Heparin-like structures on respiratory syncytial virus are involved in its infectivity in vitro

Abstract

Addition of heparin to the virus culture inhibited syncytial plaque formation due to respiratory syncytial virus (RSV). Moreover, pretreatment of the virus with heparinase or an inhibitor of heparin, protamine, greatly reduced virus infectivity. Two anti-heparan sulfate antibodies stained RSV-infected cells, but not noninfected cells, by immunofluorescence. One of the antibodies was capable of neutralizing RSV infection in vitro. These results prove that heparin-like structures identified on RSV play a major role in early stages of infection. The RSV G protein is the attachment protein. Both anti-heparan sulfate antibodies specifically bound to this protein. Enzymatic digestion of polysaccharides in the G protein reduced the binding, which indicates that heparin-like structures are on the G protein. Such oligosaccharides may therefore participate in the attachment of the virus.

FIG. 1

Effects of various concentrations of heparin, Fragmine, and heparan sulfate on RSV-induced plaque formation. Various concentrations of heparin (•), Fragmine (⧫), and heparan sulfate (▴) were incubated for 1 h at 37°C with 700 PFU of RSV per ml, and the ability to reduce virus infectivity was then assessed. mUI, international milliunits.

FIG. 2

Effect of the treatment of cells with various concentrations of heparin (○) and heparan sulfate (▵) on RSV infectivity. Cells were washed before infection. mUI, international milliunits.

FIG. 3

Effects of heparin added to RSV during different periods before inoculation or during inoculation. Symbols: ⧫, virus preincubated at 37°C for different times without heparin before inoculation; •, virus preincubated at 37°C for different times with heparin before inoculation; ■, heparin added at different times during inoculation, followed by washing of the cells before addition of agarose.

FIG. 4

Percent plaque reduction was determined per well after adsorption for 1.5 h at 37°C (A), 2 h at 37°C (B), 1 h at 4°C (C), 2 h at 4°C (D), and 2.5 h at 4°C (E). Heparin was added after adsorption and left during culture under agarose in the assays. No heparin was added after adsorption of the virus in the controls.

FIG. 5

Effect of heparinase treatment on RSV before adsorption. Symbols: ○, virus treated with heparinase before infection of cells with RSV; •, cells pretreated with heparinase and then washed before infection with RSV.

FIG. 6

Effect of protamine treatment on RSV before adsorption. Symbols: ⧫, virus treated with protamine before infection of cells with RSV. Symbols: •, cells pretreated with protamine and then washed before infection with RSV; ▵, virus treated with protamine solvent (glucose-cresol solution) before infection of cells with RSV; ○, cells pretreated with protamine solvent (glucose-cresol solution) and then washed before infection with RSV. UI, international units.

FIG. 7

Neutralizing effect of anti-HSPG antibody on RSV infectivity (▴) or on parainfluenza 3 virus infectivity (■).

FIG. 8

RSV-infected (A) or noninfected (B) HEp-2 cells stained with anti-HSPG antibody (left), F58-10E4 (middle), or HepSS-1 (right). Magnification; ×400. A positive reaction is indicated by green fluorescence.

FIG. 9

Binding of purified G protein (A), heparinase-treated G protein (B), and heparitinase-treated G protein (C) to antibody F58-10E4. The signal/background ratio was above 2.