Capsid region involved in hepatitis A virus binding to glycophorin A of the erythrocyte membrane

Abstract

Hepatitis A virus (HAV) has previously been reported to agglutinate human red blood cells at acidic pHs. Treatment of erythrocytes with different enzymes and chemical reagents indicated that HAV attachment is mediated through an interaction with sialylglycoproteins. HAV hemagglutination could be blocked by incubating the virus with glycophorin A, indicating that this sialylglycoprotein is the erythrocyte receptor. The number of receptors used was estimated to be around 500 per cell. At the same time, HAV-induced hemagglutination could also be blocked by either monoclonal antibody H7C27 or an anti-VP3(102-121) ascitic fluid, indicating that lysine 221 of VP1 and the surrounding VP3 residues lining the capsid pit are involved in HAV binding to erythrocytes.

FIG. 1.

Effect of pH on HAV hemagglutination. Two different human type-O erythrocytes were assayed with twofold dilutions of 150S or 70S HAV particles at pH 7.2 and 5.5.

FIG. 2.

Immuno-flow cytometry detection of adsorbed HAV 150S particles on human erythrocytes. (A) Typical immunodetection experiment. Two curves are superimposed. The open curve on the left corresponds to erythrocytes in the absence of viruses but immunoassayed for HAV; the shaded curve on the right corresponds to erythrocytes with adsorbed viruses that were immunoassayed for HAV. Cursor M includes the subpopulation of erythrocytes with the highest fluorescence labeling. (B) The number of HAV receptors on the erythrocyte membrane and the percentage of erythrocytes with adsorbed HAV particles are plotted versus the viral inoculum added, or MOA. Asterisks indicate mean values that are significantly different (P < 0.05).

FIG. 3.

Effects of several enzymatic and oxidation treatments of human erythrocytes on HAV-induced hemagglutination.

FIG. 4.

Effect of glycophorin A virus-blocking treatments on HAV-induced hemagglutination (and hemagglutination units).

FIG. 5.

Effects of several antibody blocking treatments on HAV-induced hemagglutination. Experiments used 4 (A) or 8 (B) hemagglutination units.

FIG. 6.

Reduction of antibody virus recognition by glycophorin A. Four hemagglutination units of 150S HAV particles was preincubated with soluble glycophorin A at 100 μg/ml and subsequently tested for recognition by MAbs H7C27 and K34C8 or the anti-VP3(102-121) antibody.