The Human Homolog of HAVcr-1 Codes for a Hepatitis A Virus Cellular Receptor

Hepatitis A virus (HAV), a hepatotropic picornavirus, causes a medically important acute hepatitis. The first step in the life cycle of HAV is binding to a cell surface receptor that in African green monkey kidney (AGMK) cells is coded by HAVcr-1 (7). Monoclonal antibody (MAb) 190/4, which was used as a probe to molecularly clone the HAVcr-1 cDNA from an expression cDNA library of AGMK cells, blocks the binding of HAV and protects AGMK cells against HAV infection (7). Nucleotide sequence analysis showed that HAVcr-1 cDNA codes for a class I integral membrane glycoprotein, termed havcr-1, of unknown natural function. The extracellular domain of havcr-1 consists of an N-terminal cysteine-rich (Cys-rich) region followed by a threonine-, serine-, and proline-rich (TSP-rich) region. The havcr-1 Cys-rich region displays homology to members of the immunoglobulin (Ig) superfamily (7), and the TSP-rich region has the characteristics of mucin-like glycoproteins (15). The putative “lollipop-on-a-stick” structure (6) of havcr-1 suggested that the extended O-glycosylated TSP-rich region presents the Cys-rich globular domain above the cell surface and makes it accessible for interactions with extracellular molecules. We have recently shown that the Cys-rich region of havcr-1 and its first N-glycosylation site are required for the binding of HAV and protective MAb 190/4 (19), and we are currently analyzing whether the Cys-rich region of havcr-1 is sufficient for HAV receptor function. Receptor-negative cell lines that are otherwise fully susceptible to HAV replication have not yet been identified. Although we devoted considerable effort to isolating one, we have not been able to do so (4). We have previously shown that mouse and dog cells, which contain internal blocks to HAV replication, gain limited susceptibility to HAV infection upon transfection with the HAVcr-1 cDNA (7, 19). Infection of these mouse and dog cell transfectants with HAV resulted in low levels of the characteristic granular cytoplasmic fluorescence of HAV-infected cells, which lasted for several days but became undetectable after 1 month postinfection. This limited level of susceptibility of the mouse and dog cell transfectants to HAV infection resulted in only a <10-fold increase in HAV titers and a <2-fold increase in HAV-specific RNA (7, 19); therefore, its is likely that the input virus internalized through havcr-1 contributed to the HAV-specific fluorescence observed in the mouse and dog cell transfectants. Although further analysis of the HAV receptor function of havcr-1 awaits the isolation of nonsusceptible cells that could fully support HAV replication upon transfection of the HAVcr-1 cDNA, the mouse and dog cell transfectants allowed us to characterize the havcr-1-mediated binding of HAV, its internalization, and the limited level of susceptibility to HAV infection (5, 7, 19). Initial studies revealed that protective MAb 190/4 reacted with the cell surfaces of clone GL37 AGMK cells but not with the cell surfaces of HeLa cells (7). Therefore, it was of great interest to ascertain the existence of the human homolog of HAVcr-1 and determine its function as an HAV receptor and its role in the pathogenesis of HAV in humans. In this report, we describe the molecular cloning of the cDNA coding for the human homolog of HAVcr-1 (huHAVcr-1). Nucleotide sequence analysis revealed that the huHAVcr-1 cDNA codes for a glycoprotein, termed huhavcr-1, that is 79% identical to havcr-1. Northern blot analysis showed that huHAVcr-1 is expressed in every human organ analyzed, including the liver, small intestine, colon, and spleen, and that it is expressed at higher levels in the kidney and testis. Dog cells transfected with the huHAVcr-1 cDNA gained limited susceptibility to HAV infection, whereas dog cells transfected with vector alone or HAVcr-1 cDNA with the Cys-rich region deleted were resistant to HAV infection. These results suggest that huhavcr-1 is a receptor for HAV which may play a role in the pathogenesis of HAV in humans.