dissemination of human cytomegalovirus

Kathryn R Stein  1 , Thomas J Gardner  1 , Rosmel E Hernandez  1 , Thomas A Kraus  1   2 , James A Duty  1   2 , Iban Ubarretxena-Belandia  3   4   5 , Thomas M Moran  1   2 , Domenico Tortorella  6
Affiliations

CD46 facilitates entry and dissemination of human cytomegalovirus

Kathryn R Stein et al. Nat Commun. .

Abstract

Human cytomegalovirus (CMV) causes a wide array of disease to diverse populations of immune-compromised individuals. Thus, a more comprehensive understanding of how CMV enters numerous host cell types is necessary to further delineate the complex nature of CMV pathogenesis and to develop targeted therapeutics. To that end, we establish a vaccination strategy utilizing membrane vesicles derived from epithelial cells to generate a library of monoclonal antibodies (mAbs) targeting cell surface proteins in their native conformation. A high-throughput inhibition assay is employed to screen these antibodies for their ability to limit infection, and mAbs targeting CD46 are identified. In addition, a significant reduction of viral proliferation in CD46-KO epithelial cells confirms a role for CD46 function in viral dissemination. Further, we demonstrate a CD46-dependent entry pathway of virus infection in trophoblasts, but not in fibroblasts, highlighting the complexity of CMV entry and identifying CD46 as an entry factor in congenital infection.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Physical attributes of ARPE-19 cell-derived vesicles and their humoral immune response in mice. a Increasing amounts of ARPE-19 membrane fraction (μg) were resolved on a SDS-polyacrylamide gel (12.5%) and visualized using Gel Code Blue Reagent (ThermoFisher Scientific). b Transmission electron microscopy (TEM) images of the membrane fraction. Representative images demonstrate the range in vesicle sizes within the fraction. Arrows indicate observed protein aggregates surrounding vesicles. c The distribution of vesicle diameters (nm) (n = 29). Bounds of the box span 25–75% percentile, the center line represents median, and whickers visualize 5 and 95% of the data points. d Dilutions of serum from five mice (M1–M5) immunized with the ARPE-19 membrane fractions were subjected to flow cytometry with ARPE-19 cells. Normal mouse serum (NMS), PBS, and the mAb W6/32 (targeting MHC class I) were used as controls. e Two dilutions of serum from the ARPE-19 vesicle immunized mice were subjected to a high-throughput infectivity assay (HTI) with TB40/EFLAG YFP infection of ARPE-19 cells using YFP fluorescence as readout for infection. The % infection was determined using virus incubated with normal mouse serum (NMS) as 100%. Infection data was performed in triplicate. s.d. is depicted in the experiment. **P < 0.01(Student’s two-tailed t test)
Fig. 2
Fig. 2
High-throughput screening for cell-surface binding clones. a Hybridoma supernatants across 32 96-well plates were incubated with ARPE-19 cells, with binding detected through flow cytometry. Clones that bound with mean fluorescent intensity (MFI) two fold over background (~5 k) or higher were designated as cell-surface binders. Darkening red hues are relative to increasing MFI. Wells without clones are represented in gray. b Supernatant from ARPE-19 cell-surface binders was subjected to high-throughput flow cytometry using against Jurkat, HEK293T and A549 cells to evaluate specificity. Fold change of MFI was determined on a cell-type basis compared to a known non-binder (anti-gH 5C3) and is represented by darkening red hues relative to its increase
Fig. 3
Fig. 3
Identification of inhibitory monoclonal antibodies and their cellular target. a Supernatants from cell-surface binding clones were subjected to a high-throughput infectivity assay (HTI) with TB40/EFLAG YFP infection of ARPE-19 cells using YFP fluorescence as readout for infection. The % infection was determined using virus incubated with media alone as 100%. b Clones demonstrating reduced infection were validated using the TB40/EFLAG YFP/ARPE-19 cells HTI with varying amounts (%) of supernatant. c Purified mAbs (10 μg ml−1) were analyzed using the TB40/EFLAG YFP/ARPE-19 HTI in technical triplicates. d Polypeptides recovered with mAb 2E7 and 12H8 (arrows) from ARPE-19 cells metabolically labeled with 35S-methinionine (6 h) were resolved on a SDS-polyacrylamide gel and visualized on a radiographic film. Beads only was used as a control. e Polypeptides recovered with mAb 2E7 and 12H8 from ARPE-19 cells were subjected to immunoblot analysis using anti-CD46 antibodies. mAb W6/32 and total cell lysates (TCL) were included as controls. The polypeptides and molecular weight markers are indicated. s.d. is depicted in the experiment. *P < 0.05, **P < 0.01 (Student’s two-tailed t test)
Fig. 4
Fig. 4
CD46-dependent CMV entry in epithelial cells. a TB40/E wt was subjected to a mAb inhibition HTI in ARPE-19 cells with mAb 5C3 (anti-gH), mAb 2E7 (anti-CD46), mAb PY102 (non-binding control) or no-antibody control (20-0.01 μg ml−1, in threefold dilutions). Virus infection from PY102-treated cells represents 100% infection. b Total cell lysates from uninfected, TB40/E wt and AD169BADrUL131 infected ARPE-19 cells treated with mAbs 5C3 or 2E7 (10 and 2 μg ml−1), mAb PY102 (10 μg ml−1), and no mAb were subject to immunoblot analysis for IE1 and GAPDH. (c) CD46 protein model represents four consensus repeats (SCRs) where complement proteins C4b, C3b, and C4b bind, a serine/threonine/proline (STP)-rich region, an uncharacterized segment (U), a transmembrane domain (TM), and a cytoplasmic tail. Alternative splicing accounts for common isomers expressing either STP regions BC or just C and either a short cytoplasmic tail (CYT1) or long cytoplasmic tail (CYT2). d Using flow cytometry, anti-CD46 mAbs TRA-2–10 and GB24 (2 μg ml−1) were analyzed on ARPE-19 cells in comparison to PY102. e ARPE-19 cells were incubated with labeled 2E7647 (2 μg ml−1) and increasing concentrations (6.7–0.22 μg ml−1, in 3-fold dilutions) of mAbs TRA-2-10 or GB24. The mean fluorescence intensity (MFI) of anti-mouse IgGAlexa647 was measured by flow cytometry with PY102 (−) and non-labeled 2E7 (+) as controls. f TB40/E wt and AD169BADrUL131 infection of ARPE-19 cells treated with mAbs 5C3, 2E7, TRA-2-10, GB24, and PY102 (6.7–0.74 μg ml−1, in threefold dilutions) and a no-antibody control were analyzed using an HTI. PY102-treated cells represents 100% infection. g ARPE-19 cells transfected with non-targeting siRNA (SCR) or siRNA targeting CD46 (CD46) were infected with TB40/EFLAG YFP and AD169BADrUL131 and analyzed for infection (YFP fluorescent intensity) by flow cytometry. SCR-transfected cells represent 100% infection. h Cell lysates from ARPE-19 cells transfected with no siRNA, SCR, or CD46 targeting siRNA and infected with AD169BADrUL131 or TB40/E wt (MOI:0.5) were subjected to anti-IE1 and GAPDH immunoblots. The polypeptides and molecular weight markers are indicated for the immunoblots. Infection experiments were performed in triplicate. s.d. is depicted in the experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s two-tailed t test)
Fig. 5
Fig. 5
CD46 is an important factor for CMV infection and spread. a CD46 cell surface expression of wild-type ARPE-19 cells (Ewt), β2m Knock-out (KO) ARPE-19 cells (Eβ1), and three CD46-KO ARPE-19 cell clones (EC1, EC2, and EC3) was assessed by flow cytometry with mAbs against CD46 (2E7 and GB24), MHC class I (W6/32), and PY102 as a non-binding control. Following incubation with anti-mouse IgGAlexa647, the normalized cell number was plotted based on Alexa647 fluorescence intensity. b The respective KO cells (a) were subjected to anti-CD46 and GAPDH immunoblots. c ARPE-19 wt and CD46- and β2m-KO cells infected with TB40/E wt or AD169BADrUL131 were analyzed for infection by an HTI. Total number of infected cells was determined using anti-IE1 antibodies. d ARPE-19 wt and CD46- and β2m-KO cells infected with TB40/E wt, TB40/EMC UL99-eGFP, or AD169BADrUL131 were subjected to a plaque assay, counted 14 dpi. e Supernatant from Ewt, Eβ1, and EC2 cells infected (E: epithelial cells) with AD169BADrUL131 (MOI:0.1) from days 0–12 was added to MRC5 and analyzed for infection at 24 hpi using YFP fluorescent with a cytometer. The virus titre (infectious units (IU)/ml) was plotted for up to 12 dpi. f Cell lysates of wt HTR-8/SVneo (Twt) cells, β2m-KO HTR-8/SVneo (Tβ1) cells, and CD46-KO HTR-8/SVneo cell clones (TC1 and TC2, T: trophoblasts) were subjected to immunoblot using anti-CD46 and anti-GAPDH antibodies. g Cell lysates of AD169BADrUL131 (MOI:2) infected Twt, Tβ1, TC1 and TC2 were subjected to anti-IE1 and GAPDH immunoblots. h ARPE-19 wt and Tβ1, TC1, and TC2 clones infected with AD169BADrUL131 at varying MOIs were analyzed for infection by an HTI assay. The total number of infected cells was determined using a cytometer. i Representative cytometer images of (h) (overlay: Hoechst stain for nuclear stain (blue) and GFP expression (green) for virally infected cells) (MOI 2). The polypeptides and molecular weight markers are indicated for the immunoblots. Infection experiments (excluding westerns) were performed in triplicate. s.d. is depicted in the experiment. **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s two-tailed t test)
Fig. 6
Fig. 6
CD46 is involved in a post-binding entry step. a TB40/E wt infection of ARPE-19 cells untreated or treated at −0.5, 0.5, and 5hpi with mAbs targeting HCMV gH (5C3) and CD46 (2E7), heparin, a non-binding antibody control (PY102), or a no-antibody control (10 μg ml−1) was analyzed with a HTI. PY102-treated cells was used as 100% infection. b TB40/E wt infection of ARPE-19 cells and (c) AD169BADrUL131 infection of HTR-8/SVneo cells incubated with mAbs 5C3, 2E7, and anti-Nrp2 (αNrp2), or soluble CD46 and Nrp2 proteins (P) (20–0.24 μg ml−1, in threefold dilutions) were subjected to the HTI assay. The % infection was determined using incubation with PY102 as 100% infection. Experiments were performed in triplicate. s.d. is depicted in the experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s two-tailed t test)
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