The distal short consensus repeats 1 and 2 of the membrane cofactor protein CD46 and their distance from the cell membrane determine productive entry of species B adenovirus serotype 35

Abstract

The human regulator of complement activation membrane cofactor protein (CD46) has recently been identified as an attachment receptor for most species B adenoviruses (Ads), including Ad type 3 (Ad3), Ad11, and Ad35, as well as species D Ad37. To characterize the interaction between Ad35 and CD46, hybrid receptors composed of different CD46 short consensus repeat (SCR) domains fused to immunoglobulin-like domains of CD4 and a set of 36 CD46 mutants containing semiconservative changes of single amino acids within SCR domains I and II were tested in binding and in Ad35-mediated luciferase transduction assays. In addition, anti-CD46 antibodies and soluble polypeptides constituting various CD46 domains were used in binding inhibition studies. Our data indicate that (i) CD46 SCR I or SCR II alone confers low but significant Ad35 binding; (ii) the presence of SCR I and II is required for optimal binding and transgene expression; (iii) transduction efficiencies equivalent to that of full-length CD46 are obtained if SCR I and II are at an appropriate distance from the cell membrane; (iv) ablation of the N-glycan attached to SCR I has no influence on receptor function, whereas ablation of the SCR II N-glycan results in about a two- to threefold reduction of binding and transgene expression; (v) most putative Ad35 binding residues are located on the same solvent-exposed face of the SCR I or SCR II domain, which are twisted by about 90 degrees ; and (vi) the putative Ad35 binding sites partly overlap with the measles virus binding surface.

FIG. 1.

CD46-expressing cells infected by chimeric adenoviruses carrying B group fibers. Parental mouse B16 melanoma cells or stably transfected B16-CD46 cells expressing the BC1 splice form were incubated for 2 h with luciferase-expressing Ad5-Luc or Ad5-chimeric viruses at an MOI of 1,000 vp/cell. Cells were washed, and transgene expression was analyzed 2 days p.i. Luciferase activity is expressed in relative light units.

FIG. 2.

Inhibition of Ad35 binding to CD46 by anti-CD46 antibodies and SCR domain polypeptides. (A) Ad35 binding inhibition by CD46-specific antibodies. BHK-CD46 cells were incubated with the indicated CD46-specific antibodies, control anti-CAR antibody E1-1, or a 30-fold excess of cold Ad35, followed by the addition of [³H]Ad35 at 4°C. Virus binding was measured by liquid scintillation counting. The data were normalized to the amounts of virus bound in the absence of inhibitors and are represented as averages and standard deviations for triplicate determinations. The asterisks indicate the level of significance (P < 0.05 [single asterisk] and P < 0.005 [double asterisk] for comparison with the E1-1 negative control). (B) Summary of binding inhibition data with monoclonal antibodies. Percentage of residual binding is depicted as follows: >90%, □; 76 to 90%, □▪; 51 to 75%, □▪; <25%, ▪. Amino acid residues mainly involved in antibody binding sites and their positions are indicated. For further details see Fig. 5 and reference . (C) Ad35 binding inhibition by SCR domain polypeptides. BHK-CD46 cells were incubated with the indicated SCR domain polypeptides or control CARex-Fc, followed by the addition of [³H]Ad35. For the lower five measurements, equimolar amounts of the different proteins were applied. The subsequent processing was as described for panel A.

FIG. 3.

Ad35 binding efficiencies and transgene expression mediated by CD46-CD4 hybrid proteins. Proteins were transiently expressed in Ltk⁻ cells by using the VV-T7 system. CD46 surface expression levels of the various CD46-CD4 hybrid constructs were normalized to the surface expression levels of CD46-BC1 (data from one representative experiment). Binding efficiencies were calculatedby dividing the [³H]Ad35 binding values by surface expression levels and are shown in relation to that of the CD46-BC1 standard. Likewise, luciferase transgene expression measured by infection of the CD46-expressing cells with Ad35.ΔE3.Luc at 24 h posttransduction was determined by normalizing to protein concentration and surface expression of the CD46 proteins, attributing 100% to the BHK cells expressing CD46 (BC1). Measurements were done in triplicate and repeated at least three times. Schematics indicate the following: SCR I, black; SCR II, crosshatched; SCR III, light grey; and SCR IV, white. The CD4 Ig-like domain is drawn as a loop. N-linked oligosaccharides of SCR domains are indicated, whereas the single oligosaccharide of the CD4 Ig-like domain is omitted, as this sugar is present in all constructs. (A) Function of four hybrid proteins containing SCR I-IV fused to increasing numbers of CD4 Ig-like domains. (B) Function of six hybrid proteins containing SCR I-II fused to increasing numbers of CD4 Ig-like domains or combined with mutations ablating N-linked oligosaccharide in SCR I (dg1) or SCR II (dg2). (C) Function of seven hybrid constructs consisting of single SCR domains, or containing duplicated or exchanged intracysteine segments, fused to a constant CD4 Ig-like domain.

FIG. 4.

Ad35 binding efficiencies mediated by 36 single-amino-acid SCR I-II mutant proteins. (A) Nineteen single-amino-acid mutants plus the exchange mutant P39-T44, containing six alanines replacing the endogenous sequence localized in SCR I, were analyzed as described for Fig. 3. (B) Seventeen single-amino-acid mutants of SCR II were similarly analyzed. The asterisks indicate the level of significance (P < 0.05 compared to wt CD46).

FIG. 5.

Localization of Ad35 (A to D) or MV hemagglutinin (E to H) binding to the CD46 SCR I-II structure. Four views, each twisted by 90°, of a space-filling drawing of the CD46 SCRI-II structure (6) were prepared with the program MOLMOL (31), using the Brookhaven protein data bank (code 1ckl). Data for MV binding sites are taken from references (, , , and 46). Light blue, SCR I; dark blue, SCR II; green, N-glycans; light brown, residues involved in antibody binding of 13/42 (SCR I) and M177 (SCR II); pink, 80% to 100% loss of MV binding in ER24/25AA and P39-44T mutant; red and dark yellow, 31% to 50% and 20% to 30% loss of binding in single point mutants, respectively; bright yellow, binding stretches defined by peptide inhibition studies for MV. For details, see the text.