Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis - PubMed (original) (raw)

Membrane cofactor protein is a receptor for adenoviruses associated with epidemic keratoconjunctivitis

Eugene Wu et al. J Virol. 2004 Apr.

Abstract

Subgroup D adenovirus (Ad) types 8, 19, and 37 (Ad8, -19, and -37, respectively) are causative agents of epidemic keratoconjunctivitis and genital tract infections. Previous studies showed that Ad37 binds to a 50-kDa membrane glycoprotein expressed on human ocular (conjunctival) cells. To identify and characterize the role of the 50-kDa glycoprotein in Ad37 infection, we partially purified this molecule from solubilized Chang C conjunctival cell membranes by using lentil lectin chromatography and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Liquid chromatography coupled to nano-electrospray ionization-tandem mass spectrometry was subsequently used to identify four Ad37 receptor candidates: CD46, CD87, CD98, and CD147. Immunodepletion analyses demonstrated that the 50-kDa protein is identical to CD46 (also known as membrane cofactor protein). The Ad37, but not Ad5, fiber knob bound to the extracellular domain of CD46, demonstrating a direct interaction of an Ad37 capsid protein with CD46. An antibody specific for the N-terminal 19 amino acids of CD46 also blocked Ad37 infection of human cervical carcinoma and conjunctival cells, indicating a requirement for CD46 in infection. Finally, expression of a 50-kDa isoform of human CD46 in a CD46-null cell line increased cell binding by wild-type Ad37 and gene delivery by an Ad vector pseudotyped with the Ad37 fiber, but not by a vector bearing the Ad5 fiber. Together, these studies demonstrate that CD46 serves as an attachment receptor for Ad37 and shed further light on the cell entry pathway of subgroup D Ads.

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Figures

FIG. 1.

FIG. 1.

Identification of the Ad37 receptor. (A) Membrane proteins from Chang C cells were probed on an Ad37-specific VOPBA in the presence (lanes 1 and 3) and absence (lanes 2 and 4) of 2 mM EDTA as described in Materials and Methods. Proteins in lanes 3 and 4 were reduced with β-mercaptoethanol prior to SDS-PAGE, while proteins in lanes 1 and 2 were not. The 45-, 50-, and 60-kDa proteins that bind Ad37 are indicated by arrows. (B) Soluble Chang membrane proteins (lane 1); proteins immunodepleted with antibodies directed against CD46, CD87, CD98, or CD147 (lanes 2 to 5, respectively); or proteins recovered in the immunoprecipitate (CD46, lane 6; CD87, lane 7; CD98, lane 8; CD147, lane 9) were probed with the Ad37 VOPBA. (C) The domain structure of CD46 is presented schematically. Horizontal lines in each SCR domain represent pairs of conserved disulfide bonds in the CD46 structure. Hexagonal chains represent locations of glycosylation. Four major isoforms (BC1, BC2, C1, and C2) are generated by alternative splicing of coding regions for serine/threonine/proline (STP)-rich domains (squares) and two possible cytoplasmic tails.

FIG. 2.

FIG. 2.

Purification and analysis of sCD46 binding to Ad fiber knobs. The extracellular domain of the C isoform of CD46 with a C-terminal hexahistidine tag was purified from 293EBNA cell culture medium by Ni-agarose affinity chromatography. Culture medium (lane 1), column flowthrough (lane 2), wash fraction (lane 3), and elution fractions 1 to 5 (lanes 4 to 8, respectively) were analyzed by SDS-PAGE (A) and anti-CD46 Western blotting (B). Binding of purified sCD46 to immoblized BSA or Ad37 or Ad5 fiber knobs in an ELISA (C). Data represent the averages and standard deviations of quadruplicate determinations.

FIG. 3.

FIG. 3.

Antibody specific for CD46 blocks Ad37 infection of Chang conjunctival and HeLa cervical epithelial cells. Chang conjunctival cells were preincubated with various concentrations of an antibody (Ab) specific for the N-terminal 19 amino acids of CD46 or CD55 (DAF) and then infected with 1,000 particles per cell of Ad vector pseudotyped with the Ad37 fiber (A). HeLa cells were preincubated with 100-μg/ml CD46 antibody or 1 mM V. cholerae neuraminidase prior to infection with Ad37 vector (B). Infection was measured as expression of GFP transgene by flow cytometry. Data represent the averages and standard deviations of triplicates.

FIG. 4.

FIG. 4.

CD46 expression selectively promotes Ad37 infection. CHO cells expressing the human DAF (CHO-DAF) (A) or the C2 isoform of CD46 (CHO-C2) (B) were analyzed by flow cytometry for CD46 (dark gray) and CD55 (light gray) expression with monoclonal antibodies. CHO-C2 cells or CHO-DAF cells were infected with Ad vectors carrying the fibers of Ad5 or Ad37 in the absence or presence of 100 μg of anti-CD46 antibody (Ab) per ml (C). GFP transgene expression was measured 24 h postinfection by flow cytometry. Data represent the averages and standard deviations of quadruplicates. CHO-DAF and CHO-C2 cells were incubated with wild-type Ad37 virus particles at 4°C for 1 h to measure virus binding to cells (D). Bound virus particles and cell counts were determined by quantitative PCR. Data represent the averages and standard deviations of triplicate or quadruplicate determinations of virus counts divided by cell counts (number of particles bound per cell).

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