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.Hepatitis A virus (HAV) is a nonenveloped, positive-strand RNA virus classified as the type species of the genus Hepatovirus within the family Picornaviridae (45). Three types of intact antigenic HAV particles have been described: (i) RNA-containing capsids composed of proteins VP1, VP2, VP3, and possibly VP4 (virions); (ii) RNA-containing capsids composed of proteins VP1, VP0, and VP3 (provirions); and (iii) empty capsids with the same capsid composition as provirions but lacking RNA (procapsids) (8).HAV has a limited number of antigenic sites. The immunodominant site, composed of closely clustered epitopes, is defined by two major groups of escape mutants that include residues 70, 71, and 74 of VP3 and residues 102, 171, and 176 of VP1 (29, 30). There is another, apparently distinct antigenic site represented by mutants at residue 221 of VP1, and an additional and still undefined third antigenic site to which no escape mutant has so far been isolated (29,30). Several monoclonal antibodies (MAbs), such as K34C8, K24F2, and B5B3, are directed toward the immunodominant site, while MAbs H7C27 and MAK-4E7 are directed against the other two antigenic sites, respectively. Some of the epitopes contained in the immunodominant site, such as those defined by MAb K24F2, are detected in the first stages of capsid formation on the pentameric subunits, while others, such as those defined by MAb K34C8, are formed by structural changes during the assembly of pentamers into intact particles (40).A class I integral membrane glycoprotein of unknown natural function has been independently isolated by two groups from AGMK cells (22) and from a hybrid marmoset-Vero cell line (2) and has been characterized as a receptor for HAV (havcr-1). Additionally, a human homologue of this HAV receptor (huhavcr-1) has been isolated from human liver and kidney cells (15), and although nothing is known about its natural biological function, it has been reported to play a major role in T-cell helper differentiation and as an asthma determinant gene (27,28). On the other hand, like other picornaviruses, HAV binds to the surfaces of human erythrocytes, causing hemagglutination (13,14). This hemagglutination is optimally observed at pH 5.5.In the present work, we describe the nature of the receptor an...

Hepatitis A Virus Mutant Spectra under the Selective Pressure of Monoclonal Antibodies: Codon Usage Constraints Limit Capsid Variability

“…The immunodominant site is composed of multiple discontinuous epitopes defined by different MAbs. The glycophorin A binding site epitope defined solely by the H7C27 MAb is relevant to the antigenic structure of the capsid and also to its involvement in HAV spread in the host (36).…”

“…In the present work, the HAV mutant spectra at different passages under the immune pressure of two MAbs (K34C8 and H7C27) have been analyzed. These MAbs represent two distinct antigenic sites: the immunodominant site (K34C8) (31,41,42) and the glycophorin A binding site (H7C27) (36). The immunodominant site is composed of multiple discontinuous epitopes defined by different MAbs.…”

A Single Mutation in the Glycophorin A Binding Site of Hepatitis A Virus Enhances Virus Clearance from the Blood and Results in a Lower Fitness Variant

“…Erythrocyte glycoproteins may function as decoy receptors, attracting pathogens to the erythrocyte and keeping them away from target tissues (12). HAV interacts with glycophorin A of human erythrocytes, and the capsid region involved in this interaction is located around the putative pit area and coincides with the H7C27 MAb binding site (33). However, this interaction is optimal under acidic conditions and impaired at neutral physiological pH, suggesting that the actual HAV capsid conformation would allow escape from erythrocyte attachment.…”

“…Finally, pooled samples were concentrated by ultracentrifugation at 229,600 ϫ g for 4 h, and the pellet was recovered in a final volume of 500 l. Erythrocyte purification. Human type O erythrocytes (provided by the blood bank of the Hospital Vall d'Hebron, Barcelona, Spain) and erythrocytes from adult female Wistar rats were purified on preformed self-generated gradients of Percoll (Pharmacia Biotech) following the manufacturer's specifications, as previously described (33). Briefly, a gradient was formed by spinning 10 ml of a 70% solution of Percoll in 150 mM NaCl at 2,000 ϫ g for 15 min.…”

“…Hemagglutination assays were performed in 96-well U-bottom plates as previously described (33). Briefly, serial 2-fold dilutions of 150S particles were made in phosphate-buffered saline (PBS) at pH 5.5 or 7.2.…”