Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks

Abstract

Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus-induced hepatocellular carcinoma (HCC). Woodchucks that were chronically infected with HBV-related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery, the WHV carrier woodchucks were super-infected with WHV-enveloped HDV (wHDV). Six weeks later, the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real-time PCR assay and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues

Conclusion

Cells of WHV-induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Since others previously hypothesized that hepadnavirus-induced HCCs are resistant to re-infection with a hepadnavirus in vivo, our data suggest that if such a resistance exists, it likely occurs via a block at the post-entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection.

HDV is a natural subviral agent of HBV. HDV uses the envelope proteins of HBV to form virions and to infect susceptible hepatocytes. HBV and HDV utilize the same so far unidentified receptor(s). With the exception of the envelope proteins, the HDV life cycle is independent of HBV. HDV encodes the only protein, delta antigen (δAg), which is essential for HDV replication through the RNA-directed RNA synthesis catalyzed by host RNA polymerase II. The HDV genome is a 1700 nucleotides-long single-stranded circular RNA. Apart from the envelope proteins and δAg, HDV acquires all factors necessaryf for its life cycle from the host. In nature, HDV always co-exists with HBV in infected liver (1–2).

In a natural setting, only humans can acquire HDV either by co-infection with HBV, or by super-infection of HBV carriers with HDV (1–2). There are about 400 million chronic HBV carriers worldwide, of whom one million die every year from advanced liver disease, including HBV-induced hepatocellular carcinoma (HCC). Chronic HBV infection increases the HCC risk by about 100-fold and causes 50–80% of all HCCs (3–4). There are approximately 20 million HDV carriers worldwide. Concomitant HDV infection usually enhances HBV-induced liver pathogenesis. Super-infection of HBV carriers with HDV results in 70–90% of cases in chronic delta hepatitis that is the more severe form of chronic viral hepatitis, leading to accelerated and more frequent cirrhosis. Persistent HDV replication is a predictive factor for liver-associated mortality. Only a subset of HDV carriers is reported to benefit from interferon-α (including peginterferon) treatment, the only approved anti-HDV therapy. Currently, there are no drugs in use to directly target HDV, and a number of anti-HBV drugs do not block HDV infection (1–2, 5–8). In Europe, HDV-induced disease is frequent among immigrants from regions of higher HDV endemicity. HDV remains a serious problem in Vietnam, Iran, Pakistan, India, Tajikistan, Mongolia, Tunisia, Brazil and other South American countries (1, 6, 9). Despite reports suggesting that chronic carriers of HBV/HDV have a 3-fold increased risk of HCC, and develop HCC approximately 14 years earlier than carriers of HBV only, there is no consensus opinion on the relationship between HDV infection and liver cancer. The molecular basis of HDV pathogenesis is poorly understood, and the role of HDV in HCC induction/development has yet to be elucidated (1, 5–6, 10–13).

To advance our understanding of the mechanism of HDV infection and its relation to liver carcinogenesis, we determined whether HDV could infect in vivo the cells of hepadnavirus-induced HCCs. To accomplish this goal, we used woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus that is closely related to HBV. The WHV carrier woodchucks are very valuable surrogate animal model to study HBV infection, hepadnavirus-induced HCC, and to test anti-HBV and anti-HCC drugs. While chronic HBV carrier chimpanzees and wild-type HBV full genome transgenic mice do not develop HCCs, 100% of chronic WHV carrier woodchucks develop HCCs usually within 12–36 months post-infection (4, 14). Furthermore, in the laboratory HDV can be coated with WHV envelope proteins, because the HDV-binding site is conserved within the orthohepadnavirus subfamily. Therefore, numerous in vivo studies on HDV infection were conducted using WHV carrier woodchucks super-infected with HDV (1–2, 7). For HDV, a putative entry receptor (the receptors for WHV and HBV are currently not identified) and the host range are defined by the origin of the hepadnavirus (i.e., HBV or WHV) envelope proteins forming the virion’s coat. For this reason, HDV is often used as a tool to study the mechanism of hepadnavirus attachment and entry (1–2). Unlike previous studies, we for the first time have super-infected WHV carriers with WHV-enveloped HDV (wHDV) during the late stage of chronic WHV infection, when WHV-induced HCCs were already developed. Three out of three HDV-negative WHV carriers with formed HCCs were successfully super-infected with wHDV. All HCCs harvested upon the completion of the experiment were infected with wHDV. Therefore, one possible mechanism how HDV may modulate liver carcinogenesis is to infect already formed HCCs and influence their fate by replicating in tumor cells and changing their gene expression profile. Although, the effect of HDV replication in HCC cells on a fate of a tumor needs to be elucidated in future studies. Our results clearly show that WHV-induced HCCs during chronic hepadnavirus infection are susceptible to wHDV infection, and thus, express functional putative WHV receptors and support the steps of attachment and entry that depend on functioning of the WHV envelope proteins. Therefore, because several studies suggested that HCCs induced by either HBV or WHV are resistant to subsequent re-infection with a hepadnavirus and appear free of hepadnavirus replication markers (15–17), we conclude that if such resistance exists, it is mediated by a block at the post-entry step.

EXPERIMENTAL PROCEDURES

Super-infection of WHV carrier woodchucks with wHDV

The animals were housed at the animal facilities of Cornell University (Ithaca, NY). All experimental manipulations of animals were performed under protocols approved by the Institutional Animal Care and Use Committee. We used two males and one female WHV carrier (M7724, M7788 and F7807, respectively), which were produced by neonatal infection with the strain WHV7 at three days of age (14). Numbers and locations of WHV-induced HCCs within the livers were initially determined by ultrasound imaging. One week before wHDV super-infection, an open surgery was performed and biopsies from one HCC (referred further to as HCC1s) and from normal tissue of the left liver lobe (LL) were obtained from each animal. Thus, the findings of the ultrasound imaging were confirmed and the exact locations of established HCCs were determined. Following complete recovery from surgery, each woodchuck was inoculated via the sublingual vein with 0.5 ml of woodchuck-derived serum wHDV, which equaled to 8×10⁹ HDV genome-equivalents (GE)/animal. The administered HDV dose corresponds to a multiplicity of infection (MOI) of ~0.27 HDV GE/hepatocyte, because the average liver of an adult woodchuck contains about 3×10¹⁰ hepatocytes (18). Woodchucks were monitored for six weeks after wHDV super-infection, and blood was taken weekly. After six weeks, the animals were sacrificed and blood, normal liver tissues, and HCCs were analyzed for markers of HDV and WHV infections. To avoid the presence of normal tissues, the center masses of HCCs were used for analysis.

RNA isolation and HDV RNA standards

Total RNA from all tissues was isolated using the TRI reagent (Molecular Research Center). For the HDV strand-specific Northern analysis and real-time PCR assays (qPCRs), the standards of genomic and antigenomic unit-length RNAs were in vitro transcribed and subsequently gel-purified as previously described (19–20).

HDV strand-specific Northern analysis

Northern analysis was performed essentially as previously described (20). The RNAs were glyoxalated and then resolved using 1.7% agarose gels. The hybridization solution used was Ultrahyb™ Ultrasensitive Hybridization Buffer, containing 0.1 mg/ml of sheared salmon sperm DNA (Applied Biosystems). The HDV RNAs were detected using ³²P-labeled riboprobes, produced by in vitro transcription of either pTW108 or pTW107 plasmid linearized with HindIII. Plasmid pTW108 bears 1.1× unit-length HDV genome (G), while plasmid pTW107 contains 1.1× of the HDV antigenome (αG) (20–22). Radioactivity was visualized using a biomolecular imager (Typhoon FLA 9000, GE Heath Care). Images were acquired and quantified using ImageQuant TL and prepared for publication using Power Point and Adobe Photoshop software.

HDV strand-specific qPCR

The procedure was described in detail elsewhere (19). The primers used were: (i) forward primer, 312-GGACCCCTTCAGCGAACA-329; and (ii) reverse primer, 393-CCTAGCATCTCCTCCTATCGCTAT-360. The TaqMan probe was 332-AGGCGCTTCGAGCGGTAGGAGTAAGA-357. The HDV numbering was according to Kuo et al. (23). To assay G RNA, the above reverse primer was used in the reverse transcription (RT) reaction. To assay αG RNA, the above forward primer was used for the RT reaction. The copy numbers were quantified using a 10-fold dilution series of either G or αG RNA standards (range: 20–200,000 GE of HDV). We deduce that 1 million of HDV RNA molecules (G or αG) is equal to 1 pg of the corresponding HDV RNA standard. The 7500 Real Time PCR instrument (Applied Biosystems) was used for all qPCR assays according to the instructions of the manufacturer.

Quantification of WHV cccDNA copy numbers using qPCR

The cccDNA-enriched fraction that is mostly free of cellular genomic DNA was isolated using previously described protocols (24–26) and was further treated with RNase A (Roche), Hpa I (New England Biolabs), and Plasmid-Safe-ATP-dependent DNase (Epicentre Technologies) to eliminate RNA, linear WHV DNA intermediates, double-stranded (ds) linear WHV genomic DNA, residual genomic cellular DNA and certain forms of relaxed circular (rc) WHV DNA. The resulting DNA was subsequently treated with Mung Bean nuclease (New England Biolabs) to eliminate remaining rcDNA. The qPCR to quantify cccDNA assayed the dsDNA region unique for cccDNA. Forward primer, 1701-GGTCCG TGTTGCTTGGTCT-1719; reverse primer, 1977-GGACATGGAACACAGGCAAAAACA-1954; and TaqMan probe, 1846-AATGGGAGGAGGGCAGCATTGATCCT-1871 were used. The numbering corresponds to the WHV7 sequence (27). qPCR was performed with the Applied Biosystems TaqMan Gene Expression Mastermix using each primer at a concentration of 600 nM, and the TaqMan probe at a concentration of 250 nM. The reaction conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 60 s at 60°C. To calculate cccDNA copy numbers, a 10-fold dilution series of NheI-linearized plasmid PUC-CMVWHV (28) was used (range: 20–200,000 GE of WHV). The cccDNA copy numbers were expressed per µg of total DNA.

Quantification of WHV pre-genome(pg)/pre-core RNA copy numbers using qPCR

For eliminating WHV DNA, total RNA from normal liver tissues or HCCs was treated with Turbo DNase (Ambion) (6 units of DNase/1µg of RNA) for 2 hours at 37°C. The cDNA was synthesized with high Capacity cDNA Reverse Transcription Kit (Applied Biosystems) using the reverse primer for qPCR, 2579-TGGCAGATGGAGATTGAGAGC-2559 that is located in a region exclusively present on WHV pre-genome (pg)/pre-core RNAs. For the subsequent qPCR (that we have developed) forward primer, 2504-AGAAGACGCACTCCCTCTCCT-2524; reverse primer (also used for the RT step as described above); and a TaqMan probe, 2531- AGAAGATCTCAATCACCGCGTCGCAG-2556 were used. The numbering corresponds to the WHV7 sequence (27). qPCR was carried out with the Applied Biosystems TaqMan Gene Expression Mastermix using each primer at a concentration of 900 nM, and the TaqMan probe at a concentration of 250 nM. The reaction conditions were 10 min at 95°C, followed by 40 cycles of 15 s at 95°C, and 60 s at 60°C. To quantify WHV pgRNA copy numbers, a 10 fold dilution series of NheI-linearized plasmid PUC-CMVWHV was used (range: 20–200,000 GE of WHV). The pgRNA copy numbers were expressed per µg of total RNA.

Immunohistochemistry

Normal liver tissues from LL, left medial liver lobe (LM) and right lateral liver lobe (RL) and HCCs were harvested at the end of the study and were processed together with the samples biopsied one week prior to wHDV super-infection. Paraffin sections of formalin-fixed tissues were immunostained with polyclonal rabbit antibodies against recombinant small δAg (1:8000 dilution) followed by immunoperoxidase detection and hematoxylin-eosin post-staining (29).

RESULTS

Super-infection with wHDV of WHV carrier woodchucks with HCCs

To determine whether hepadnavirus-induced HCCs are susceptible to HDV infection, three WHV carriers (M7724, M7788 and F7807) were used at the late stage of chronic infection, when HCCs already had developed. WHV carriers were super-infected with wHDV, using a low MOI of 0.27 HDV GE/hepatocyte. Six weeks after wHDV super-infection, woodchucks were euthanized and blood, normal liver tissues, and HCCs were examined for markers of HDV and WHV infections. Serum samples were assayed for HDV genomic RNA and WHV DNA using qPCRs as described previously (19). As shown in Figure 1, all woodchucks quickly developed HDV viremia, and the serum HDV titers reached the WHV titers within two to four weeks. The increase of HDV titers coincided with a transient 4–10 fold decrease in WHV titers. The serum concentrations of HDV and WHV remained relatively high for the duration of the experiment. Thus, all WHV carriers were successfully super-infected with HDV.

Figure 1.

Susceptibility of WHV-induced HCCs to HDV infection

Woodchucks were monitored for six weeks following HDV super-infection assuming that this period is long enough to develop detectable HDV infection, and short enough, so new HCCs likely will not develop. During necropsy at the end of the study, one HCC was recovered from the liver of woodchuck M7724, five HCCs – from M7788, and two HCCs – from F7807. All HCCs with exception of HCC3 of the M7788 and HCC2 of the F7807 were identified in the WHV-infected livers prior to wHDV super-infection via ultrasound imaging and open surgery, and therefore were developed before super-infection.

We examined total RNA from normal liver tissues and HCCs by Northern analysis. For discrimination between G and αG HDV RNAs, HDV strand-specific hybridizations were employed (Fig. 2). The detection of αG RNA is an ultimate proof of infection, since αG RNA is absent from the virions and only appears via RNA replication following HDV infection of hepatocyte (2). At the top panel of Figure 2, it is shown that all three RNA samples extracted from normal LL tissues of either woodchuck M7724 (lane 1) or F7807 (lane 3), and from normal LM tissue of woodchuck M7788 (lane 2) were positive for αG RNA. Also, all HCCs assayed (i. e., HCC1 from woodchuck M7724 (lane 4); HCC1, HCC3, HCC4 and HCC5 from woodchuck M7788 (lanes 5, 6, 7 and 8, respectively); and HCC1 and HCC2 from woodchuck F7807 (lanes 9 and 10, respectively)) were positive for αG RNA, and thus were infected with wHDV. The levels of αG RNA in HCCs and in normal liver tissues were comparable. As anticipated, all RNA samples that tested positive for αG RNA, were also positive for G RNA (Fig. 2, bottom panel). No αG and G RNA were detected in total RNA extracted from the HCCs of two control WHV carriers M7746 and M7747, which were not super-infected with wHDV (lanes 11 and 12).

Figure 2.

These findings obtained by Northern analyses were confirmed and extended using HDV strand-specific qPCR (Table. 1). The RNAs from all normal and HCC tissues obtained from the super-infected animals during necropsy tested positive for both strands of HDV RNA. As anticipated, the G RNA levels were ~7–27-fold higher than those of αG (2). In animal M7724, the HDV RNA levels in HCC1 were higher than those in normal LL, LM and RL tissues. In woodchuck M7788, in the HCC3, HCC4 and HCC5 the levels of HDV RNA accumulation were higher than in surrounding normal liver tissues. The HCC1 (M7788) had HDV RNA levels comparable to those of normal tissues. Only HCC2 from the same animal had the lowest HDV RNA levels amongst all tissues analyzed. This may reflect a difference in the susceptibility to infection related to the differentiation state of HCC. The levels of G RNA accumulation in HCCs of woodchuck M7807 were ~4-fold lower than the average G RNA level in normal tissues. As expected, total RNA from HCC1s of WHV carriers M7746 and M7747 that were not super-infected with wHDV, and also the RNAs from the normal liver tissues and HCC1s of woodchucks M7724, M7788 and F7807, which were obtained during laparotomy prior to wHDV super-infection, were negative for HDV RNAs (data not shown). All HCCs including those identified prior to wHDV super-infection, became HDV-positive following super-infection, and most of them appeared to be infected at least as efficiently as normal liver tissues.

Table 1.

Quantitative analysis of HDV genomic (G) and antigenomic (αG) RNAs in normal liver tissues and WHV-induced HCCs using HDV strand-specific real-time PCR assays.

Woodchuck	Normal liver tissue	HDV RNA (×104 GE/µg total RNA)			HCC	HDV RNA (×104 GE/µg total RNA)
		G	αG	G/αG ratio		G	αG	G/αG ratio
M7724	LL	113d	7.73a	14.6	HCC1	252d	15.8b	15.9
	LM	81.1b	6.69a	12.1
	RL	102b	9.30a	11.0
M7788	LL	48.7d	1.99a	24.5	HCC1	78.1b	5.07a	15.4
	LM	107c	11.7a	9.15	HCC2	0.87b	0.10b	8.4
					HCC3	354d	13.0a	27.2
					HCC4	312c	16.8c	18.6
					HCC5	743b	37.0a	20.1
F7807	LL	1090c	73.9a	14.7	HCC1	215c	23.1c	9.3
	LM	827a	59.7a	13.9	HCC2	278c	39.1a	7.1
	RL	1020b	67.4a	15.1

LL – left liver lobe, LM – left medial liver lobe, RL – right lateral liver lobe. The number of HDV genome equivalents (GE) was determined using HDV strand-specific qPCR assays. To calculate copy numbers of G and αG HDV RNA, we used the in vitro transcribed gel-purified unit-length HDV RNA standard of genomic or antigenomic polarity, respectively.

The letters a, b, c, or d indicate the average number based on the analysis of two, three, four or five independent RNA isolations, respectively. In each qPCR assay, each RNA sample was analyzed in triplicate.

Next, formalin-fixed sections of normal liver tissues and HCCs were examined by immunohistochemistry, using antibodies against δAg. As anticipated, all normal liver tissues from woodchucks super-infected with wHDV displayed δAg -positive hepatocytes. Figure 3 demonstrates the staining for newly synthesized δAgs in several normal liver tissues of woodchucks M7724, M7788 and F7807. The intracellular δAg distribution is typical for HDV infection. The staining is mainly nuclear with apparent nucleolar exclusion (Fig. 3B–E). Occasionally, HDV-infected hepatocytes display additional cytoplasmic staining (Fig. 3F) that was observed previously and likely represents the appearance of δAgs with mutation(s) in the nuclear localization signal and/or the formation of complexes between δAgs and WHV envelope proteins (19, 30–31). Similarly, all the HCCs except HCC2 of woodchuck M7788 displayed HDV-positive cells. This observation correlates with the finding that HCC2 had the lowest level of HDV replication (Table 1). Figure 4 demonstrates HDV-positive cells from five different HCCs. The intracellular δAg distribution patterns (Fig. 4B–F) are virtually the same as those observed in normal hepatocytes (Fig. 3B–F). The unperturbed morphology of normal hepatocytes and the tumorous phenotype of HCC cells (including HDV-positive cells in both kinds of tissues) were confirmed by a pathologist (IT), thus verifying that both normal hepatocytes (Fig. 3) and HCC cells (Fig. 4) were infected with wHDV. The samples of normal liver tissues and HCCs that were obtained via surgery prior to wHDV super-infection were negative for δAgs (Fig. 3A, and Fig. 4A, respectively), as expected. The number of HDV-positive cells (with the exception of the HCC2 of woodchuck M7788) ranged between 0.09 and 6.7 cells/per 1000 analyzed cells (Table 2) that reflects the consequences of low MOI infection (0.27 HDV GE/hepatocyte) as early as six weeks post-inoculation. Within each set of tissues obtained from an individual animal a higher number of HDV-positive cells did not always correspond to the higher level of HDV RNA accumulation (Tables 1 and 2), likely because different subpopulations of hepatocytes may support the same efficiency of wHDV entry, but different levels of HDV RNA replication. Based on the numbers of HDV-infected cells (Table 2) and the qPCR data (Table 1), we confirmed that normal hepatocytes and HCC cells have comparable susceptibilities to HDV infection in vivo.

Figure 3.

Figure 4.

Table 2.

Numbers of the cells infected with HDV in normal liver tissues and in WHV-induced HCCs.

Woodchuck	Normal liver tissue	Number of cells positive for δAg /1000 cells	HCC	Number of cells positive for δAg /1000 cells
M7724	LL	0.29 (7/24096)	HCC1	0.45 (9/20232)
	LM	0.25 (3/12177)
	RL	0.42 (6/14315)
M7788	LL	0.7 (18/25531)	HCC1	2.0 (65/31760)
	LM	0.09 (17/189300)	HCC2	0.0 (0/28000)
			HCC3	1.7 (16/9600)
			HCC4	6.7 (116/17423)
			HCC5	3.1 (60/19360)
F7807	LL	3.1 (33/10750)	HCC1	0.4 (9/21677)
	LM	2.2 (23/10450)	HCC2	0.8 (34/44160)
	RL	4.4 (67/15120)

LL – left liver lobe, LM – left medial liver lobe, RL – right lateral liver lobe. Paraffin sections of formalin-fixed tissue were stained by immunoperoxidase technique using antibodies against delta antigen (δAg). The numbers of δAg-positive cells per total number of analyzed cells are shown in parentheses.

Markers of WHV infection in normal liver tissues and HCCs

Detection of cccDNA, which is not present in virions, is the ultimate evidence of hepadnavirus infection. The quantification of WHV cccDNA by qPCR is summarized in Table 3. Overall, in HCCs the levels of cccDNA accumulation were ~5 to 360-fold lower than in normal liver tissues. Several recent studies analyzed HCCs induced by HBV or WHV using immunostaining for the core antigen and in situ hybridization for viral DNAs, and concluded that HCCs were free of viral replication markers (15–17). Our data suggest that during chronic infection, WHV replication is not completely blocked, but is significantly suppressed in the majority of HCCs. The only exception, HCC3 of woodchuck M7788 had 4.8 times less cccDNA as compared to normal liver tissues. Examination of tissue sections by a pathologist (IT) did not detect normal (non-tumorous) hepatocytes within HCC tissues, indicating that we indeed quantified the accumulation of cccDNA in HCCs and not in normal hepatocytes that could have been engulfed in HCCs. Overall, the susceptibility of the normal liver tissues and HCCs to HDV infection apparently does not correlate with the extent of WHV replication, because no correlation was observed between the levels of WHV and HDV replications (Tables 1 and 3).

Table 3.

Quantitative analysis of covalently closed circular WHV DNA (cccDNA) in normal liver tissues and in WHV-induced HCCs using WHV cccDNA-specific real-time PCR assay.

Woodchuck	Normal liver tissue	WHV cccDNA (GE/µg total DNA)	HCC	WHV cccDNA (GE/µg total DNA)
M7724	LL	1.45 ×105	HCC1	7.85 ×103 (15.7 fold lower)
	LM	1.31 ×105
	RL	9.29 ×104
M7788	LL	3.37 ×104	HCC1	1.57 ×103 (17.9 fold lower)
	LM	2.25 ×104	HCC2	5.15 ×102 (54.6 fold lower)
			HCC3	5.86 ×103 (4.8 fold lower)
			HCC4	4.00 ×102 (70.2 fold lower)
			HCC5	7.84 ×101 (358.6 fold lower)
F7807	LL	1.10 ×105	HCC1	1.38 ×103 (71.5 fold lower)
	LM	8.34 ×104	HCC2	2.24 ×103 (44.0 fold lower)
	RL	1.02 ×105

LL – left liver lobe, LM – left medial liver lobe, RL – right lateral liver lobe. The numbers of WHV cccDNA genome equivalents (GE) were determined using WHV cccDNA-specific real-time PCR assay and represent the average for two independent cccDNA preparations. For each animal, the fold reduction of cccDNA content in HCCs has been calculated by dividing the average number of cccDNA GE in normal tissues by the number of cccDNA GE in a particular HCC. In each qPCR assay, each DNA sample was analyzed in triplicate.

Next, RNAs extracted from normal tissues and HCCs were treated with DNase to eliminate WHV DNAs, and subsequently were assayed for WHV pre-genome/pre-core RNAs (pgRNA) using qPCR (Table 4). We found that pgRNA accumulation in HCCs was diminished within 2.5 – 120-fold range as compared to corresponding normal liver tissues. The HCCs from woodchucks M7724 and F7807 accumulated ~16 – 36 times less of pgRNA than normal tissues, while HCC2, HCC3 and HCC5 from woodchuck M7788 accumulated only ~2.5 – 7.0 times less pgRNA. The HCC1 and HCC4 from woodchuck M7788 accumulated 120.5 and 23.1 times less pgRNA, respectively (Table 4). No correlation was found between the levels of pgRNA and cccDNA in both normal liver tissues and in HCCs. Based on the cccDNA levels, WHV replication was significantly suppressed in most HCCs. At the same time, pgRNA accumulation in HCCs seemed to be less profoundly decreased (Tables 3 and 4). Similar to cccDNA levels, there was no apparent correlation between the accumulation levels of HDV RNAs and WHV pgRNA (Tables 1 and 4).

Table 4.

Quantitative analysis of WHV pre-genome/pre-core RNA (pgRNA) in normal liver tissues and in WHV-induced HCCs using WHV pgRNA-specific real-time PCR assay.

Woodchuck	Normal liver tissue	WHV pgRNA (GE/µg total RNA)	WHV pgRNA/ cccDNA	HCC	WHV pgRNA (GE/µg total RNA)	WHV pgRNA/ cccDNA
M7724	LL	4.30 ×108	2.95 ×103	HCC1	1.12 ×107 (36.4 fold lower)	1.43 ×103
	LM	2.61 ×108	2.00 ×103
	RL	5.38 ×108	5.79 ×103
M7788	LL	6.69 ×107	1.98 ×103	HCC1	4.94 ×105 (120.5 fold lower)	3.15 ×102
	LM	5.22 ×107	2.32 ×103	HCC2	2.43 ×107 (2.5 fold lower)	4.72 ×104
				HCC3	8.03 ×106 (7.4 fold lower)	1.37 ×103
				HCC4	2.58 ×106 (23.1 fold lower)	6.46 ×103
				HCC5	1.47 ×107 (4.1 fold lower)	1.87 ×105
F7807	LL	4.25 ×108	3.84 ×103	HCC1	2.43 ×107 (16.4 fold lower)	1.76 ×104
	LM	2.85 ×108	3.42 ×103	HCC2	1.85 ×107 (21.5 fold lower)	8.27 ×103
	RL	4.84 ×108	4.77 ×103

LL – left liver lobe, LM – left medial liver lobe, RL – right lateral liver lobe. The numbers of WHV pre-genome/pre-core RNA (pgRNA) genome equivalents (GE) were determined using WHV pgRNA-specific real-time PCR assay and represent the average for two independent RNA preparations. In each qPCR assay, each DNA sample was analyzed in triplicate. The ratio of WHV RNA/cccDNA was calculated by dividing the number of the pgRNA copies/per µg of total RNA by the number of cccDNA copies/ per µg of total DNA, and it indicates rate of the WHV pgRNA transcription.

In addition, we performed immunostaining for WHV core antigen. Similarly to previous reports (15–17), we found that normal liver tissues contained a considerable number of strongly core-positive hepatocytes along with hepatocytes that displayed core staining of reduced intensity (Fig. S1). The HCCs demonstrated an overall negative core staining, with only light positivity in rare neoplastic subpopulations (Fig. S2). No core-positive cells were observed in HCC1 of woodchuck M7724 and in HCC2 of woodchuck F7807.

Discussion

In the present study, we using Northern analysis, qPCR and immunohistochemistry established for the first time that HDV infects WHV-induced HCCs in vivo, which advances our understanding of the infection mechanisms of HDV and hepadnavirus, and of the relationship between the ongoing infection and development/progression of HCC. The levels of HDV replication (Table 1) and numbers of HDV-infected cells (Table 2) demonstrate that HCC cells in vivo express functional putative hepadnavirus receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins, and trafficking and replication of HDV with the efficiency comparable to that of normal hepatocytes. Taken together with detection of WHV cccDNA in tumors, which suggests WHV replication and therefore a production of the envelope proteins in HCC cells, our data lead to the speculation that HCC cells may support the entire HDV life cycle in vivo, including the assembly and egress of new virions. The fact that only HCC2 (M7788) was inefficiently infected with HDV (Table 1) may reflect differences in the differentiation status of HCCs. By inference, it is very likely that HBV-induced HCCs are susceptible in vivo to infection with HBV-enveloped HDV. Overall, it appears that the loss of hepadnavirus receptors is not an essential feature of HCC development.

A single previous study has reported detection of HDV RNA in HCCs of super-infected WHV carrier woodchucks. However, this article did not address the mechanism of how and when HDV appeared in HCCs (32). We can envision two such mechanisms. One is that HDV persists in the hepatocyte from the moment of the initial infection (i.e., before the infected hepatocyte becomes malignant) and therefore may influence the induction and development of HCC. The second is that HDV infects already established hepadnavirus-induced HCC. Previously, it has not been investigated whether HDV could infect hepadnavirus-induced HCCs in vivo. In the present study, we clearly demonstrated that in vivo HDV is able to infect WHV-induced HCCs in WHV carrier woodchucks. Because the HDV genome can accumulate up to ~300,000 copies/infected cell (33), our data suggest that the efficient HDV replication may change the gene expression profile in HCC cells following infection of the tumor, and therefore may influence further HCC development. However, the effect of HDV replication in HCC cells on further development/progression of a tumor has to be elucidated in future studies.

Previously, several reports described HCCs induced either by HBV or by WHV as apparently hepadnavirus-free (based on negative staining for the core antigen and negative in situ hybridization for viral DNAs) regardless of ongoing viremia (15–17). Accordingly, a hypothesis was proposed that HCCs, which originated from hepadnavirus-infected hepatocytes (as evidenced by presence of integrated hepadnavirus DNA in HCCs), are resistant to new re-infections with a hepadnavirus (15–16). Our results are not consistent with the absence of WHV replication, but rather suggest its significant suppression in most HCCs. Our data suggest that WHV reverse transcription and/or conversion of the rcDNA into cccDNA is suppressed, or cccDNA stability is compromised in HCCs. However, this hypothesis needs to be tested further using a larger number of HCCs and matching liver tissues from WHV carriers. Interestingly, based on the copy number ratio of pgRNA/cccDNA, which may indicate the efficiency of cccDNA-directed transcription of pgRNA, and considering that it is unlikely that pgRNA could have been transcribed from integrated WHV DNA, it seems that rates of pgRNA transcription in HCCs were either comparable to or higher than those in normal liver tissues. The only exception was HCC1 (M7788), for which the pgRNA/cccDNA ratio was 6.9-fold lower than the average ratio calculated for normal liver tissues (Table 4). Furthermore, our data demonstrating the susceptibility of HCCs to HDV infection in vivo suggest that if above proposed super-infection exclusion for hepadnavirus occurs in HCCs, it is mediated by a block at post-entry step. Currently, super-infection of hepadnavirus-induced HCCs with either HBV or WHV has yet to be demonstrated.

The observed absence of a correlation between HDV and WHV replication levels in both normal liver tissues and HCCs suggests that, because HDV requires only the envelope proteins from the helper hepadnavirus (2), then as long as sufficient supply of the envelope proteins is available, the extent of HDV infection is not directly dependent on the rate of hepadnavirus replication. This may explain at least in part why some anti-HBV drugs do not inhibit HDV infection.

Abbreviations

HDV

hepatitis delta virus

HBV

hepatitis B virus

HCC

hepatocellular carcinoma

WHV

woodchuck hepatitis virus

wHDV

HDV coated with the envelope proteins of WHV

δAg

delta antigen

LL

left liver lobe

GE

genome equivalent

MOI

multiplicity of infection

G

genomic

αG

antigenomic

qPCR

quantitative real-time PCR assay

RT

reverse transcription

cccDNA

covalently closed circular DNA

ds

double-stranded

rcDNA

relaxed circular DNA

pg

pregenomic

LM

left medial liver lobe

RL

right lateral liver lobe