Hepatitis A virus (HAV) infects African green monkey kidney cells via HAV cellular receptor 1 (havcr-1).The ectodomain of havcr-1 contains an N-terminal cysteine-rich immunoglobulin-like region (D1), followed by a mucin-like region that extends D1 well above the cell surface. D1 is required for binding of HAV, and a soluble construct containing D1 fused to the hinge and Fc portions of human immunoglobulin G1 (IgG1), D1-Fc, bound and neutralized HAV inefficiently. However, D1-Fc did not alter the virions. To determine whether additional regions of havcr-1 are required to trigger uncoating of HAV, we constructed D1muc-Fc containing D1 and two-thirds of the mucin-like region fused to the Fc and hinge portions of human IgG1. D1muc-Fc neutralized 10 times more HAV than did D1-Fc. Sedimentation analysis in sucrose gradients showed that treatment of HAV with 20 to 200 nM D1muc-Fc disrupted the majority of the virions, whereas treatment with 2 nM D1muc-Fc had no effect on the sedimentation of the particles. Treatment of HAV with 100 nM D1muc-Fc resulted in low-level accumulation of 100-to 125S particles. Negative-stain electron microscopy analysis revealed that the 100-to 125S particles had the characteristics of disrupted virions, such as internal staining and diffuse edges. Quantitative PCR analysis showed that the 100-to 125S particles contained viral RNA. These results indicate that D1 and the mucin-like region of havcr-1 are required to induce conformational changes leading to HAV uncoating.
Hepatitis A virus (HAV) is an atypical member of the familyPicornaviridae that causes acute hepatitis in humans (for a review, see reference 20). HAV has a positive-strand genomic RNA of approximately 7.5 kb that is covalently linked to a small virus-encoded VPg protein at its 5Ј end (38) and contains a poly(A) tail at its 3Ј end. The mature HAV capsid is formed by 60 copies of at least three viral proteins, VP1, VP2, and VP3. A small unmyristoylated protein, VP4, of 23 amino acids plays a signal role in capsid assembly (29) but has not been detected in mature virions. Nonstructural protein 2A remains associated with the structural proteins and serves as a signal for the assembly of pentamers, which are precursors involved in the morphogenesis of the capsid (29).Wild-type HAV usually does not grow in cell culture. The virus was adapted to in vitro growth by serial passage in cell cultures of primate origin, which resulted in the establishment of persistent infections and attenuation (7, 8, 10, 12-14, 17, 30). HAV has also been adapted to growth in guinea pig, pig, and dolphin cell cultures (11), indicating that the cellular factors required for HAV replication are not restricted to primates.Picornaviruses have different cell entry mechanisms. For instance, cellular receptors bind differently to a depression around the fivefold axis of poliovirus and the major group of rhinovirus (2, 18, 39) and induce conformational changes in the virions that result in the accumulation of 135S A particles and other uncoating intermediates (for...