Role of cellular glycosaminoglycans and charged regions of viral G protein in human metapneumovirus infection

Abstract

Human metapneumovirus (hMPV) is an important cause of lower respiratory tract disease, particularly in infants and young children. hMPV has two major glycoproteins, G and F, which are responsible for virus attachment and membrane fusion, respectively. We investigated the role of cellular glycosaminoglycans (GAGs) and G protein in hMPV infection. The pretreatment of hMPV with soluble heparin markedly inhibited the infection of HEp-2 cells. Recombinant G protein, comprising the extracellular domain of G, bound to heparin-agarose columns and also to HEp-2 cells. hMPV infection and G protein binding to HEp-2 cells was inhibited by other soluble GAGs, including chondroitin sulfates, by the enzymatic removal of cell surface GAGs with GAG lyases or by the pretreatment of cells with basic fibroblast growth factor. The role of cellular GAGs was confirmed by the binding of G protein to wild-type CHO cells but not to GAG-deficient CHO-pgsA745 cells. An analysis of the G protein sequence revealed two adjacent clusters of positively charged amino acids ((149)EKKKTRA(155) and (159)QRRGKGKE(166)). Truncated G fragments were expressed, and only the fragment containing these putative heparin binding domains retained heparin binding. The alanine mutagenesis of charged residues in either of these regions resulted in the loss of binding to heparin and to HEp-2 cells, suggesting that both sites are likely to be required for hMPV attachment. These results, taken together with the inhibition of hMPV infection by soluble G protein, indicate an important role for G protein and cellular GAGs in hMPV infection.

FIG. 1.

Schematic diagram of recombinant G protein constructs. The putative heparin binding regions are indicated by the white bars. G-mut1 contained alanine substitutions of K151A, K153A, and R155A, and G-mut2 included alanine substitutions of R161A, K163A, and K165A. The lysine and arginine residues mutated to alanine are underlined. The truncated G constructs hMPV-G1 and hMPV-G2 used in this study also are shown.

FIG. 2.

Effect of heparin on hMPV infectivity. (A) HEp-2 cells were incubated for 2 h with heparin pretreated hMPV. Control cells were incubated with untreated virus (no heparin). Infectivity was assessed by ELISA 48 h postinoculation using a MAb against hMPV matrix protein. Uninfected Hep-2 cells also were treated with heparin or left untreated and analyzed by ELISA. Results shown are for triplicate wells of a representative experiment performed four times. OD 490 nm, optical density at 490 nm. (B) Dose-dependent inhibition of hMPV infectivity by heparin. Results are expressed as percent inhibition relative to values HEp-2 cells infected with untreated virus.

FIG. 3.

Western blot analysis following heparin chromatography of recombinant hMPV G. Recombinant proteins were applied to the heparin column four times, the unbound material was collected (fall-through), and the columns were washed with 10 column volumes of PB. The final wash (1 ml) was collected, and bound protein was eluted with a stepwise salt gradient. Fractions were analyzed by SDS-PAGE on a 12.5% gel under reducing conditions, followed by Western blot analysis. The lane designations are as follows: 1, start material; 2, fall-through; 3, final wash; 4 to 9, elution fractions.

FIG. 4.

Binding of G protein to HEp-2 cells. Confluent HEp-2 cell monolayers were incubated with increasing concentrations of biotinylated G protein at 37°C for 1 h in the presence or absence of heparin. After being washed, bound G protein was detected by incubation with streptavidin-HRP. OD 490 nm, optical density at 490 nm.

FIG. 5.

Effect of HS, CS-A, CS-B, CS-C, dextran sulfate (DS), and dextran (D) on hMPV infectivity and G protein binding. (A) hMPV was pretreated with various concentrations of GAGs prior to the inoculation of HEp-2 cells. Infectivity was assessed at 48 h. (B) Biotinylated G protein was added to HEp-2 cell monolayers in the presence or absence of soluble GAGs, and the binding of G protein was determined after 1 h. Results are expressed as the percent inhibition relative to that of untreated hMPV or G protein binding in the absence of GAGs.

FIG. 6.

Effect of the GAG lyase treatment of HEp-2 cells on virus infectivity and G protein binding. HEp-2 cells were treated with heparinase I, heparitinase, or chondroitinase ABC at 37°C for 1 h. Cells were washed three times with serum-free medium before infection with hMPV (A) or the addition of biotinylated G protein (B). hMPV infectivity and the binding of G protein was determined by ELISA. Results are expressed as the percent inhibition of infection or binding relative to that of untreated cells.

FIG. 7.

Effect of chemically modified heparins and bFGF on hMPV infection and G protein binding to HEp-2 cells. (A) hMPV was incubated with 10 or 500 μg/ml heparin (H), chemically modified heparins (de-O-sulfate [H-de-O] or de-N-sulfate [H-de-N]), or bFGF (0-5 μg/ml) for 30 min at 37°C prior to the inoculation of HEp-2 cells. Infectivity was examined 48 h postinoculation. (B) Biotinylated G protein was mixed with 10 or 500 μg/ml of heparin, chemically modified heparins, or bFGF (0 to 5 μg/ml) and incubated with HEp-2 cells. Binding was detected using streptavidin-HRP.

FIG. 8.

Binding of hMPV-G protein to wild-type CHO (CHO-wt) and mutant CHO-pgsA745 cells. The cells were incubated with increasing concentrations of biotinylated G protein at 37°C for 1 h in the presence or absence of heparin. After being washed, bound G protein was detected by incubation with streptavidin-HRP. OD 490 nm, optical density at 490 nm.

FIG. 9.

Binding of hMPV-G (G-wt), hMPV-G1 (G1), hMPV-G2 (G2), G-mut1, and G-mut2 to heparin and HEp-2 cells. (A) Proteins were passed over heparin columns, the unbound material was collected (fall through), and the columns were washed with PB. Bound protein was eluted with a stepwise salt gradient. Fractions were analyzed by SDS-12.5% PAGE and Western blotting. The lane designations are as follows: 1, start material; 2, fallthrough; 3, final wash; 4 to 9, elution fractions. (B) HEp-2 cells were incubated with biotinylated hMPV-G, hMPV-G1, hMPV-G2, G-mut1, or G-mut2 proteins at 37°C for 1 h. Bound G protein was detected by incubation with streptavidin-HRP. OD 490 nm, optical density at 490 nm.