Avidity binding of human adenovirus serotypes 3 and 7 to the membrane cofactor CD46 triggers infection

Abstract

The species B human adenoviruses (HAdVs) infect cells upon attaching to CD46 or desmoglein 2 (DSG-2) by one or several of their 12 fiber knob trimers (FKs). To test whether DSG-2 and CD46 simultaneously serve as virus receptors for adenovirus type 3 (Ad3), we performed individual and combined CD46/DSG-2 loss-of-function studies in human lung A549 and 16HBE14o cells. Our results suggest that in these cells, DSG-2 functions as a major attachment receptor for Ad3, whereas CD46 exerts a minor contribution to virus attachment and uptake in the range of ∼10%. However, in other cells the role of CD46 may be more pronounced depending on, e.g., the expression levels of the receptors. To test if avidity allows Ad3/7 to use CD46 as a receptor, we performed gain-of-function studies. The cell surface levels of ectopically expressed CD46 in CHO or human M010119 melanoma cells lacking DSG-2 positively correlated with Ad3/7 infections, while Ad11/35 infections depended on CD46 but less on CD46 levels. Antibody-cross-linked soluble CD46 blocked Ad3/7/11/35 infections, while soluble CD46 alone blocked Ad11/35 but not Ad3/7. Soluble Ad3/7-FKs poorly inhibited Ad3/7 infection of CHO-CD46 cells, illustrating that Ad3/7-FKs bind with low affinity to CD46. This was confirmed by Biacore studies. Ad3/7-FK binding to immobilized CD46 at low density was not detected, unlike that of Ad11/35-FK. At higher CD46 densities, however, Ad3/7-FK bound to CD46 with only 15-fold-higher dissociation constants than those of Ad11/35-FK. These data show that an avidity mechanism for Ad3/7 binding to CD46 leads to infection of CD46-positive cells.

Fig 1

Loss-of-function studies for individual contribution of CD46 and DSG-2 to Ad3 infection. (A and B) Inhibition of Ad3 (A) and Ad35 (B) binding to A549 cells by anti-CD46 and DSG-2 antibodies. A549 cells were incubated with the indicated concentrations of CD46 (MEM258), DSG-2 (6D8 and 8E5), a mix of the two types of antibodies, control CAR antibodies, or PBS, followed by the addition of atto488-labeled Ad3 or Ad35 at 4°C. Virus binding was assessed by cytofluorometric analysis. The data were normalized to the amounts of virus bound when using PBS. Mean values and standard deviations of triplicates from one representative experiment are shown. (C to H) Effect of siRNA-mediated downregulation on virus infection in A549 (C to E) and 16HBE14o (F to H) cells. (C and F) Cells were transfected with the indicated siRNAs, resulting in specific downregulation of CD46 and DSG-2 but not of unrelated CAR. Shown is one representative analysis. (D and G) Binding of atto488-labeled Ad2 (control), Ad3, and Ad35 to siRNA-transfected cells was assessed as described above. (E and H) Transduction of siRNA-transfected cells with eGFP-expressing control Ad5, Ad3, and Ad35 vectors at 500 vp/cell. eGFP expression values were analyzed 2 days p.i. by flow cytometry and are expressed as MFI.

Fig 2

Binding and transduction of adenovirus species B viruses in CD46 gain-of-function cells negative for DSG-2. (A) For Ad binding assays, 5 × 10⁵ human A549 cells and the different rodent CHO-CD46 cells were incubated on ice with 1,000 vp of the indicated ³H-labeled species B serotypes. After incubation for 2 h, the cells were washed and cell-associated radioactivity was determined. Mean values and standard deviations of triplicates from one representative experiment are shown. Asterisks indicate here and in the experiments described below the level of significance (*, P < 0.05; **, P < 0.005; ***, P < 0.0005, for comparisons of corresponding Ad binding [infection] in parental CHO versus CD46-transfected cells). (B) Transduction assays of human A549, parental CHO, and CHO-CD46-expressing cells. Cells (10⁵) were incubated with eGFP-expressing Ad3, Ad7, Ad11, and Ad35 vectors at increasing virus concentrations of 10, 100, and 1,000 vp/cell. eGFP expression was analyzed 2 days p.i. by flow cytometry and is expressed as MFI. (C) Transduction assays of human parental and stable CD46-transfected M010119 melanoma cells. Cells were transduced and tested as described above. Background fluorescence intensity for uninfected M010191-eGFP-CD46 cells was higher due to the eGFP-tagged CD46 in these cells.

Fig 3

Cross-linking of CD46ex-huFc strongly increases blocking of Ad3/7 infection in CHO-CD46#2 and A549 cells. CHO-CD46#2 cells (A) or A549 cells (B) were preincubated for 1 h in the cold using the indicated concentrations of adapter CD46ex-huFc alone or in combination with a 2-fold-increasing series of goat anti-human Fc antibody. Following addition of the different eGFP-expressing vectors for another 1 h, cells were washed and analyzed 48 h p.i. Asterisks indicate the levels of significance (*, P < 0.05; **, P < 0.005; ***, P < 0.0005, for comparisons of corresponding Ad infection using CD46ex-huFc versus control CARex-huFc of Fig. S2 in the supplemental material).

Fig 4

Inhibition of Ad3-, Ad7-, Ad11-, and Ad35-eGFP transduction in CHO-CD46#2 and A549 cells by recombinant Ad fiber knobs. Cells were preincubated for 1 h in the cold using a 5-fold dilution series of the individual FK proteins, followed by addition of the different eGFP-expressing vectors for another 1 h. (A) For CHO-CD46#2 cells, the viral inputs amounted to 29,600, 8,200, 657, and 1,088 vp/cell for Ad3-, Ad7-, Ad11-, and Ad35-eGFP, respectively. The virus input concentrations had been determined in preceding experiments and were chosen such that the unblocked transgene expression values amounted to fluorescence intensity values of about 200. eGFP analysis was performed 48 h p.i. FKs are color coded as follows: Ad5-FK in green, Ad3-FK in red, Ad7-FK in purple, Ad11-FK in cyan, and Ad35-FK in dark blue. (B) For A549 cells, virus inputs amounted to 14,800, 8,200, 1,314, 2,540, and 2,825 vp/cell for Ad3-, Ad7-, Ad11-, Ad35-, and Ad5-eGFP, respectively. Otherwise, the procedure was the same as that described above.

Fig 5

Subtracted SPR sensorgrams for Ad3-, Ad7-, Ad11-, and Ad35-FK interacting with CD46. Soluble receptor CD46ex-huFc was immobilized on a CM5 chip at high density in Fc2 (2,630 RU) and low density in Fc4 (345 RU). Control CARex-huFc was immobilized at high density in Fc1 (3,431 RU) and low density in Fc3 (278 RU). (A to D) FK analytes were injected over the sensor surface at 18.75 and 150 nM concentrations for Ad3-FK (A), Ad11-FK (C), and Ad35-FK (D) and at 11.07 and 88.59 nM for Ad7-FK (B), respectively. Association times were either 240 or 280 s. (E to H) Overlay of analyte responses for Ad3-, Ad7-, Ad11-, and Ad35-FK at different concentrations. Measurements were performed using the CD46 high-density 2,630-RU CM5 chip. Serial concentrations of 0.27, 0.82, 2.47, 7.41, 22.22, and 66.67 nM Ad3-FK (E), Ad7-FK (F), Ad11-FK (G), and Ad35-FK (H) were injected in HBS-P+ buffer at 30 μl/min under a contact time of 300 s and a dissociation time of 3,600 s (for better visibility, only four of the six binding curves are shown). Data evaluation was fitted globally by a two-stage reaction model with Biacore T100 evaluation software, and resulting kinetics/affinity results (listed in Table S1 in the supplemental material and summarized in Table 4).