Woodchuck Hepatitis Virus Enhancer I and Enhancer II Are Both Involved in N-myc2 Activation in Woodchuck Liver Tumors

Direct activation of the N-myc2 oncogene by insertion of woodchuck hepatitis virus (WHV) DNA is a major oncogenic step in woodchuck hepatocarcinogenesis. We previously reported that WHV enhancer II (We2), which controls expression of the core/pregenome RNA, can also activate the N-myc2 promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2 promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the win locus in tumor 2260T1 and mapping approximately 175 kb 3′ of N-myc2. This viral insert was made of 11 concatemerized WHV fragments, 5 of which overlapped with We2 sequences and 1 with WHV sequence homologous to that of hepatitis B virus enhancer I (We1). In transient transfection assays in hepatoma-derived cells, the We2 activator was found to be fully effective only when inserted in close proximity to the N-myc2 promoter whereas the We1 element by itself was apparently devoid of activity. In contrast, the 2260T1 viral insert exhibited a potent enhancer capacity that depended both on multimerized We2 and on We1 sequences. In a survey of different woodchuck hepatomas, both elements were commonly found within integrated viral sequences involved in long-range N-myc2 activation.     

Nucleotide sequence of the woodchuck hepatitis virus surface antigen mRNAs and the variability of three overlapping viral genes

A cDNA library was constructed from the liver of a woodchuck chronically infected with woodchuck hepatitis virus (WHV). A clone, pWS23, encompassing the entire surface and X genes of WHV was isolated. Comparison of the complete nucleotide (nt) sequence of pWS23 with those of genomic DNAs from two different WHV isolates showed that it contained a nearly full-length copy of the major mRNA encoding the viral surface antigen (5 mRNA). It was colinear with the WHV genome over 1858 nt and terminated 22 nt downstream from the variant polyadenylation signal within the core gene. Evidence for heterogeneity of the 5′ -terminal region of the S mRNA came from direct sequencing of the 5′ extremities of 20 cDNA inserts, similar to that of pWS23, isolated from a second cDNA library of the same woodchuck liver. In agreement with previous mapping studies of hepadnaviruses, two main initiation regions of S mRNA were localized 27–30 nt upstream and 22–49 nt downstream from the pre-S2 initiation codon. Further analysis of the amino acid sequences of the surface, polymerase and X genes of WHV showed a high conservation among three WHY isolates and a similar distribution of conserved and variable regions in woodchuck and human hepatitis B viruses.     

Analysis of integrated ground squirrel hepatitis virus and flanking host DNA in two hepatocellular carcinomas.

We cloned the integrated ground squirrel hepatitis B virus (GSHV) sequences from two hepatomas showing a single viral insertion. The GSHV inserts shared structural features with integrated DNAs of other hepadnaviruses. Insertional activation of a cellular gene appears unlikely: the integrated GSHV sequences lacked the known viral enhancers and were not expressed in the tumors, and we found no evidence for the presence of a gene at the integration site. Our results, together with those earlier studies, suggest that GSHV does not behave as an extensive insertional mutagen, in sharp contrast with the closely related woodchuck hepatitis virus. GSHV may thus cause carcinogenesis by more indirect mechanisms, as does the human hepatitis B virus.     

Repeated passage of wild-type woodchuck hepatitis virus in lymphoid cells does not generate cell type-specific variants or alter virus infectivity

Woodchuck hepatitis virus (WHV), which is closely related to human hepatitis B virus, infects the liver but also invariably establishes persistent infection in the lymphatic system. Although the dose of invading virus appears to be the main factor in determining whether WHV infection is restricted to the lymphatic system or also engages the liver, the nature of WHV lymphotropism remains unclear and a role for a specific lymphotropic variant was not excluded. The availability of woodchuck lymphocyte and hepatocyte cultures susceptible to WHV infection allows investigation of this issue in vitro. We hypothesized that repeated passage of wild-type WHV in lymphoid cells should lead to enrichment of a lymphotropic virus variant, if in fact such a variant exists. For this purpose, wild-type WHV with a homogeneous sequence was used as the inoculum, while lymphoid cells from a single healthy woodchuck donor and a normal woodchuck WCM-260 hepatocyte line served as infection targets. The serial passage of the wild-type virus repeated up to 13 times for both cell types did not lead to the emergence of cell type-specific WHV variants, as revealed by sequence analysis of the virus envelope and the core and X gene sequences. Moreover, the virus passaged in both cell types remained infectious for naive woodchucks, produced infection profiles that depended upon virus dose but not on virus cellular origin, and retained its initial DNA sequence. These results imply that WHV lymphotropism is a natural propensity of the wild-type virus and is not a consequence of infection with a viral variant.     

Failure to detect polyalbumin-binding sites on the woodchuck hepatitis virus surface antigen: implications for the pathogenesis of hepatitis B virus in humans.

Binding sites for polymerized albumin on hepatitis B virus components were reported in human hepatitis B virus chronic carriers predominantly with active viral replication (HB e antigen positive). The presence of comparable albumin-binding sites in the woodchuck hepatitis virus (WHV) model was examined on WHV components obtained from woodchucks with active viral replication (DNA polymerase positive). Binding sites for polymerized woodchuck serum albumin were not detected on the intact WHV virion, on 22-nm woodchuck hepatitis surface antigen (WHsAg), or on WHsAg polypeptides. Woodchuck albumin was not detected in purified 22-nm WHsAg, and anti-albumin antibodies were not detected in WHV chronic-carrier woodchucks. Our results in the WHV model argue against a role for viral polyalbumin-binding sites in tissue- and host-specific virus infectivity.     

荧光定量PCR检测土拨鼠肝炎病毒核酸方法的建立

目的建立土拨鼠肝炎病毒（woodchuck hepatitis virus,WHV）核酸的荧光定量PCR（Real-time PCR）检测方法,应用于土拨鼠肝炎病毒模型的研究。方法分别根据土拨鼠肝炎病毒核心抗原（WHcAg）和表面抗原（WHsAg）的DNA序列设计13对扩增引物,从中筛选无非特异性扩增及引物二聚体且灵敏度高的引物,用于土拨鼠血清中WHV DNA的Real-time PCR检测。建立感染土拨鼠肝炎病毒的土拨鼠血清中WHV核酸的Real-timePCR检测方法。结果根据WHsAg基因的5＇端设计的一对引物WHVSF1与WHVSR1,检测灵敏度可达1×101拷贝/μL,病毒拷贝数与Real-time PCR Ct值的标准曲线的R2值为0.997,且电泳未见明显非特异性条带及引物二聚体。结论建立了土拨鼠血清中WHV DNA的Real-time PCR检测方法,该方法为进一步研究土拨鼠肝炎病毒模型奠定了基础。     

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.     

Inhibition of cellular proteasome activities enhances hepadnavirus replication in an HBX-dependent manner

The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.     

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.     

Quantitative detection of hepadnavirus-infected lymphoid cells by in situ PCR combined with flow cytometry: implications for the study of occult virus persistence

The detection of small amounts of viral pathogens in infected cells by classical PCR is hampered by a partial loss of virus nucleic acid due to extraction and by difficulties in discrimination between truly intracellular virus genome material and that possibly adhered to the cell surface. These impediments limit reliable identification of virus traces within infected cells, which are typically encountered in latent and persistent occult infections. In this study, hepadnavirus-specific in situ PCR combined with the enzymatic elimination of extracellular virus and flow cytometry permitted detection of viral genomes in lymphoid cells without nucleic acid isolation and allowed quantification of infected cells during the course of persistent infection with woodchuck hepatitis virus (WHV). The validity of the procedure was confirmed by hybridization analysis of the in situ-amplified viral sequences. The results showed that hepadnavirus can be directly detected within lymphoid cells not only in serologically accountable infection, but also years after recovery from viral hepatitis and in the course of primary occult virus carriage. Percentages of infected peripheral lymphoid cells in symptomatic WHV hepatitis fluctuate between 3.4 and 20.4% (mean +/- standard error of the mean, 9.6% +/- 1.7%), whereas those in persistent, serologically mute WHV infection range from 1.1 to 14.6% (mean +/- standard error of the mean, 4.8% +/- 0.8%) (P = 0.005). The data obtained provide further evidence that WHV infection continues indefinitely in the lymphatic system independently of whether it is symptomatic or concealed. They document that hepadnavirus can be detected in a significant proportion of circulating lymphoid cells in both immunovirologically apparent as well as occult persistent infection.     

Monitoring of early events of experimental woodchuck hepatitis infection: Studies of peripheral blood mononuclear cells by cytofluorometry and PCR

Abstract The peripheral blood mononuclear cells (PBMC) of woodchucks experimentally infected by woodchuck hepatitis virus (WHV) were examined simultaneously for the presence of membrane associated WHV antigens by cytofluorometry, and for WHV DNA and RNA sequences by the polymerase chain reaction (PCR). Four woodchucks were inoculated: two with a well-defined infectious inoculum and two with an inoculum obtained from an animal at the late incubation phase, which was positive for WHV DNA by PCR but still devoid of WHV markers. Infection was demonstrated in all four inoculated woodchucks by the appearance at different times of WHV DNA and WHV antigens in both leucocytes and serum. WHV DNA was first detected by PCR either in the serum (two cases) or in leucocytes (two cases). The mean percentage of cells positive for membrane associated WHsAg or WHcAg detected by cytofluorometry were 37%±25 and 17%±15 respectively. After 8 weeks, all inoculated animals were WHsAg positive in serum. These data suggest that PBMC are involved in the early events of hepadnavirus infection. They also show that sera which are positive by PCR for WHV DNA may transmit viral infection even while still seronegative for WHV markers and for WHV DNA by dot blot.     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.     

Natural history of woodchuck hepatitis virus infections during the course of experimental viral infection: molecular virologic features of the liver and lymphoid tissues.

In this study, the kinetic patterns of woodchuck hepatitis virus (WHV) infection were monitored in the liver and the five primary components of the lymphoid system (peripheral blood lymphocytes, lymph nodes, bone marrow, spleen, and thymus). Groups of woodchucks experimentally infected with a standardized inoculum of WHV were sacrificed at different times over a 65-week period beginning in the preacute phase of viral infection and continuing to the period of serologic recovery or the establishment of chronic infections and subsequent hepatocellular carcinoma. Infection by WHV was not limited to the liver but involved the major components of the lymphoid system during all stages of virus infection. A complex series of kinetic patterns was observed for the appearance of WHV DNA in the different lymphoid compartments and the liver during the entire course of viral infection. A progressive evolution of different WHV genomic forms related to the replicative state of WHV was also observed. Lymphoid cells of the bone marrow were the first cells in which WHV DNA was detected, followed in order by the liver, the spleen, peripheral blood lymphocytes, lymph nodes, and finally the thymus. Several differences were observed in the cellular WHV DNA patterns between woodchucks that developed chronic WHV infections and those that serologically recovered from acute WHV infections. The observations compiled in this study indicate that the host lymphoid system is intimately involved in the natural history of hepadnavirus infections from the earliest stages of virus entry.     

Topoisomerase I-mediated integration of hepadnavirus DNA in vitro.

Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.