Glycine Decarboxylase Mediates a Postbinding Step in Duck Hepatitis B Virus Infection

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Hepatitis B e antigen (HBeAg) is a secreted version of hepatitis B virus (HBV) core protein that promotes immune tolerance and persistent infection. It is derived from a translation product of the precore/core gene by two proteolytic cleavage events: removal of the amino-terminal signal peptide and removal of the carboxylterminal arginine-rich sequence. Four RXXR motifs are present at the carboxyl terminus of the HBeAg precursor, with the first two fused as 151 Approximately 2 billion people worldwide have been exposed to hepatitis B virus (HBV), and 350 million of them remain chronically infected. The long-term consequences of HBV infection include liver cirrhosis and hepatocellular carcinoma (21). The risk of developing chronic HBV infection correlates inversely with the age of transmission. In areas where HBV infection is endemic, such as China where over 10% of people are chronic carriers of the virus, HBV is transmitted both horizontally during early childhood and vertically from hepatitis B e antigen (HBeAg)-positive mothers during birth (68). In countries of intermediate HBV incidence such as South Africa, infection is acquired primarily horizontally during early childhood (28). In Western countries, HBV is transmitted mostly by drug abuse or unprotected sex during adulthood, and the majority of carriers will recover from the acute infection. In this regard, HBeAg is implicated as a major viral factor contributing to chronic infection following neonatal transmission (41).HBeAg was discovered by Magnius and Espmark over 30 years ago as a new serological marker for HBV infection (38), supplementing the then available assays for HBcAg (HBV core antigen or core protein) and HBsAg (HBV surface antigen or envelope protein). Soon after its discovery, the presence of HBeAg was found to correlate with liver diseases, whereas the loss of HBeAg followed by the rise of corresponding antibody (anti-HBe) was associated with normalization of liver function (22,65). Later studies further revealed a correlation of HBeAg with high viremia titer and anti-HBe with reduced viral load.

Section: Methodsmentioning

confidence: 99%

Characterization of Genotype-Specific Carboxyl-Terminal Cleavage Sites of Hepatitis B Virus e Antigen Precursor and Identification of Furin as the Candidate Enzyme

Ito

Kim

Lok

et al. 2009

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“…Although the LT1.4 mutant was shown to be able to bind to CPD, the block to infection could still result from the inability to bind to one of the proposed DHBV coreceptors (18). To provide further evidence that the infection block occurs at the stage of fusion, we monitored entry of mutant and wild-type colocalizes with transferrin after the 2-h infection period, indicating that the mutant enters the cell (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Hydrophobic Domain in the Large Envelope Protein Is Essential for Fusion of Duck Hepatitis B Virus at the Late Endosome

Chojnacki

Anderson

Grgacic³

2005

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three ␣-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.In order to initiate productive cell infection, enveloped viruses need to fuse to a host cell membrane in order to release a capsid into the cytoplasm. Details of this process have been well described for viruses such as the influenza A virus, human immunodeficiency virus, or tick-borne encephalitis virus. However, details of the virus fusion process in the Hepadnaviridae, a family of enveloped viruses that replicate in the liver, remain unknown. Studies of the early infection steps of hepatitis B virus (HBV) have been hampered by the lack of a suitable infection system. However, the study of duck hepatitis B virus (DHBV), which provides a primary cell culture infection system, has enabled elucidation of some viral-entry mechanisms, most notably the identification of the attachment receptor, carboxypeptidase D (CPD) (15,16,33,35).The DHBV envelope is comprised of two transmembrane proteins, the large (L) and small (S) surface proteins, that assemble into virions and subviral particles (SVPs). These proteins are translated by differential initiation from a single pre-S/S open reading frame. Hence, the L protein contains a 161-amino-acid (aa) pre-S extension to the common 167-aa C-terminal S domain sequence which comprises the S protein. Secondary-structure predictions suggest that the S protein and consequently the L protein have three ␣-helical, membranespanning domains, termed transmembrane domains one, two, and three (TM1, TM2, and TM3, respectively). The S protein forms the main structural component of the envel...

“…On the other hand, our experiments cannot rule out the existence of a further receptor recognition sequence that is exposed by CT treatment but remains masked when T is used. In this respect, the demonstration of a further cellular protein able to bind truncated pre-S molecules much more efficiently (43)(44)(45) opens the possibility of a receptor recognition cascade for viral entry of DHBV. Interestingly, it was not possible to infect human or chicken hepatoma cell lines with CT-treated DHBV, even though these cell lines are well known to allow the virus to replicate when they are transfected with DHBV expression constructs.…”

Section: Discussionmentioning

confidence: 99%

“…So, it was speculated that the initial bindingand-internalization event between L and gp180 must be followed by a species-and cell type-specific interaction with so-far-unknown cellular factors to allow infection (7, 64). Interestingly, another protein, the duck glycine decarboxylase, whose expression is restricted to the liver, kidney, and pancreas, coincides well with the organotropism of DHBV and binds truncated L more efficiently than the full-length polypeptide (43)(44)(45).From this point of view, it is not surprising that several observations indicate that cleavage of L, followed by exposure of a fusogenic region downstream of the cleavage site, could be a prerequisite step before infection can occur (54-56), as published for a couple of other viruses (10,19,21,26,46,59,60,65). Direct cell permeability of virus-like particles carrying the translocation motif of HBV on their surface could also be demonstrated (5).…”

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confidence: 99%

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confidence: 99%

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

Maenz

Chang

Iwanski

et al. 2007

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...