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, DNA-containing, 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 internalization of DHBV but not production of viral progeny. So, it was speculated that the initial binding-and-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 (43-45).

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). Whether a comparable event takes place with DHBV at the hepatocellular surface or whether fusion occurs during trafficking from early to late endosomes, as recently demonstrated (13), remains enigmatic, as the data so far published have a drawback in common. (i) All analyses were performed in the presence of so-called subviral particles (SVPs), which are always present in a vast majority of virions; (ii) because of the nearly identical ratio of the small and large envelope proteins (S and L) in both particles, it is automatically assumed that the localizations of these proteins are also identical; and (iii) as a consequence of the last argument, a common pathway for DHBV virions and SVPs is postulated.

For a better understanding of the early events of infection with the aim to find out whether fusion between viral and cellular membranes or translocation through cellular membranes could signify one essential step in this process, we altered DHBV virions by digesting intact particles with the protease chymotrypsin (CT). This enzymatic treatment resulted in particles with an exposed putative fusogenic region that infected not only primary duck hepatocytes (PDHs) but also primary duck kidney cells (PDKs), which are insusceptible to wild-type virus infection. This implies a mechanism by which the viral receptor region of L may have to be altered in the process of infection to expose its fusogenic region for viral entry into the cytoplasm. As other hepatoma cell lines and primary chicken hepatocytes (PCHs) cannot be infected by CT-treated virus, overcoming of the host cell membrane appears to be an organ-specific restriction to infection by DHBV, whereas species-specific restrictions are supposedly regulated by other mechanisms.

MATERIALS AND METHODS

Cells and virus.

Preparation of PDHs from fetal ducks was performed according to a method already described (8, 37), where the PDHs were isolated by digestion of dissociated liver tissue with 0.5% collagenase (Sigma, Deisenhofen, Germany) for 1 h at 30°C and resuspended in Williams’ medium E (WME; GIBCO, Eggenstein, Germany) (2, 66). Thereafter, the cultures were maintained in WME with 1.5% dimethyl sulfoxide (DMSO; Sigma) and supplements and cultivated at 37°C with 5% CO₂ as previously described (8, 31, 52).

Isolation of PDKs was carried out after removal of the liver and the gut by scraping off the small segmented kidneys from the inner back of the abdomen. The dissociated kidney tissues were trypsinized for 0.5 h at room temperature, washed, and divided into equal aliquots to be cultivated and passaged (i) in WME in the presence of DMSO, (ii) in Dulbecco's modified Eagle medium (D-MEM; GIBCO) (15) supplemented with 10% fetal calf serum (FCS), or (iii) in D-MEM with 10% fetal duck serum (FDS). The addition of DMSO was a good way to prevent possible contamination with hepatocytes because this supplement, used at a concentration that is beneficial for liver cell cultures, proved to be toxic for other cell types. PCHs were prepared according to the method described for the isolation of PDHs, except that chicken eggs were hatched for only 14 days.

The cells (PDHs, PDKs, and PCHs) were infected for 4 h on a rocking table at room temperature with 0.2 ml WME containing purified wild-type DHBV isolate 3 (DHBV-3) or overnight at 37°C with 0.5 ml WME containing DHBV-26 deletion mutants (17). The infection studies, if not mentioned otherwise, were usually performed with purified DHBV at a multiplicity of infection (MOI) of 1. After virus adsorption, cells were washed twice and then incubated for 7 days, whereupon supernatants and cells were harvested separately.

Enzyme treatment of purified viral particles.

Virus purification from sera of carrier ducks was performed as described earlier (8, 18). In some experiments, virus was obtained from the supernatants of the chicken hepatoma cell line LMH (33) transfected with tandem-DNA constructs of wild-type DHBV-3 (WT3T) or with the different genome length DNAs of DHBpreS deletion mutants (see below).

Purified DHBV particles were, if not stated otherwise, treated with 100 μg/ml CT in phosphate-buffered saline (PBS), pH 7.5, for 0.5 h at room temperature. Thereafter, digested virus particles were ultracentrifuged in a 0 to 70% sucrose gradient at 200,000 × g for 4 h at 4°C in an SW55 rotor (Beckman) to remove degraded products. For dephosphorylation, purified CT-treated or mock-treated virus particles were alternatively transferred to 1 U/ml acid phosphatase or buffer, similar to the protocol already described (18, 24). In order to diminish protease activity, it turned out to be beneficial to precipitate the samples directly after phosphatase treatment by addition of 10% ice-cold trichloroacetic acid and then wash them two times with ice-cold ethanol. In some cases, CT at different concentrations was, together with virus, directly added to PDHs. All enzymes were purchased from Sigma.

Preparation and analysis of DHBV DNA.

Viral DNA replicative intermediates were isolated from infected cells essentially as previously described (8, 17). For detection of DHBV, DHBV-3 plasmid DNA was labeled with [γ-³²P]dCTP (Megaprime; Amersham Buchler) and used for hybridization at >10⁶ cpm/ml. The same hybridization protocol was used for DNA dot blot assays, where a serial dilution of DHBV-3 containing plasmid DNA was used as a mass standard and where the number of viral genome molecules was considered equivalent to the number of virions since viremic sera seem to contain very few defective genomes (32).

Detection of DHBV envelope proteins.

Purified virus and DHBV-infected cells were analyzed after dissolving the samples in double-concentrated disruption buffer containing 4% sodium dodecyl sulfate and 10% 2-mercaptoethanol, whereupon proteins were separated by polyacrylamide gel electrophoresis essentially according to the method published by Laemmli (40), in 5 to 20% gradient slab gels with a stacking gel on top. For Western blot analysis, proteins separated by electrophoresis were transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA) and viral antigens were detected by antibodies against recombinant DHBpreS (17) and DHBs (35), followed by peroxidase-conjugated secondary antibodies (Dianova, Hamburg, Germany). The bands were visualized by the enhanced chemiluminescence method according to the manufacturer's protocol (Pierce, Rockford, IL).

Immunofluorescent staining of infected cells.

Indirect immunofluorescence studies were carried out with PDHs 3 days after infection. Cell layers grown on coverslips were washed with cold PBS, fixed for 5 min in methanol and for 4 s in acetone at −20°C, and air dried. Fixed cells were blocked with 1% bovine serum albumin in 0.1% Tween 20 in PBS (PBS-Tween), incubated for 1 h with anti-DHBpreS antiserum, washed three times with PBS-Tween, and then incubated with Alexa Fluor 488-conjugated secondary antibody. Cell nuclei were counterstained with Hoechst 33342 (Invitrogen, Karlsruhe, Germany). Cells were mounted with Kaiser's gelatin (Merck, Darmstadt, Germany).

Transfection experiments.

Conditions for the production of wild-type DHBV (WT3T) or mutant DHBV particles (D813, D864, D984, D1020, D1053, D1119, and D1176) were exactly as described earlier (8, 17). In brief, 10 μg of the plasmids previously linearized by EcoRI digestion were transfected by calcium phosphate precipitation (14, 23) into LMH cells grown in D-MEM-F-12 medium (GIBCO) (28) in the presence of 10% FCS. The cell culture media were harvested 4 and 6 days after transfection, and virus particles were concentrated by ultracentrifugation. After resolubilization, virus was used directly for infection studies or separated into virions and SVPs as previously described (8, 18).

RESULTS

CT-treated SVPs inhibit infection of CT-treated DHBV.

Previous experiments have shown that treatment of purified DHBV virions with CT leads to complete removal of pre-S subunits from the viral surface (18), thereby exposing an area designated transmembrane domain 1 overlapping a putative fusogenic region (see Fig. 5a) (54-56). It is recognized that in HBV, a similar sequence, a translocation motif, directs translocation through cellular membranes (5). If the same were true of transmembrane domain 1, infection efficacy should be raised by its exposure. We added, in a first attempt, different concentrations of CT directly together with a native DHBV particle population of virions and SVPs to the culture medium of PDHs according to a method where trypsin (T) was included in the medium of chicken embryonic cells during infection with influenza virus (34, 41). It became evident that addition of 10 μg CT per well totally inhibited infection, although the relatively large amount of protease did not diminish the quantity of PDHs (Fig. 1a). One possible explanation for the reduced infection in the presence of CT is that the detached pre-S bound the receptor but lost interaction with the virion for its entry (16, 29, 30, 38, 39, 45). Other data also demonstrated an inhibitory effect of pre-S peptides on the infection of PDHs with DHBV by competition of receptor binding (64; M. Bruns, unpublished data). The presence of CT during infection could also have destroyed a cellular receptor protein needed for binding of DHBV. However, when mixed populations of purified enzyme- or buffer-treated DHBV virions and SVPs were used after separation from free peptides and proteins through a sucrose gradient (Fig. 1b), infection was only obtained with the latter preparation (Fig. 1c). Thus, manipulation of the cellular receptor by CT or the excess of free pre-S cannot be the explanation for the inhibition of infection observed.

FIG. 5.

Infectivity of CT- and T-treated DHBV in PDHs. (a) Sequence alignment of the putative fusogenic regions within the envelope proteins of different avian hepadnaviruses and HBV. The displayed sequences, starting 2 aa before the initiation site for P18, harbor recognition sites for T (lysine, K) and CT (phenylalanine, F) at the transition of the pre-S and S domains. The start of the transmembrane domain and the highly conserved putative fusogenic GILAGLIGLLV/g region of some avihepadnaviruses and the GFLGPLLVLQAG region of HBV strain ayw are shown. Note the high conservation of amino acids between the sequences of avian hepadnaviruses illustrated with capital letters. The bold capital M represents the starting point for S, whereas the bold capitals A and G symbolize the minimal requirement of amino acids for fusogenic activity within the members of the Hepadnaviridae family. (b) Purified virus was incubated with CT (lanes 2 and 3) or T (lanes 4 and 5). Following ultracentrifugation, two DNA peaks of different densities for each treatment could be identified, which were again classified as A and B. The collected DNA-containing fractions from peaks A and B were incubated with PDHs for the analysis of infectivity by Western blotting for the expression of L. Infectivity was low for peak A (lane 2) and high for peak B (lane 3) of CT-treated virions. Almost no infectivity was found for peak A (lane 4) or peak B (lane 5) of T-treated DHBV. DHBV-positive duck serum (DS) and uninfected PDHs (lane 1), as well as PDHs infected with DHBV at an MOI of 1 (lane 6), served as controls.

FIG. 1.

Influence of CT treatment on the infection of PDHs with DHBV. (a) PDHs were infected 1 day after plating in a six-well plate with DHBV from duck serum at an MOI of 0 (mock infection; lanes 1, 4, and 7), an MOI of 0.1 (lanes 2, 5, and 8), and an MOI of 1 (lanes 3, 6, and 9) with CT added at concentrations of 0 (lanes 1 to 3), 1 (lanes 4 to 6), or 10 (lanes 7 to 9) μg/well. Each well contained, in total, 10 μl of duck serum. Cells were harvested 7 days after infection, and viral protein synthesis was studied by Western blot assay with anti-DHBpreS antiserum (left). Thereafter, blots were stained with amido black to compare the amounts of cellular proteins in the lysates (right). (b) Mixtures, at a ratio of 1:1, of gradient-purified virions and SVPs were treated with buffer (B), 100 μg CT, or T and then repurified over a sucrose gradient; thereafter, the fractions were analyzed for DHBV proteins by Western blotting with anti-DHBpreS antiserum. (c) DHBV-positive fractions were pooled and examined for infectivity in cultures of PDHs. Note that only a buffer-treated DHBV mixture was infectious. DS, DHBV-positive duck serum.

In purified DHBV virions, the majority of the pre-S domains of L are located on the outside.

In contrast to findings obtained with purified SVPs (18), the analysis of L of purified DHBV virions after CT digestion showed almost all of the P36 cleaved, suggesting an outward orientation of the vast majority of pre-S domains (Fig. 2). For the following infection experiment, we used purified DHBV virions and incubated them in PBS (Fig. 2a), CT (Fig. 2b), or a combination of 2% NP-40 and 1 M KCl (Fig. 2c), which dissolves the viral envelope (9). After the incubation period, the virus was run through a 0 to 70% sucrose gradient to separate detached peptides and envelopes from capsid-containing particles. The DNA peak of PBS-treated virus was concentrated in fractions 10 and 11 at a density of 1.17 g/cm³ (Fig. 2a, top), formerly shown to be typical for intact virions (11). When the same gradient was analyzed with an anti-DHBs antiserum by Western blotting, S and L could be detected in the same fractions, as expected (Fig. 2a, middle). Thus, fractions 10 and 11 contained infectious virus, which could be proven by the detection of newly synthesized L in PDHs 7 days after infection (Fig. 2a, bottom). In the fractions of the CT gradient, nearly no P36 but major digestion products of 21 and 17 kDa and some intermediate fragments could be detected (Fig. 2b, middle). Furthermore, the greatest amount of viral DNA could be found in fractions 10 and 11 (peak A), with a density of 1.13 g/cm³, which was slightly lower than the density of the peak fraction of buffer-treated virions; fractions 12 to 14 contained less DNA (peak B) at a density of 1.10 g/cm³ (Fig. 2b, top). Surprisingly, CT-treated DHBV virions showed a high level of infectivity, with most of the infectious virus originating from the DNA peak B, which contained about 10-fold less DNA than the DNA peak of PBS-treated virus (Fig. 2b, bottom).

FIG. 2.

Comparative analysis of DHBV DNA and envelope protein peaks in sucrose gradients and infectivity of differently treated DHBV virions. Purified virions were incubated with buffer (a), CT (b), or a combination of a high salt concentration and detergent (c). After treatment, the virus was run through a 0 to 70% sucrose gradient and fractionated. Two times, 1 μl of each fraction was measured for its content of viral DNA as shown in the diagrams (upper row), Ten microliters of each fraction was subjected to protein analysis by polyacrylamide gel electrophoresis with anti-DHBs antiserum in Western blot assays (middle row), and 100 μl was administered to PDHs grown on six-well plates for examination of viral infection with anti-DHBpreS antiserum (lower row; the exposure times of the three membranes were identical). The asterisks above the Western blots (middle row) mark the fractions with the highest DNA contents. S, viral marker proteins from DHBV-positive duck serum; H, viral marker proteins from infected duck hepatocytes.

In order to find out whether minor amounts of nondigested full-length L could be responsible for infection, we used buffer- or enzyme-treated DHBV in a neutralization test with anti-DHBpreS antiserum (Fig. 3). The results revealed that only infectivity of buffer-treated DHBV could be inactivated by neutralizing antibodies. After treatment of purified virions with a high salt concentration and detergent (Fig. 2c), the DNA peak migrated during ultracentrifugation to a position of high density (1.22 g/cm³), where, according to our experience (11), viral nucleocapsids are found (Fig. 2c, top). Detached viral envelope components migrated to a position of less than 1.07 g/cm³ (Fig. 2c, middle). Infectivity of any of the fractions of NP-40-treated virions could not be observed (Fig. 2c, bottom). This point supported the general idea that parts of the DHBV envelope proteins are indispensable for the infection process and nucleocapsids by themselves do not lead to productive infection.

FIG. 3.

Neutralization tests with anti-DHBpreS antiserum. Mock-treated (lanes 1 and 2) or CT-treated (lanes 3 and 4) purified virions (each lane containing 10⁸ viral genome equivalents) were incubated for 1 h at 37°C with either 2 μl of normal rabbit serum (lanes 2 and 4) or anti-DHBpreS antiserum (lanes 1 and 3) and thereafter used for infection of PDHs; note that the infectivity of enzyme-treated virions could not be neutralized with anti-pre-S antiserum; CO, uninfected PDHs.

The envelopes of peak A and B DHBV virions are different.

The next test (Fig. 4) was performed to find an explanation for the unexpected finding that incubation with CT obviously produced two kinds of DNA-containing viral particles, differing in density and infectivity. We figured that a reason for their different behavior should be disclosed by comparing the surface features of the particles because we could never observe different virion peaks when buffer-treated DHBV was analyzed. Furthermore, we were particularly interested in phosphorylation since it is known that L is phosphorylated at different positions (4, 18, 24, 57). Therefore, purified, untreated DHBV and the two different peaks of CT-treated DHBV were analyzed in parallel by incubation with buffer or acid phosphatase (Fig. 4). The subsequent Western blot assay with anti-DHBs antibody disclosed that in mock-treated peak A, bands could be detected that corresponded to phosphorylated P28 (a proteolytic cleavage product of P36) and at least three other presumably phosphorylated species at 17.5, 18, and 23 kDa (Fig. 4, lane 3), which were not detectable after phosphatase treatment (Fig. 4, lane 4). Significantly, these bands were not visible in peak B before and after phosphatase treatment (Fig. 4, lanes 5 and 6). Residual phosphorylation of truncated L could thus lead to different densities of virions. On the basis of these findings, the reason for the elevated infectivity of virions in peak B could have been the lack of phosphorylated cleavage products of L.

FIG. 4.

Dephosphorylation of untreated DHBV and of peaks A and B of CT-treated DHBV. Collected DNA-positive fractions of untreated virions (lanes 1 and 2) or collected fractions of peak A (lanes 3 and 4) and peak B (lanes 5 and 6) of CT-treated virions were either mock treated (lanes 1, 3, and 5) or incubated with acid phosphatase (lanes 2, 4, and 6). Proteins were examined by Western blotting with anti-DHBs antiserum. Possible phosphorylated products were designated PP36 and PP28. PP36 could be found in the original preparation of DHBV (lane 1) and directly eliminated by phosphatase treatment (lane 2), whereas PP28 could only be dephosphorylated after incubation of the virions with CT (lane 4). Nontreated peak A contained other phosphorylated fragments (lane 3, arrowheads) that were no longer visible after phosphatase treatment (lane 4). Corresponding phosphorylated proteins were not present in peak B, either incubated with phosphatase (lane 6) or not (lane 5). Although the same quantity of virus was used for electrophoresis, a slight reduction in the amount of proteins after phosphatase treatment was observed, which can be interpreted as the influence of some contaminating protease activities.

T treatment does not conserve the infectivity of DHBV.

T is another serine protease with a DHBpreS cleavage site in close proximity to the CT cleavage site at amino acid (aa) 167 (Fig. 5a). A comparison of the expected recognition sites for T and CT at the transition sequence from the pre-S domain to the S domain of L was performed for different selected avihepadnaviruses (Fig. 5a), i.e., isolates 26 and 3 of DHBV, clones 1 to 5 of snow goose HBV (11), Ross goose HBV (J. Newbold, personal communication), isolate 4 of heron HBV (51, 58), and clone 21 of stork HBV (53). CT preferentially cleaves after aromatic and large hydrophobic side chains of phenylalanine (Phe, F), tyrosine (Tyr, Y), and tryptophan (Trp, W). T cuts after positively charged lysine (Lys, K) and arginine (Arg, R) side chains and, in all of the listed avihepadnaviruses, directly in front of methionine (M), leaving P18 intact, whereas CT further removes the first four amino acids of P18, creating a cleavage product of 17 kDa (compare the cleavage patterns of CT- and T-treated DHBV in Fig. 1b). If CT was able to expose the fusogenic region of S, then this could also be the case for T. So we compared the infectivities of purified DHBV virions after incubation with T and CT by infecting PDHs with gradient DNA peaks A and B. Again, especially peak B of CT-treated virions showed strong infectivity, whereas peak A, as well as peaks A and B of T-treated virus, revealed only very poor infectivity, if any at all (Fig. 5b). Altogether, our observations suggest that the fusogenic site can only be presented properly when the pre-S domain and a small part of S are cleaved of, as is the case for cleavage with CT, leaving a 17-kDa fragment behind. The additional amino acids of L left after incubation with T could hinder entry of the virions.

CT-treated DHBV is not able to infect hepatoma cell lines or PCHs.

If the assumption of fusion at the cell membrane is true, then one should expect that the infectivity created enzymatically is rather unspecific, as the pre-S subunit of L, which is reported to be responsible for the recognition of the cellular DHBV receptor (29, 30, 38, 39, 45, 63), would be lost. Hence, in that case, viral uptake would not be receptor mediated but should take place via fusion of the cellular membrane with the viral envelope. Thus, we anticipated that if host range specificity and/or organotropicity are in fact conveyed by pre-S, then treatment of virus with CT should confer “infectivity” for a variety of other cells. We first attempted to infect the human hepatoma cell lines Huh7 (50) and HepG2 (36) (not shown) and the chicken hepatoma cell line LMH (33). We could not observe virus replication in these cells, although we used MOIs of up to 100 (Fig. 6a). This let us believe that for replication of DHBV in HuH7, HepG2, and LMH cells, either certain species-specific factors are missing or there is an excess of factors that inhibit, e.g., the necessary transport of the viral DNA to the nucleus.

FIG. 6.

Infection studies with untreated or CT-treated DHBV in cells of a hepatoma cell line or primary hepatocytes. (a) Infection of LMH cells with CT-treated DHBV virions. Purified virions were treated with 100 μg (lanes 1 to 3), 10 μg (lanes 5 to 7), or 1 μg (lanes 9 to 11) of CT or mock treated (lanes 13 to 15) and used for infections at MOIs of 1, 10, and 100; MOI 0 (lanes 4, 8, 12, and 16), uninfected control cells. (b) PCHs (left) and PDHs (right) were infected with untreated DHBV (lane 3) or with peak A (lane 4) or B (lane 5) of CT-treated virus. As controls, mock-infected cells (lane 6) and cells incubated with about 1,000-fold greater amounts of untreated (lane 1) or CT-treated SVPs (lane 2) were included to demonstrate that no viral input was still bound to the cellular surface at harvest. The strong background bands in PCHs and LMH cells detectable with anti-DHBpreS antiserum are the consequence of an extended exposure time. DS, DHBV-positive duck serum.

In order to find out whether the primary hepatocyte expression state, rather than species-specific factors, is necessary for CT-treated DHBV to infect, we prepared PCHs from embryonated chicken eggs and compared their susceptibility to infection with that of PDHs (Fig. 6b). This experiment demonstrated that although the pre-S domain was eliminated, species specificity of DHBV could not be overcome for a relatively closely related species like chickens. As especially LMH cells very efficiently replicate DHBV once the genome has been established in the nucleus via transfection, the crucial step that prevents infection of these cells by CT-treated DHBV must be situated between uptake of virions and establishment of the complete viral genome (covalently closed circular DNA) within the nucleus.

CT-treated DHBV can infect PDKs.

Next, we asked whether fusion of CT-treated DHBV with duck cells other than hepatocytes like PDKs leads to infection. To assess the culture conditions best suited for PDKs, we seeded comparable cell numbers in medium containing either DMSO, FCS, or FDS (for details, see Materials and Methods) and monitored the cultures over several passages (Fig. 7a). PDKs did not survive the third passage in WME plus DMSO. In contrast, culture in D-MEM containing serum led to the growth of homogeneous cell layers with strong similarities in growth characteristics and microscopic appearance. FDS was used as an alternative to FCS because in our experience and as reported by others, the appearance and infectivity of PDHs depend on the culture medium (52; Bruns, unpublished).

FIG. 7.

Analysis of the infectivity of CT-treated DHBV for PDKs. (a) Comparison of cell growth for PDKs of the second passage maintained in WME plus DMSO (left), D-MEM plus FDS (middle), or D-MEM plus FCS (right) by light microscopy. Note that in the WME-plus-DMSO culture only a minority of the cells survived. (b) Cells were maintained for more than three passages in medium containing FDS (lanes 1 to 8) or FCS (lanes 9 to 16) and infected in parallel with untreated (lanes 3 and 11) and CT-treated DHBV virions (lanes 4 to 7 and 12 to 15). As controls, PDKs were either not infected (lanes 8 and 16), transfected with full-length DHBV DNA (tr), or incubated with an about 1,000-fold greater amount of untreated and CT-treated SVPs for detection of unspecific adsorption (lanes 1 and 9 and 2 and 10, respectively). The Western blot with anti-DHBpreS antibodies (top) demonstrates the appearance of newly synthesized P36 after infection with peak B of CT-treated DHBV virions only in PDKs maintained in medium with FDS supplement (lanes 5 and 7). The amido black stain of the blot (bottom) revealed that cellular growth was not affected in cells cultured in the presence of FCS and therefore could not explain the missing signals of P36 in the corresponding experiments (lanes 13 and 15); DS, DHBV-positive duck serum; M, 10-kDa ladder of marker proteins.

Our infection experiments were performed with PDKs cultured in D-MEM together with FDS (Fig. 7b, left) or FCS (Fig. 7b, right). Infection was carried out in duplicate with peaks A and B of CT-treated or mock-treated virions. Expression of L by the PDKs was taken as proof of viral infection, as the viral input was never detectable and P36 was virtually absent in CT-treated virions. Only PDKs cultured in the presence of FDS and infected with peak B of CT-treated DHBV virions expressed L (Fig. 7b, lanes 5 and 7). These results demonstrate the general possibility of infecting nonliver tissues with DHBV, possibly via the exposed fusogenic site, circumventing the receptor-mediated uptake conveyed by the pre-S domain of L.

CT treatment allows infectivity of a receptor-incompetent DHBV mutant.

Finally, to support the evidence that DHBV can enter and infect cells via fusion or translocation, pre-S deletion mutants (17) which had been tested for the ability to bind to PDHs and induce the phenomenon of enhancement were used. The deletions in the different mutants span the region from aa 5 to aa 139 of L (Fig. 8a), of which part (aa 22 to 90) is also designated the host range-determining region (29). Two of the mutants, D1053 and D1119, were earlier found to be unable to bind to hepatocytes and induce enhancement (7, 8). They harbor deletions from aa 85 to aa 96 and from aa 107 to aa 125, respectively. All others could perform both tasks. To test the infectivity of the mutant DHBVs, they were incubated with PDHs. UV irradiation was performed prior to infection to inactivate the virions and thus control for detection of the input virus. When the cells were harvested 7 days after infection, no input virus could be detected (Fig. 8b, right). Expression of P36 in cells was thus taken as proof of viral infection. It was found that besides wild-type DHBV-3, mutant viruses D813, D864, and D984,with deletions of aa 5 to 14, 22 to 30, and 62 to 73, respectively, could also infect PDHs (Fig. 8b, left). Mutants D1020 and D1176, although able to bind to PDHs and enhance infection, were not able to initiate viral replication in PDHs (compare Fig. 8a). Not surprisingly, as they were also unable to bind, mutants D1053 and D1119 did not infect. In order to study whether noninfectious pre-S deletion mutants can replicate after penetration of the hepatocytic membranes via the proposed fusion, we chose for comparison deletion mutants D984 and D1053. While the former was still infectious, the latter was not, most probably because of its lack of a receptor recognition motif. The virions of mutants D984 and D1053, as well as of wild-type DHBV-3, were purified and either incubated with CT or mock treated. Viral replication was monitored by indirect immunofluorescence of L after 3 days (Fig. 8c) and Southern blotting of viral DNAs after 7 days (Fig. 8d). Already at an early time point, expression of L could be detected in PDHs infected with CT-treated mutant D1053, similarly as after infection with untreated or CT-treated wild-type virus (not shown), whereas at the same time no viral protein synthesis could be observed in the untreated mutant (Fig. 8c). Likewise, single-stranded viral DNA could be detected as replicative intermediates alongside the relaxed-circular full-length DHBV DNA in PDHs infected with CT-treated DHBV-3, mutant D984, and also mutant D1053 (Fig. 8d).

FIG. 8.

Analysis of the infectivity of selected pre-S deletion mutants of DHBV before and after CT treatment. (a) Illustration of the DHBpreS transcript (gray bar on top) demonstrating the starting points for L (nucleotide 801) and S (nucleotide 1287) with the positions of neutralizing epitopes, the host range-determining region, and putative receptor binding sites indicated by dashed bars. Different deletion mutants of DHBpreS that have formerly been tested for their binding and enhancement capacities (references 7 and 8) are illustrated as black bars with the numbers of deleted amino acids in square brackets above. (b) Western blot assays with anti-DHBpreS antibodies showing the infectivity of purified deletion mutants (lanes 1 to 7) and WT3T (lane 8) for PDHs before (−) and after (+) UV irradiation of the virus. Mock-infected cells (lane 9) and cells infected with virus from positive duck serum (lane 10) not inactivated by UV light (−) were used as controls. Deletions upstream of aa 74 led to loss of infectivity. (c) Indirect immunofluorescence of PDHs with anti-DHBpreS antiserum performed 3 days after incubation of the cells with the noninfectious deletion mutant D1053 of DHBV without (top) or with (middle) prior CT treatment demonstrates a gain of infectivity with enzymatic treatment. A higher magnification of PDHs infected with CT-treated D1053 is presented at the bottom. (d) For the examination of viral replication, PDHs were harvested 7 days after incubation with the infectious deletion mutant D984 (lanes 1 and 2) or the noninfectious deletion mutant D1053 (lanes 3 and 4) or WT3T (lanes 5 and 6) with or without prior CT treatment of the virions. Viral DNA was extracted and subjected to Southern blot analysis. The DNA of uninfected PDHs was run in parallel as a control (lane 7). The positions of the relaxed circular (RC) and single-stranded (SS) DNAs and the positions of marker DNAs are indicated on the left and the right, respectively.

The last experiments demonstrated that exposure of the putative fusogenic site of S makes it possible for receptor-incompetent DHBV mutants to enter and replicate in PDHs. These results show further that pre-S of L harbors a domain including aa 85 to 125 which is solely responsible for binding the cellular receptor as a prerequisite for viral uptake and serves no further obvious function in the infection or replication process of DHBV in vitro.

DISCUSSION

Viral infection is often restricted to certain cell types by the recognition of specific receptors on the cellular surface. When this hurdle is overcome, several viruses can replicate within cells they normally do not infect. However, many viruses face intracellular barriers that make it impossible for them to replicate within alien cell types. When expression plasmids for hepadnaviruses are transfected, replication can be observed in cells that are usually insusceptible, like hepatoma cell lines, across species. The arsenal of factors involved in host range specificity and organotropicity is, however, unresolved. One of them is certainly a so-called host range-determining region within the sequence of S. So it was observed that treatment of HBV and woodchuck hepatitis virus with V8 protease changed the respective viral surface components in a way that allowed infection of the hepatoma cell line HepG2, which is usually insusceptible to these viruses (47, 48). It was conceived by the authors that the exposure of a specific site that allowed entry was responsible rather than just stripping off the pre-S domains, because treatment of HBV with CT, removing part of a putative fusogenic site near the amino terminus of S, did not produce infectivity (47). Accordingly, it was demonstrated that a chimera of influenza virus hemagglutinin and the putative fusion peptide of HBs was able to initiate hemifusion as a first step of viral entry (3). We found that, analogous to these observations, the protease CT, instead of V8, seems to be able to cleave DHBs correctly, thereby exposing a region that confers infectivity for otherwise insusceptible PDKs. Comparing the exposed site in DHBs after CT treatment with the putative fusogenic site of the corresponding molecule of HBV revealed similarities like the orientation of two or three hydrophobic amino acids and an AG motif. Those conformities, which were predicted for fusogenic sites, could also be observed in rodent HBV and in several other viruses not related to hepadnaviruses like human immunodeficiency virus types 1 and 2, measles virus, and influenza virus (47).

In contrast, when we left a longer piece of the amino acid chain upstream of the fusogenic site undamaged by using the protease T instead of CT, the putative fusion activity was abridged, which was indicated by the loss of infectivity (Fig. 5). The different cleavage sites could explain why only CT digestion creates infectious virions. In the case of CT, the obviously highly conserved region GGILAGLIGLLV/g, whose fusogenic activity could be demonstrated by using the corresponding peptides (56), would be more directly exposed than after the use of T.

The presence of CT-treated SVPs interfered with infection when the enzyme was added directly to culture medium containing DHBV-positive duck serum with the naturally occurring viral particle mixture (Fig. 1a) or when added to a mixture of purified DHBV SVPs (Fig. 1b and c). One could assume that pre-S domains of SVPs oriented toward the viral interior during CT treatment translocate to the outside of the particles in the course of time, as already argued by Guo and Pugh (25), and saturate the gp180 binding sites of PDHs. We have no conclusive explanation for the inhibiting effect, as it is not to be expected that receptor competition would interfere with the proposed fusogenic activity of truncated S, especially as the amount of naturally occurring SVPs does not hinder the infectivity of native virus preparations. We suspect that the putative second receptor glycine decarboxylase (p120) that more efficiently binds truncated pre-S molecules, which may be the prerequisite for fusion or translocation, exists only in very close proximity to gp180 and may be blocked in a competitive manner by binding of SVPs or pre-S peptides to carboxypeptidase D. Alternatively, a very large amount of SVPs entering PDHs artificially via fusion may overstrain the capabilities of the cells for intracellular transport and correct direction of the viral particles.

Another curiosity was the appearance of two virion peaks of CT-treated DHBV differing in infectivity in rate-zonal gradient centrifugation (Fig. 2). Treatment with phosphatase demonstrated a major difference between virions of the two distinct peaks, A and B. The viral envelope proteins of the former seemed to be partially phosphorylated, whereas those of the latter lacked phosphorylation. However, we cannot rule out phosphorylation at sites whose modifications did not lead to electrophoretic shifts (Fig. 4). Nevertheless, the reason for the elevated infectivity of virions in peak B could have been the lack of certain phosphorylated cleavage products of L. Hence, residual pre-S sequence on virions of peak A stemming from reduced sensitivity of phosphorylated envelope proteins to CT may reroute the virus for the conventional receptor-mediated endocytosis. The existence of peak A and B virions also suggests that the viral progeny of DHBV is inherently diverse in the phosphorylation state of pre-S.

The elevated infectivity of the virus treated with CT was remarkable because most of the external parts of the surface proteins, including the putative receptor recognition site, as revealed by a neutralization test (Fig. 3), were removed. The high infectivity is likely to be explained by receptor-independent fusion or translocation. 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 (43-45) 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. Although there is explicit information about the host range-determining region located in the pre-S domain of L (29) and taking into account that CT-treated virions entered the cells via fusion, our observations give an indication that species specificity of infection is not determined by only one receptor located on the surface of cells. Moreover, infection seems to be more or less dependent upon additional cellular factors on or inside the cell, which may participate in further binding, in transporting the capsid to the nucleus, or in supporting repair and replication of the viral genome.

In order to find out whether the lack of a liver-specific factor rather than a species-specific factor was responsible for the insusceptibility of hepatoma cells to infection with CT-treated DHBV, we set up cell cultures of PCHs and tried to infect them but without success (Fig. 6). Yet, this experiment would not tell us whether the additional factors required for infection were particularly liver specific or rather broadly distributed but species specific. However, we managed to grow cultures of PDKs and demonstrated that CT-treated DHBV was able to infect them (Fig. 7). From that, we can conclude that species-specific factors necessary for certain steps of DHBV infection following penetration are present not only in hepatocytes but also in kidney cells and, as described by others, in primary bile duct epithelial cells (42), in the yolk sacs of embryos (61), or in the pancreases of duck embryos and ducklings (27, 42, 61).

Finally, we could show that the pre-S deletion mutant D1053, whose incompetence for binding, enhancement, and infection had been shown in several instances (7, 8, 64), infected PDHs after treatment with CT (Fig. 8). Strong virus replication was apparent when using the pre-S deletion mutant with a missing receptor recognition motif and therefore added a further argument to our suspicion that viral entry is a multistep process in which fusion with either the cellular or endosomal membrane or translocation through membranes, as suggested by others (20), represents one important step.

In summary, removing the external parts of the viral surface proteins exposed a putative fusogenic site, which enabled the virus to penetrate the cellular membrane and replicate even in cells that are usually not susceptible. In contrast, CT treatment of DHBV virions would not allow infection of the hepatoma cell lines regularly used for transfection experiments and also not of primary hepatocytes from chickens, another species. Although the experiments presented here cannot differentiate whether entry of CT-treated virions occurred via fusion with or translocation through the cellular membrane or the endosomal membrane, as demonstrated for SVPs of DHBV (13), it stands to reason that virions and SVPs take the same entry route although their exteriors appear to be significantly different. Combining the data presented by others with our infection experiments performed with artificially modified virions, we come to the hypothesis that three steps with different “receptor” elements might be minimally required for the infection of PDHs with DHBV, i.e., (i) binding of L to the cellular glycoprotein gp180 as a ubiquitous carboxypeptidase also present in other cell types and other hosts, (ii) cleavage of bound L by a CT-like enzyme as an unknown organotropic component located on the surface of either liver cell membranes or endosomes, and (iii) switching of modified L to glycine decarboxylase p120, which possibly confers the necessary close contact between the viral envelope and cellular membrane for fusion or translocation.