Entry of Duck Hepatitis B Virus into Primary Duck Liver and Kidney Cells after Discovery of a Fusogenic Region within the Large Surface Protein

Abstract

Hepatitis B viruses exhibit a narrow host range specificity that is believed to be mediated by a domain of the large surface protein, designated L. For duck hepatitis B virus, it has been shown that the pre-S domain of L binds to carboxypeptidase D, a cellular receptor present in many species on a wide variety of cell types. Nonetheless, only hepatocytes become infected. It has remained vague which viral features determine host range specificity and organotropicity. By using chymotrypsin to treat duck hepatitis B virus, we addressed the question of whether a putative fusogenic region within the amino-terminal end of the small surface protein may participate in viral entry and possibly constitute one of the determinants of the host range of the virus. Addition of the enzyme to virions resulted in increased infectivity. Remarkably, even remnants of enzyme-treated subviral particles proved to be inhibitory to infection. A noninfectious deletion mutant devoid of the binding region for carboxypeptidase D could be rendered infectious for primary duck hepatocytes by treatment with chymotrypsin. Although because of the protease treatment mutant and wild-type viruses may have become infectious in an unspecific and receptor-independent manner, their host range specificity was not affected, as shown by the inability of the virus to replicate in different hepatoma cell lines, as well as primary chicken hepatocytes. Instead, the organotropicity of the virus could be reduced, which was demonstrated by infection of primary duck kidney cells.The family Hepadnaviridae, with the human hepatitis B virus (HBV) as the prototype member, consists of small, DNAcontaining, enveloped viruses which cause liver diseases in humans, rodents, and birds (22). A characteristic feature of HBV and other hepadnaviruses is a strong species specificity where, e.g., HBV infects humans and chimpanzees (1) and duck HBV (DHBV) infects only certain duck and goose species (49). Moreover, all of the hepadnaviruses show relatively strict organotropicity, infecting mainly hepatocytes.Facilitated by the identification of neutralizing epitopes (12) and the allocation of host range-specific determinants to the pre-S region of L (29), it was possible to demonstrate the binding of DHBV to a putative hepatocellular receptor molecule via a certain domain on pre-S (7,8,30,39,63). This receptor molecule has been identified as gp180, a transmembrane protein of the carboxypeptidase D family that, however, has also been found in nonliver duck tissues that are not susceptible to DHBV infection (16,38,62). Furthermore, it has been shown that LMH cells, a chicken hepatoma cell line which resists infection but is capable of virus production after transfection of full-length DHBV DNA, also express gp180 (38). In addition, it was demonstrated that this molecule is concentrated in the Golgi apparatus, to cycle to and from the plasma membrane, where it colocalizes with pre-S (6, 7). Expression of duck gp180 in HuH7 cells, a human hepatoma cell line, led merely to internalizatio...

Structures and Mechanisms of Viral Membrane Fusion Proteins: Multiple Variations on a Common Theme

“…(j)These are tentative assignments based in part on observations suggesting a need for proteolytic processing within S during virus entry(Glebe and Urban, 2007;Li et al, 2004;Maenz et al, 2007).Crit Rev Biochem Mol Biol. Author manuscript; available in PMC 2009 May 1.…”

Requirement of Activation for Hepatitis B Virus Infection

“…2b). In this study we could find out three further details: first, an incubation time of HBV with LSECs of 24 h instead of 4 h did not lead to an increase of infectivity; second, transfer of HBV-incubated activator cells instead of activated virus was also able to initiate HBV-infection; third, to employ another cell type, primary duck kidney cells 8 , as activator cells would not support subsequent infection of 5 hepatoma cell lines. In a next experiment we replaced every 24 h the culture medium of HBV infected HepG2 cells for the calculation of the amount of the viral surface proteins with a commercially available anti-HBs-coated micro particle enzyme immunoassay (Abbott AXSYM system, Germany) as a measure of virus production per day (Fig.…”

Spectroscopic Characterization and Fusogenic Properties of PreS Domains of Duck Hepatitis B Virus

“…Little is known about the role of the different envelope proteins in the viral fusion mechanism. In HBV, a peptide comprising the 16 amino acids at the N-terminus of S protein has been shown to interact with model membranes, promoting liposome destabilization in a pH-dependent manner and adopting an extended conformation during the process. , The destabilization properties observed for the HBV fusion peptide could be extended to other members of the hepadnavirus family, such as DHBV and woodchuck hepatitis B virus. , Evidence for the role of the N-terminal S peptide in fusion has also been obtained by others, , who suggest that the exposure of this consensus fusion motif is important in hepadnavirus entry. The HBV preS domain is involved in the fusion of this virus with the plasma membrane of target cells.…”