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Previous Article | Next Article 
Journal of Virology, August 2004, p. 7925-7937, Vol. 78, No. 15
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.15.7925-7937.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Michael A. Joyce, William R. Addison, Karl P. Fischer, and D. Lorne J. Tyrrell*
Department of Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada
Received 28 January 2004/ Accepted 19 March 2004
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The virion is an icosahedral capsid made up of a core protein surrounded by a lipid bilayer that contains the viral envelope proteins. Contained within the capsid is the viral genome with the polymerase protein covalently attached to the 5' terminus of the minus strand. The hepadnavirus genome is organized into overlapping reading frames that encode the precore, core, polymerase, and surface proteins. The mammalian hepadnaviruses, as well as the majority of the avian hepadnaviruses, contain an additional open reading frame that encodes the X protein (10). Infection is initiated by the interaction of the virus with a receptor present on the surface of hepatocytes. Carboxypeptidase D (CPD) has been identified as a receptor for DHBV, although it appears that additional coreceptors are required (4, 7, 35). Following attachment, the virus enters the cell, likely by endocytosis, and nucleocapsids are released into the cytoplasm (28). Transport of the nucleocapsids to the nuclear membrane is mediated by a nuclear localization signal present in the core protein (6, 20, 39). Disassembly of the nucleocapsids occurs either in the cytoplasm or at the nuclear membrane and is followed by release of the viral DNA into the nucleus. The relaxed circular genome then is converted into covalently closed circular DNA (cccDNA), which serves as the template for virus transcription. The viral genome is transcribed by host RNA polymerase II, and the transcripts are transported to the cytoplasm (10, 28). Following translation of the viral gene products in the cytoplasm, the pregenomic RNA is packaged along with the viral polymerase into the nucleocapsids, where DNA synthesis occurs (31). Reverse transcription of the pregenomic RNA followed by DNA-dependent DNA polymerization results in the relaxed circular, partially double-stranded genome. At this point, the nucleocapsids either are targeted to the nucleus or, alternatively, bud into the endoplasmic reticulum (ER) lumen and exit the cell through the secretory pathway as enveloped, infectious virions (37). This process is partially regulated by the level of expression of the large (L) surface antigen (19, 32, 33). Early in infection, when there is minimal surface antigen expression, the mature nucleocapsids are directed to the nucleus to contribute to the amplification of cccDNA. Later, when a threshold level of surface antigen expression is achieved, the mature nucleocapsids attach to ER membranes containing surface antigen, resulting in envelopment and secretion (19, 32, 33).
Superinfection exclusion is a phenomenon whereby a cell infected with a virus is resistant to superinfection by the same virus. Superinfection exclusion is observed during infections by a broad range of viruses, including human immunodeficiency virus (HIV) (17), vesicular stomatitis virus (VSV) (38), vaccinia virus (5), and alphavirus (14). There is some evidence to suggest that superinfection exclusion occurs in hepadnavirus infections as well. When liver transplant and nontransplant patients with chronic HBV infections were treated with lamivudine, differences in the patterns of development of lamivudine resistance could be seen. The average duration of lamivudine monotherapy before resistant HBV variants emerged was longer in nontransplant patients than in transplant patients, 562 days versus 371 days, respectively (11). In addition, the rates of resistance appeared to be higher in transplant patients than in nontransplant patients (8, 16, 23). These results suggest that lamivudine-resistant HBV establishes an infection more readily in an uninfected liver than in an HBV-infected liver.
Recent studies with the DHBV animal model have also suggested that superinfection exclusion occurs in avian hepadnavirus infections. Studies of viral kinetics in ducks have shown that enrichment of wild-type DHBV over replication-defective mutants is rapid during the initial spread of infection. Thereafter, the enrichment rate is slower and appears to be dependent on the generation of new, uninfected hepatocytes (40, 41). In a similar study, the emergence of wild-type DHBV in competition with either cytopathic or noncytopathic DHBV was found to be dependent on cell death caused by the cytopathic virus, because the wild-type virus did not emerge in competition studies with the noncytopathic variant (18).
In this study, the DHBV animal model was used to investigate superinfection exclusion in hepadnavirus infections. The results show that superinfection exclusion occurs in DHBV infection, is mediated by the L surface antigen, and does not involve the down-regulation of CPD, a known receptor for DHBV.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For studies involving UV-inactivated virus, 100 µl of high-titer DHBV-positive serum was irradiated for 1 h at 4°C by using a Southern New England UV Co. RPM200 UV box (reactor wavelength, 350 nm) and then used immediately.
Preparation and infection of PDHs. Primary duck hepatocytes (PDHs) from 14- to 21-day-old ducklings were prepared by using collagenase as previously described (34). The resulting PDHs were plated at 750,000 cells per well in six-well plates and cultured at 37°C in Leibovitz 15 medium supplemented with 1.2 µg of insulin/ml, 1.7 µg of glucose/ml, 11 µM hydrocortisone hemisuccinate, 15 mM HEPES, 5% fetal bovine serum, 50 IU of penicillin/ml, 10 µg of streptomycin/ml, and 25 µg of nystatin/ml (PDH medium). At 1 day postplating, the medium was replaced with serum-free medium, and the cells were cultured for an additional 2 days. At 3 days postplating, the cells were infected with DHBV-positive serum at a multiplicity of infection (MOI) of 100 to 200 in medium containing 1.5% dimethyl sulfoxide overnight at 37°C. The medium was replaced with fresh serum-free medium every second day.
Extraction of extracellular viral DNA from serum. Twenty microliters of serum was added to 80 µl of 50 mM Tris-HCl (pH 8)-150 mM NaCl- 10 mM EDTA-0.1% sodium dodecyl sulfate (SDS)-800 µg of proteinase K/ml and incubated at 42°C for a minimum of 4 h. The sample was extracted with an equal volume of phenol-chloroform. DNA was precipitated by adding a 0.10 volume of 3 M sodium acetate, 10 µg of yeast tRNA, and 2 volumes of 95% ethanol. The DNA was resuspended in 20 µl of water. Ten microliters was used for a subsequent PCR.
Isolation of intracellular viral DNA from PDHs. Monolayers of PDHs were rinsed with phosphate-buffered saline (PBS) and lysed in 10 mM Tris-HCl (pH 7.5)-50 mM NaCl- 1 mM EDTA- 0.25% NP-40- 8% sucrose. The nuclei and cellular debris were pelleted by centrifugation at 13,000 x g for 4 min and discarded. To digest cellular nucleic acids, 6 mM MgCl2, 100 µg of DNase I/ml, and 10 µg of RNase A/ml were added to the lysates, and the mixtures were incubated at 37°C for 30 min. The samples were centrifuged as described above, and the virus was precipitated from the supernatants by the addition of 0.3 volume of 26% polyethylene glycol 6000- 1.4 M NaCl- 25 mM EDTA and incubation overnight at 4°C, followed by centrifugation at 13,000 x g for 15 min at 4°C. The pellets, containing the virus, were resuspended in 50 mM Tris-HCl (pH 8)-150 mM NaCl- 10 mM EDTA. To digest the capsid and polymerase, 800 µg of proteinase K/ml and 0.1% SDS were added, and the mixture was incubated at 42°C overnight. The samples were extracted with phenol-chloroform. Ten micrograms of yeast tRNA was added as a carrier, and DNA was precipitated as described above and resuspended in 20 µl of water. Five to 10 µl was used for subsequent PCRs or Southern blots.
Analysis of viral DNA. The extracted viral DNA was amplified by PCR with Taq polymerase (Gibco BRL) according to the manufacturer's specifications, 1.5 mM MgCl2, and the following primers at 0.25 µM: 5'-CTCAAGAGATTCCTCAGCC-3' and 5'-GTCATACCATTCTCCTACT-3'. Cycling conditions were as follows: 95°C for 4 min; 30 cycles of 95°C for 30 s, 50°C for 30 s, and 72°C for 1 min; and 72°C for 7 min. To distinguish between wild-type DHBV and DHBV-ClaI, the PCR products were digested with restriction enzyme ClaI at 37°C for at least 1 h. The digestion products were separated on 1.3% agarose gels and visualized with ethidium bromide. To distinguish between wild-type DHBV and DHBV-M512V, the PCR products were sequenced.
Cell sorting and single-cell PCR. Primary cells growing in cultures were washed twice with PBS, treated with glycine buffer (50 mM glycine, 150 mM NaCl [pH 2.2]) for 1.5 min to remove bound virus (3), and washed twice more with PBS. The cells then were removed from the culture dish by trypsin digestion, pelleted by centrifugation, and washed twice more with PBS. The cells were counted and checked for viability by trypan blue exclusion. Single-cell PCR was performed by using a modified version of a previously described protocol (36). The cells were sorted into 0.2-ml PCR tubes containing 10 µl of lysis solution (200 mM KOH, 50 mM dithiothreitol), heated at 65°C for 10 min, and cooled briefly, and the KOH was neutralized with 5 µl of 0.2 N HCl and 5 µl of 400 mM Tris (pH 8.3). The cells then were heated at 93°C for 15 min and cooled briefly, and a PCR mixture was added to a final volume of 100 µl. The PCR and subsequent analysis were performed as described above, except that 40 cycles were used for the single-cell PCR.
Virus stocks and infections. John Elliot (University of Alberta) kindly provided a recombinant adenovirus which expresses a ß-galactosidase with a nuclear localization signal. PDHs were infected with this adenovirus at an MOI of approximately 1 at 3 to 5 days postplating. PDHs were washed with PBS and incubated with 0.5 to 1 ml of adenovirus for 1 h at 37°C. The adenovirus then was removed and replaced with PDH medium. The number of adenovirus-infected cells was determined 24 to 48 h postinfection by staining for ß-galactosidase activity. Briefly, the cells were washed with PBS and fixed for 5 min at 4°C with 0.25% glutaraldehyde. The cells were washed three times with PBS and incubated with X-Gal solution (1 mg of 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal]/ml, 2 mM MgCl2, 100 mM each potassium ferricyanide and potassium ferrocyanide in PBS) overnight at 37°C. The cells were washed twice with PBS, and the number of ß-galactosidase-positive cells was counted.
James Smiley (University of Alberta) kindly provided VSV. For infection of PDHs, cell monolayers were incubated with culture medium containing VSV at an MOI of 0.1 to 0.15 at 37°C overnight. VSV then was removed and replaced with fresh medium. Cells were monitored daily for cytopathic effects. When the majority of cells were dead, the cells were washed with PBS and fixed and stained with Wright's solution.
Herpes simplex virus type 1 (HSV-1) subtype KOS 1.1 was also provided by James Smiley and used in infection studies. PDHs were infected at 3 days postplating with HSV-1 at an MOI of 10 and were monitored daily for cytopathic effects.
Generation of recombinant adenoviruses. Recombinant adenoviruses were generated by using the AdEasy system. Bert Vogelstein (Johns Hopkins Oncology Center) kindly provided vectors pAdtrack-CMV and pAdEasy-1 and Escherichia coli strain BJ5183.
The nucleotide sequences encoding the L surface antigen, the small (S) surface antigen, and the core genes of DHBV were first amplified by using an Expand high-fidelity PCR system (Roche, Laval, Quebec, Canada) according to the manufacturer's specifications. The primers used were as follows: for the L surface antigen, 5'-CAGATATCACCATGGGGCAACATCCAGCAAAATCAATGG-3' and 5'-CAGATATCCTAACTCTTGTAAAAAAGAGC-3'; for the S surface antigen, 5'-CAGATATCACCATGTCTGGTACCTTCGGG-3' and 5'-CAGATATCCTAACTCTTGTAAAAAAGAGC-3'; and for the core, 5'-CTTGGGATCCGATGGATATCAATGCTTCTAGAGC-3' and 5'-GCAAAGCTTTTATTTCCTAGGCGAGGGAG-3'. The L surface antigen PCR product was digested with EcoRV and cloned directly into EcoRV-digested pAdtrack-CMV to generate Adtrack-CMV-LsAg. The S surface antigen PCR product was first subcloned into pCR2.1 (Invitrogen, Carlsbad, Calif.), which was then digested with EcoRV; the resulting fragments were cloned into EcoRV-digested pAdtrack-CMV to generate pAdtrack-CMV-SAg. The core PCR product was blunt ended by filling in with T4 DNA polymerase (Invitrogen) and cloned into EcoRV-digested pAdtrack to generate pAdtrack-CMV-core. To generate an L surface antigen with a deletion of amino acids 83 to 109 (L surface antigen 
83-109), the overlap extension method was used (13). The primers used were as follows: the 5'-flanking primer was 5'-CAGATATCACCATGGGGCAACATCCAGCAAAATCAATGG-3', the 3'-flanking primer was 5'-CAGATATCCTAACTCTTGTAAAAAAGAGC-3', and the internal primers were 5'-CTCTTGAGGAGTCGGATTTGATAATCC-3' and 5'-CGACTCCTCAAGAGGAAACCACCACCATTCCTCCGTCTTCC-3'. The resulting PCR products were treated in the same manner as the S surface antigen PCR product to generate Adtrack-CMV-LsAg83-109. The integrity of the sequences generated by PCR amplification was confirmed by DNA sequencing. Recombinant adenoviruses Ad-LsAg, Ad-LsAg83-109, Ad-SsAg, Ad-core, and Ad-GFP were generated as previously described (12).
Infection of PDHs with recombinant adenoviruses. Two-day-old cultures of PDHs were incubated with recombinant adenoviruses at an MOI of 50 at 37°C overnight. The cells then were washed once with PBS, and fresh medium was added. At 4 days postinfection, the efficiency of infection was estimated by using fluorescence microscopy to detect green fluorescent protein (GFP)-expressing cells. At 4 days after adenovirus infection, DHBV antigen expression within the PDHs was examined by Western blotting, and the PDHs were infected with DHBV as previously described. One week later, intracellular virus was harvested and analyzed by Southern blotting.
Fluorescent labeling of DHBV stocks. DHBV virions from duck serum were first partially purified on a 20% sucrose cushion. Approximately 20 to 30 ml of DHBV-positive serum was layered over 5 to 6 ml of 20% sucrose and centrifuged at 76,000 x g for 18 h at 4°C. The resulting pellet was resuspended in 500 µl of PBS, followed by the addition of an equal volume of 400 mM Na2BO3 (pH 8.5) and 0.04 mg of 5 (and 6)-carboxy-X-rhodamine (succinimidyl ester)/ml. The mixture was incubated at room temperature for 1 h. Unreacted rhodamine label was removed by using a PD-10 gel filtration column (Bio-Rad, Mississauga, Ontario, Canada). To remove any contaminating labeled serum albumin, rhodamine-labeled DHBV (approximately 1.5 ml) was incubated at 4°C with 250 µl of Affi-Gel Blue (Bio-Rad) for 2 h. The sample then was centrifuged briefly to remove the Affi-Gel Blue beads. Serum from an uninfected animal was labeled in the same manner as a control.
Binding studies and confocal microscopy. PDHs from uninfected and congenitally DHBV-infected ducks were prepared as described above. The cells were incubated with rhodamine-labeled DHBV either at room temperature or at 4°C for 5 to 6 h, washed a minimum of six times with PBS to remove unbound virus, and analyzed by fluorescence microscopy. For blocking experiments, PDHs were incubated with labeled DHBV preincubated with monoclonal antibodies specific for either the L or the S surface antigen. Alternatively, the cells were incubated with labeled DHBV in the presence of 25 µl of concentrated subviral particles. The subviral particles were obtained from 50 ml of culture supernatant from Huh-7 cells that were infected with both Ad-LsAg and Ad-SsAg; the supernatant was concentrated to 3 ml by using a Millipore 50K MWCO Centriplus concentrator. Serum albumin was removed by incubation with 500 µl of Affi-Gel Blue as described above. For confocal microscopy, PDHs were grown on glass coverslips, incubated with rhodamine-labeled DHBV for 18 h at 37°C, and washed six times with PBS. The nuclei were stained with Hoechst 33342 dye (Molecular Probes Inc.) (500 ng/ml) for 5 min at room temperature. The coverslips were mounted on slides with 50% glycerol, and the cells were examined with a Zeiss LSM 5 confocal microscope.
SDS-PAGE and Western blot analysis of viral antigens and CPD. The cells were washed with PBS, harvested from 12-well cell culture dishes by using a cell scraper, and resuspended in 100 µl of 6x loading buffer (125 mM Tris [pH 6.8], 5% SDS, 10% ß-mercaptoethanol, 15% glycerol, 0.1% bromophenol blue). Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) with 8 or 10% polyacrylamide. The separated proteins were transferred to nitrocellulose (Hybond ECL; Amersham Biosciences, Little Chalfont, United Kingdom), processed according to the manufacturer's specifications, and visualized by chemiluminescence. Rabbit anti-CPD antisera were kindly provided by Heinz Schaller (University of Heidelberg), rabbit anti-core antisera were provided by Jesse Summers (University of New Mexico), and monoclonal antibodies to L and S surface antigens were obtained from Pat Nakajima (Fox Chase Institute, Philadelphia, Pa.). Horseradish peroxidase- goat-anti-mouse and horseradish peroxidase- goat-anti-rabbit antibodies were obtained from Cappel Rockland Inc., Gilbertsville, Pa.
Nucleotide sequence accession number. The sequence of the Alberta strain of DHBV type 16 has been deposited in GenBank under accession number AF047045.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Serum from the congenitally infected animals (Fig. 1A, lanes 1 to 5) showed no evidence of DHBV-ClaI superinfection at 14 days postinfection, since no 820-bp product was detected. However, the virus present in the serum from uninfected animals was entirely DHBV-ClaI (Fig. 1A, lanes 6 to 10), indicating that the DHBV-ClaI stock was infectious and that the ClaI digestion was complete. Analysis of serum taken at all other time points postinfection yielded the same results (data not shown). In total, 13 congenitally infected and 12 uninfected animals were studied. All 12 uninfected animals were infected with DHBV-ClaI, whereas none of the 13 congenitally infected animals showed DHBV-ClaI infection. Four of the congenitally infected ducklings were monitored for 12 weeks, with no evidence of DHBV-ClaI infection. The PCR assay was able to detect 2% of DHBV-ClaI in a background of wild-type DHBV (data not shown). These results suggest that the preexisting infection in these animals prevented superinfection by DHBV-ClaI.
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Single-cell PCR was performed to determine whether individual hepatocytes were dually infected with both viruses or whether DHBV and DHBV-ClaI replicated exclusively in separate cells. Newborn ducklings were infected with serum containing an approximately 1:1 ratio of DHBV to DHBV-ClaI and were monitored for coinfection by PCR of serum viral DNA. At 2 weeks postinfection, the liver of a coinfected duckling was perfused, and the hepatocytes were cultured for 1 week. The cells then were sorted into single cells by fluorescence-activated cell sorting, and viral DNA was amplified and analyzed as for previous experiments.
Some of the results of the single-cell PCR analysis for the coinfected duckling are shown in Fig. 2A. In total, 89 of 105 cells showed the presence of both viruses. Five cells were infected exclusively with DHBV, while 11 cells contained only DHBV-ClaI. Interestingly, analysis of serum viral DNA from this animal also showed slightly more DHBV-ClaI than DHBV. These results indicate that the majority of the cells contained both viruses. When cells infected with wild-type DHBV and with DHBV-ClaI were prepared separately and mixed prior to cell sorting, only one virus was detected in each well (Fig. 2B). In total, 48 of 50 positive cells showed the presence of either DHBV or DHBV-ClaI. These results confirm that cells were sorted into single cells which were not contaminated by extracellular virus. Therefore, the presence of both DHBV and DHBV-ClaI in the hepatocytes of the coinfected duckling was due to a dual infection.
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) and IFN-
/ß receptor knockout mice (22). This finding suggests that these cytokines inhibit HBV replication to some extent. In addition, DHBV replication has been shown to be inhibited by both IFN-/ß and IFN- (26, 27). However, the effect is greatest when IFN is present at or before the time of infection. It is possible that infection with DHBV induces a low level of IFN expression which does not affect the replication of the established DHBV infection but which inhibits the establishment of a second infection, in this case, a DHBV-ClaI infection. The response of VSV to DHBV infection was used to test this hypothesis. VSV is extremely sensitive to both duck IFN-/ß and duck IFN- (26, 27). If a low level of IFN-/ß is expressed in DHBV-infected hepatocytes, then they should be protected against VSV-mediated lysis. Both DHBV-infected and uninfected PDHs were infected with VSV at an MOI of 0.1 and monitored daily for cytopathic effects. The majority of both uninfected and DHBV-infected hepatocytes were killed by day 4 after VSV infection, indicating that they were equally susceptible to VSV infection (Fig. 4C). This result suggests that the mechanism of DHBV exclusion is unlikely to be mediated by IFN. The results from the adenovirus superinfection experiment support this conclusion, as the entry and possibly the gene expression of recombinant adenoviruses have also been shown to be inhibited by IFN expression (22). DHBV gene expression is required for the exclusion of DHBV-ClaI. The need for gene expression by DHBV for the exclusion of DHBV-ClaI was examined by determining the effect of treating DHBV with UV radiation prior to infection of PDHs. UV irradiation results in cross-linking of the viral DNA, which inhibits transcription. PDHs were mock infected, infected with wild-type DHBV, or infected with UV-treated DHBV. The cells then were incubated with DHBV-ClaI at 3, 7, or 9 days later (Fig. 5). Intracellular virus was harvested 1 week after the last DHBV-ClaI infection (day 16) and analyzed. As expected, when cells were initially infected with wild-type DHBV, DHBV-ClaI was almost completely excluded by day 9 (Fig. 5A, lanes 19 to 21). However, there was evidence of DHBV-ClaI infection in cells that were initially infected with irradiated DHBV (Fig. 5A, lanes 22 to 24) or mock infected (lanes 25 to 27). The small amount of the PCR product corresponding to the size of wild-type DHBV in cells that were either mock infected or infected with UV-treated DHBV is likely due to incomplete digestion of the PCR product with the ClaI enzyme. Figure 5B shows a Western blot revealing the expression of two envelope proteins from mock-infected cells, cells infected with UV-treated DHBV, or cells infected with wild-type DHBV at various times postinfection. The expression of both surface antigens could be detected only in cells infected with wild-type DHBV. Neither of these antigens could be detected in cells infected with UV-treated DHBV, indicating that this virus was incapable of gene expression. A small amount of each surface antigen could be detected 3 days postinfection but not at subsequent time points and therefore likely represents the virus inoculum. Therefore, the ability of DHBV to exclude DHBV-ClaI is dependent on viral protein expression.
Identification of the DHBV protein responsible for superinfection exclusion.
The results of the previous experiment suggested that gene expression is required for the exclusion of a second infection, in this case, DHBV-ClaI infection. However, they do not indicate which viral protein is involved. To determine which protein was responsible for the observed superinfection exclusion, individual DHBV proteins were tested for their ability to exclude DHBV infection. In addition, a DHBV L surface antigen with a deletion of the CPD binding domain was also analyzed for its ability to exclude DHBV infection. Recombinant adenoviruses were used to express DHBV proteins, since the transfection efficiency of PDHs is inefficient (approximately 1 to 5%). Recombinant adenoviruses Ad-core, Ad-LsAg, Ad-LsAg83-109, Ad-SsAg, and Ad-GFP were used to infect PDHs at 2 days postplating. At 4 days after adenovirus infection, the cells were analyzed by fluorescence microscopy to determine the percentages of cells infected by the recombinant adenoviruses (Fig. 6A). As shown in Fig. 6A, the efficiencies of the adenovirus infections (measured as the percentages of GFP-expressing cells) were similar and ranged from 60 to 80% between experiments. In addition, on day 4, some cells were harvested to confirm the expression of the DHBV antigens and to examine the expression of CPD (Fig. 6B and C, respectively). Figure 6B shows that the appropriate proteins were expressed, and Fig. 6C shows that only Ad-LsAg decreased the expression of CPD. Also on day 4, the adenovirus-infected PDHs were infected with DHBV. One week later, intracellular viral DNA was harvested and analyzed by Southern blotting (Fig. 6D). Evidence of DHBV infection, as indicated by the presence of viral replicative intermediates, was seen in cells infected with Ad-GFP, Ad-core, and Ad-SsAg. Conversely, the level of DHBV replication was significantly reduced in cells infected with either Ad-LsAg or Ad-LsAg83-109. These results indicate that the L surface antigen alone is capable of inhibiting DHBV infection of PDHs and that the region of this antigen which interacts with CPD (amino acids 83 to 109) is not necessary for exclusion.
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83-109. In addition, unlike the inhibition seen with both forms of the L surface antigen, the slight inhibition of DHBV replication seen with the S surface antigen was not consistent between experiments.
Down-regulation of CPD expression does not correlate with exclusion of DHBV-ClaI.
The L surface antigen was the only DHBV protein that inhibited DHBV infection; thus, it is involved in the mechanism of superinfection exclusion. This protein was previously shown to down-regulate a DHBV receptor, CPD, at between 5 and 9 days after infection (2). This time is similar to the time at which DHBV-ClaI is excluded from hepatocytes previously infected with DHBV. Furthermore, receptor down-regulation by viral envelope proteins is a mechanism of superinfection exclusion used by a number of viruses, most notably, HIV, and it is possible that DHBV also mediates exclusion in this way. The previous experiment suggested that this is not the case, because a form of the L surface antigen which does not down-regulate CPD, L surface antigen 83-109, was still capable of inhibiting DHBV infection.
To further confirm that receptor down-regulation was not the mechanism of exclusion, the correlation between DHBV-ClaI exclusion and the decrease in CPD expression was examined. PDHs were first infected with wild-type DHBV and then, 4, 6, 7, or 8 days later, infected with DHBV-ClaI or harvested for Western blot analysis. To confirm that the cells were still susceptible to infection, PDHs that had not been infected with DHBV were infected with DHBV-ClaI at these time points. One week after the last DHBV-ClaI infection, intracellular virus was harvested and analyzed by PCR (Fig. 7A). In this experiment, partial exclusion of DHBV-ClaI was seen when DHBV-ClaI was introduced 6 days after the initial DHBV infection, and exclusion of DHBV-ClaI was almost complete by 7 or 8 days after DHBV infection (Fig. 7A). The L surface antigen was first detected at 6 days after DHBV infection (Fig. 7B); this time correlated with the time at which DHBV-ClaI exclusion first became evident. However, CPD expression remained approximately the same over the course of the experiment (Fig. 7C). A decrease in CPD levels was observed at 4 days postinfection; such a decrease was not observed in other experiments and so was presumed to be an artifact of the particular experiment shown. To confirm that equal amounts of protein were loaded in the lanes, this blot was stripped and reprobed with an antibody specific for actin (Fig. 7D).
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DISCUSSION |
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Simultaneous inoculation of DHBV and DHBV-ClaI resulted in the establishment of a dual infection in animals. Analysis of viral DNA present in individual hepatocytes indicates that the majority of cells in these ducks are infected with both viruses. This presence of dual infections within cells makes it unlikely that the mechanism of exclusion involves the first infecting virus rapidly inducing a change in the cell to exclude the second virus. Further evidence for a slow mechanism of exclusion comes from the staggered coinfection experiments in which exclusion of the second virus became apparent only 5 days after the first infection. The high MOI (100 to 200 VGE/cell) used to infect the PDHs likely results in infection of all susceptible cells by the initial inoculum. Therefore, the delay in exclusion likely reflects the time required for sufficient expression of the viral antigen in the cell. A slow onset of exclusion is also consistent with the dependence of exclusion on the expression of the L surface antigen, as evidenced by the correlation of DHBV-ClaI exclusion with the expression of the L surface antigen (Fig. 7).
Exclusion does not depend on on-going viral replication. Inhibition of DHBV replication in congenitally infected ducklings with lamivudine did not, in 12 of 14 animals, prevent exclusion of a second virus. This is despite the fact that the second virus, DHBV-M512V, contains a mutation in the polymerase which renders it resistant to lamivudine and so should have a replicative advantage in the lamivudine-treated animals. Lamivudine inhibits viral replication but does not inhibit viral gene expression from the stable pool of nuclear cccDNA. The presence of DHBV-M512V in the two remaining animals could be related to the number of DHBV-infected hepatocytes at the time they were infected with DHBV-M512V. Congenitally infected animals vary in their level of viremia and a small percentage is able to clear the infection (unpublished data). This may explain why DHBV-M512V was able to establish an infection in two ducklings.
The exclusion mechanism is specific for DHBV as the unrelated viruses HSV 1, adenovirus, and VSV were not excluded from DHBV-infected hepatocytes. Thus, exclusion is unlikely to be mediated by antiviral cytokines such as IFNs. Previous studies have indicated that the small number of liver-resident macrophages (Kupffer cells) that are present in primary duck hepatocyte cultures can be artificially stimulated by endotoxin to produce IFN (15). The level of IFN produced is sufficient to inhibit DHBV replication. However, the highly IFN-sensitive virus VSV was still capable of infecting and killing DHBV-infected hepatocytes. Therefore, the initial DHBV infection is unlikely to be producing sufficient IFN to mediate the exclusion of DHBV-ClaI.
Exclusion of DHBV requires the expression of viral gene products. UV-treated DHBV, which did not express viral proteins, as shown by Western blotting, was incapable of excluding DHBV-ClaI infection of cultured hepatocytes. This is not surprising as the majority of viral interference mechanisms involve at least some viral gene expression. Since UV-treated virus can bind to the cells (data not shown), viral interference is not likely mediated by the transient occupancy of cellular receptors by the initial virus, a mechanism of exclusion seen with retroviruses, including Rous sarcoma virus and avian leukosis virus (29, 30).
Exclusion of DHBV infection is dependent on the expression of the L surface antigen. Recombinant adenoviruses were used to express the core, the L surface antigen, or the S surface antigen in primary hepatocytes to determine which viral protein mediates exclusion. The expression of GFP alone or core protein in hepatocytes did not inhibit DHBV infection relative to the results obtained with the control adenovirus, Ad-GFP. However, the expression of the L surface antigen and, to a lesser extent, the S surface antigen, in hepatocytes did result in decreased levels of DHBV replicative intermediates, a possible result of viral exclusion.
An alternative interpretation is that the decreased level of DHBV replication in cells expressing the envelope proteins is due to cytopathic effects caused by the overexpression of these proteins. However, while infection of primary hepatocytes with adenovirus does appear to have a limited cytopathic effect, this cytopathic effect was comparable with each of the viral proteins and appeared to be related more to the MOI used for infection (the higher the MOI, the greater the cytotoxicity). Western blot analysis of actin levels at the time of DHBV infection of the adenovirus-infected hepatocytes showed no significant differences in cell numbers in cultures expressing the various proteins at the time of DHBV infection.
Recently, it was shown that one of the putative receptors for DHBV, CPD, is down-regulated in DHBV-infected hepatocytes (2). Breiner et al. (2) showed that the L surface antigen binds to CPD in the ER, causing premature degradation of the receptor. Exclusion of DHBV could be explained if the level of receptor down-regulation in an infected cell is sufficient to prevent entry of potential superinfecting virus. Receptor down-regulation as a mechanism of superinfection exclusion is known to occur in a number of viral infections. For example, the HIV receptor, CD4, is down-regulated at the translational and posttranslational levels by the envelope, Vpu and Nef proteins of the virus (1). Surprisingly, the L surface antigen-mediated down-regulation of the receptor CPD, first demonstrated by Breiner et al. (2) and repeated in the present study, does not appear to be involved in exclusion. This conclusion is based on a number of observations. First, the time of DHBV-ClaI exclusion in PDHs did not correlate with a decrease in CPD expression. DHBV-ClaI was excluded 5 to 7 days after DHBV infection. At this time, Western blot analysis indicated that there was no significant decrease in total cellular CPD levels. However, it remains possible that cell surface levels of CPD are reduced at this time. The use of cell fractionation to examine specifically the plasma membrane levels of CPD might give an indication of cell surface CPD levels. However, CPD is localized primarily to the Golgi apparatus and only transiently cycles to the cell surface, and its detection on the cell surface of even uninfected hepatocytes is difficult (4).
Second, the expression of the L surface antigen containing a deletion of the CPD binding domain was still capable of excluding DHBV infection to levels comparable to those seen with the wild-type L surface antigen. The L surface antigen mediates the down-regulation of CPD by interacting with CPD in the ER, leading to premature degradation of CPD (2). It was speculated that deletion of the pre-S domain involved in the L surface antigen-CPD interaction would eliminate the intracellular interaction of these two proteins and prevent CPD down-regulation. Western blot analysis showed that the expression of L surface antigen 83-109 did not result in the down-regulation of CPD. Despite this, L surface antigen 83-109 still inhibited DHBV infection.
Third, DHBV-infected hepatocytes were still capable of binding to rhodamine-labeled DHBV. Conversely, rhodamine-labeled control serum did not result in any significant binding, indicating that the observed binding was specific for DHBV. As well, binding was not observed when the rhodamine-labeled DHBV was incubated with cells known to be nonpermissive for DHBV infection. The binding of labeled virus was completely inhibited when cells were first incubated with an excess amount of subviral particles (data not shown). Binding was also partially inhibited when the labeled virus was preincubated with a monoclonal antibody specific for the pre-S region of the L surface antigen (data not shown).
Labeled virus was also capable of entering DHBV-infected hepatocytes, apparently by endocytosis, as indicated by the localization of fluorescence in endosome-like vesicles. It is the surface of the virus which becomes labeled with rhodamine, most likely through attachment of the rhodamine to the L and S surface antigens present in the viral envelope. Fusion of the viral and endosome membranes would disrupt the endosome membrane, allowing the release of the nucleocapsid into the cytoplasm. The fluorescence-labeled viral envelope would presumably remain associated with the disrupted endosome membrane. The fluorescence signal appears to be within endosomes in both uninfected and DHBV-infected hepatocytes.
In summary, superinfection exclusion in DHBV infection is not IFN mediated. Exclusion requires viral gene expression but not viral replication. The L surface antigen is capable of independently mediating exclusion. However, the block in superinfection occurs after attachment and entry of the virus into hepatocytes. The results of this study are consistent with a model of exclusion involving the L surface antigen and the establishment of the cccDNA pool. Establishment of the cccDNA pool occurs early in infection and is negatively regulated by the L surface antigen (32, 33, 37). Early in infection, when L surface antigen levels are low, nucleocapsids containing newly synthesized relaxed circular DNA are directed to the nucleus, where the relaxed circular DNA is converted to cccDNA. As the cccDNA pool increases, the amount of the L surface antigen also increases and the nucleocapsids are enveloped and exported from the cell as infectious virus. DHBV-infected hepatocytes likely contain sufficient levels of the L surface antigen to effectively block the amplification of the cccDNA of the "superinfecting" virus. Without establishing a pool of its specific cccDNA, the second virus would not produce detectable extracellular virus. This model is consistent with the results shown in this study. DHBV-ClaI was unable to establish an infection in either DHBV-infected cells or animals, yet rhodamine-labeled virus was able to bind to and enter DHBV-infected hepatocytes. This model also explains why a mutant L surface antigen was capable of inhibiting DHBV infection of PDHs independent of its ability to bind to CPD.
The existence of superinfection exclusion would explain the observation that the development of lamivudine resistance is more rapid and occurs at higher rates in liver transplant patients than in patients with chronic HBV infections and treated with the drug. Previous studies have shown that enrichment of wild-type DHBV over a replication-defective variant is rapid during the initial phase of infection, when DHBV is spreading within the liver. Once the majority of hepatocytes become infected, however, this enrichment of wild-type DHBV is much slower and appears to be dependent on an increase in liver mass (40, 41). This pattern is consistent with superinfection exclusion. Similarly, the spread of any variant arising in a single cell, such as lamivudine-resistant HBV, would be limited by the slow production of new, uninfected hepatocytes. In a patient undergoing a liver transplant, the uninfected hepatocytes of the new liver would be susceptible to infection by any lamivudine-resistant HBV in the viral population. Conversely, the liver of a chronically infected individual undergoing lamivudine therapy still has a low level of wild-type viral replication and a persistent pool of wild-type cccDNA, making it more difficult for the mutant to spread through the liver.
Superinfection exclusion also has implications for proposed antiviral therapy with HBV as a gene therapy vector. Infection of congenitally infected duck hepatocytes with a recombinant DHBV expressing GFP is significantly less efficient than infection of naive hepatocytes (24). In this study, more than 90% of uninfected hepatocytes were infected with the recombinant virus, compared with 1 to 4% of congenitally infected hepatocytes. Although these cells did show superinfection, it was extremely inefficient compared with infection of uninfected hepatocytes. The success of gene therapy for chronic HBV infection may therefore be limited by the ability of recombinant HBV to enter an infected cell and express the therapeutic gene.
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ACKNOWLEDGMENTS |
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K.-A.W. was supported by an Alberta Heritage Foundation for Medical Research studentship. M.A.J. was supported by a Canadian Institutes of Health Research postdoctoral fellowship. Funding for this work was provided by Glaxo Wellcome Canada and the Canadian Institutes of Health Research.
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FOOTNOTES |
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Present address: Department of Microbiology, University of Washington, Seattle, Wash.
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