Envelope proteins of many viruses are cleaved by furin-like PCs. They include HIV gp160, cytomegalovirus glycoprotein B, mouse mammary tumor virus superantigen, influenza virus A hemagglutinin, measles virus F0, Newcastle disease virus F0, Sindbis virus gpE2, and parainfluenza virus F0 (see references 36
for reviews). In the case of HIV, the cleavage is mediated by the two ubiquitous PCs, furin and PC7 (18
). The N-terminal cleavage product (surface, or SU) is involved in receptor binding, whereas the C-terminal part (transmembrane, or TM) mediates membrane fusion. The lack of processed envelope proteins in mature hepadnavirus particles could be explained by the unique mechanism of hepadnaviral morphogenesis. The pre-S domain of the large envelope protein is cytosolically oriented to serve as a matrix protein during capsid envelopment, thus precluding its accessibility to PCs (5
). Only at a later stage would the pre-S domain of some large envelope proteins be translocated onto the virion surface, where it serves as the contact site for the viral receptor for the next round of infection. Thus, cleavage activation of the fusogenic peptide of viral envelope proteins may be postponed, such as after binding with a docking receptor, for this class of viruses. Identification of DGD as a binding partner for the truncated pre-S domain of the DHBV L envelope protein and demonstration of its requirement for productive DHBV infection of PDH reinforce this hypothesis (27
). In the present study, we provide several pieces of evidence to suggest that enzymatic cleavage, possibly by a furin-like PC, plays a critical role in the DHBV life cycle.
First, introduction of DCPD into liver- or kidney-derived human cell lines led to virus binding and internalization in the form of intact virions. However, the bound and/or internalized virus particles were rapidly released to the culture supernatant (Fig. ). Furin could cleave DCPD and increase cell-associated DHBV signals (Fig. and ). The cleavage site was located at residues 166 to 169 in the N terminus, because a double amino acid substitution prevented cleavage by chicken furin. In this regard, DCPD is composed of three domains, A, B, and C, each bearing similarity to carboxypeptidase N/E. Most of the DCPD molecule is extracellular, except for a single transmembrane segment followed by a cytoplasmic tail composed of the last 58 residues of domain C. Whereas the enzymatic activities reside in domains A and B, domain C mediates DHBV binding (10
). At present we do not know the subcellular location where DCPD is cleaved, whether this cleavage occurs prior to DCPD binding to DHBV particles, or whether it increases DHBV binding to domain C or rather slows down shedding of bound and/or internalized DHBV particles. It will be of interest to determine whether a DCPD mutant rendered resistant to furin cleavage acts in a dominant negative manner to interfere with DHBV infection of primary duck hepatocytes.
Second, we found endosomal colocalization of DCPD, DGD, and viral envelope proteins in the duck liver, suggesting the possible involvement of the endosome in switching DHBV binding from DCPD to DGD followed by membrane fusion. Recent studies by others also indicated that DHBV entry depends on trafficking to the endosomal compartment, where fusion is expected to occur (3
). Indeed, the endosomal fraction obtained from the susceptible duck liver could cleave DHBV L protein (Fig. to ), whereas the endosome prepared from the resistant LMH cell line could not (our unpublished observation). The requirements for low pH, divalent cations, and inhibition by decanoyl-RVKR-chloromethylketone are consistent with furin or a furin-like PC being the enzyme involved. At least some of the cleavage sites are located in the pre-S domain (Fig. and Fig. ), where four dibasic or tribasic residues are found. Since the cleavage product often showed a smearing pattern, possibly due to degradation of the initial cleavage product, the exact cleavage site(s) remains to be established. Generation of DHBV mutants with different sets of the basic residues mutated will help solve this issue. At any rate, such a cleavage event has biological significance, because DHBV particles pretreated with an endosomal enzyme(s) gained the ability to generate a small amount of ccc DNA in LMH cells (Fig. ). This result suggests that a small proportion of the processed virus particles successfully entered LMH cells and removed their envelopes, followed by nuclear migration of the relaxed circular DNA, removal of the DNA polymerase, and DNA repair and ligation. The RCA method we used to amplify the ccc DNA is specific, as demonstrated by the negative results when virus particles, whether or not pretreated with endosome, were used directly for amplification. It is a major improvement over the previous method amplifying the gap region of the replicative DNA (23
The relative low efficiency of ccc DNA formation by the endosome-treated DHBV particles in LMH cells could be attributed to many factors, such as the metastable nature of cleaved envelope proteins, trimming of the newly exposed basic residues by a carboxypeptidase such as DCPD (R101 and R102 are critical for DGD binding), lack of ccc DNA amplification, and additional restriction factors associated with the chicken hepatoma cell line. In this regard, it has been reported that Huh7 cells are permissive to HBV replication following lipid-mediated transfer of core particles to the cytosol (39
), suggesting a block at or before uncoating. It will be of interest to perform similar experiments with LMH cells to determine whether there are additional defects subsequent to uncoating.
Finally, we demonstrated that a furin inhibitor markedly suppressed productive DHBV infection of primary duck hepatocytes if added immediately following virus incubation. The same inhibitor added 2 days later had no effect. This provided strong evidence for the involvement of furin or a furin-like protease in the initiation of DHBV infection. Since this inhibitor, decanoyl-RVKR-chloromethylketone, could block both furin cleavage of DCPD and endosomal cleavage of DHBV envelope proteins in vitro, the result obtained does not distinguish the contribution of DCPD cleavage relative to that of envelope protein cleavage in the initiation of infection. We also observed that a higher dose of this inhibitor (100 μM) is needed to efficiently inhibit DHBV infection, which could be associated with the wide intracellular distribution and stability of the inhibitor in vivo. Nevertheless, no cytotoxic effect was observed in PDH treated with 100 μM inhibitor. Use of RNA interference technology is necessary to further establish whether furin per se or another PC is required for productive DHBV infection.
The requirement of protease cleavage for hepadnavirus infection has been suggested by several previous studies. Lu et al. reported that in vitro
treatment of HBV particles with bacterial V8 protease triggered infection of HepG2 cells, a human hepatoma cell line otherwise resistant to HBV infection (31
). It was proposed that cleavage by V8 protease exposes the fusion peptide in the amino-terminal region of the HBV S protein (40
). Another study from the same group revealed that a serine protease inhibitor Kazal (SPIK) was expressed at a 1,000-fold-higher concentration in HepG2 cells than in normal human liver, which may explain the lack of envelope protein cleavage in HepG2 cells (30
). Stoeckl and colleagues reported that treatment of HBV particles with endosomal enzymes from HepG2 cells resulted in the exposure of the so-called translocation motif and viral infection of Huh7 cells (44
). Similarly, treatment of DHBV particles with endosome derived from LMH cells enabled viral infectivity in LMH cells. However, we found that endosome prepared from LMH cells failed to cleave DHBV envelope proteins (our unpublished observation). Moreover, furin failed to cleave DCPD when introduced into LMH cells (Fig. ). These findings are more in line with the observations of Lu and colleagues regarding HBV and HepG2 cells. Furthermore, two recent reports demonstrated that the presumed translocation motif in HBV is dispensable for HBV infectivity (2
We have identified DCPD as a binding partner for the intact L protein and DGD as the binding partner for several truncated versions of the L protein. Moreover, we have demonstrated the importance of DGD in productive DHBV infection. The discovery that furin and an endosomal enzyme(s) could cleave DCPD and viral envelope proteins and the demonstration that such cleavages have functional consequences for ccc DNA formation and productive infection may provide the missing link between DCPD and DGD. We propose that DCPD serves as the DHBV docking receptor on the cell surface. Subsequent cleavage of DCPD and viral envelope proteins in the endosome switches the viral binding partner to DGD, which somehow leads to membrane fusion and uncoating. Further studies are needed to test this hypothesis and to elucidate the details of the early steps in the DHBV life cycle. Such studies will also shed light on the nature of the HBV receptor and cofactors.