Woodchuck hepatitis virus is a more efficient oncogenic agent than ground squirrel hepatitis virus in a common host.

Chronic infection with hepatitis B viruses (hepadnaviruses) is a major cause of hepatocellular carcinoma (HCC), but the incubation time varies from 1 to 2 years to several decades in different host species infected with indigenous viruses. To discern the influence of viral and host factors on the kinetics of induction of HCC, we exploited the recent observation that ground squirrel hepatitis virus (GSHV) is infectious in woodchucks (C. Seeger, P. L. Marion, D. Ganem, and H. E. Varmus, J. Virol. 61:3241-3247, 1987) to compare the pathogenic potential of GSHV and woodchuck hepatitis virus (WHV) in chronically infected woodchucks. Chronic GSHV infection in woodchucks produces mild to moderate portal hepatitis, similar to that observed in woodchucks chronically infected with WHV. However, HCC developed in GSHV carriers about 18 months later than in WHV carriers. Thus, although both viruses are oncogenic in woodchucks, GSHV and WHV differ in oncogenic determinants that can affect the kinetics of appearance of HCC in chronically infected animals.     

Core particles of hepatitis B virus and ground squirrel hepatitis virus. II. Characterization of the protein kinase reaction associated with ground squirrel hepatitis virus and hepatitis B virus.

The recently described protein kinase activity in hepatitis B virus core antigen particles (Albin and Robinson, J. Virol. 34:297-302, 1980) has been demonstrated here in the liver-derived core particles of ground squirrel hepatitis virus. Both protein kinase activities were initially associated with DNA polymerase-positive heavy core particles in CsCl density equilibrium gradients and shifted to polymerase-negative cores during the course of purification. The major core-associated polypeptide of each virus was the dominant species labeled. A variable number of other polypeptide species were also labeled by this reaction. Tryptic peptide mapping of both major and minor phosphorylated polypeptides of each virus resulted in similar patterns, suggesting that many of the sites of phosphorylation were the same in the components of each core particle. Hydrolysis of these phosphorylated core particles revealed a major phosphoamino acid as serine and a minor phosphoamino acid as threonine. The products of the protein kinase reaction in both human hepatitis B and ground squirrel hepatitis virus core particles, then, share many characteristics. The possible function(s) of this protein kinase activity is discussed in the light of similarly characterized activities in other animal viruses.     

Virion DNA of ground squirrel hepatitis virus: structural analysis and molecular cloning.

The structure of the encapsidated DNA genome of ground squirrel hepatitis virus (GSHV) has been examined by restriction endonuclease cleavage, nucleic acid hybridization, and molecular cloning. GSHV virion DNA is a relaxed circular molecule of approximately 3,200 bases in length; most molecules harbor an extensive single-stranded region which is largely confined to one-half of the genome. The full-length viral DNA strand is covalently bound to protein. The single-stranded region can be repaired in vitro by the action of the endogenous virion polymerase, exogenously added DNA polymerase from avian myeloblastosis virus, or both. Restriction enzyme cleavage of viral DNA from different isolates demonstrated that multiple variants of GSHV exist in nature. The genomes of two such strains have been cloned in Escherichia coli, and their physical maps have been determined. Nucleic acid hybridization studies revealed that the strains share sequence homology with the DNA of human hepatitis B virus. Regions homologous to the coding regions for the surface and core antigens of human hepatitis B virus have been localized on the GSHV chromosome. Molecular cloning experiments have also led to the identification of a region of the viral genome which is altered in a procaryotic host.     

Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas.

Integrated hepatitis B virus (HBV) DNA is found in hepatocellular carcinomas which develop in HBV carriers. Presented here are the results of analyses of four integrants that show chromosomal rearrangements associated with the integrated HBV DNA. Two clones (p4 and C15) were found to have large inverted repeating structures, each consisting of HBV genome along with flanking cellular sequences. The structure must have arisen by duplication of the primary integrant, including the flanking cellular DNA, followed by recombination within the viral DNA. One of the two viral arms in each clone joins to the other viral arm at the "cohesive end region." Two clones (DA2-2 and DA2-6) were found to have integrated HBV sequences, each flanked by cellular DNAs from different chromosomes (chromosome X joined to 17 and chromosome 5 joined to 9). They must be the products of cellular DNA translocations using the integrated HBV DNA as the switch point. The viral DNA in each clone is a continuous stretch of a single virus genome with one end in the cohesive end region. These complex structures seem to have been produced by activation of the cohesive end of an integrant viral genome, followed by its recombination with another chromosomal DNA.     

Biological characterization of acute infection with ground squirrel hepatitis virus.

Ground squirrel hepatitis virus (GSHV) is a small DNA virus, structurally and antigenically related to the human hepatitis B virus, which occurs naturally among certain wild populations of ground squirrels (P. L. Marion et al., Proc. Natl. Acad. Sci. U.S.A. 77:2941-2945, 1980). Serum from naturally infected animals was used to transmit GSHV in the laboratory by parenteral inoculation of susceptible squirrels. Sixty percent of recipient animals developed viral surface antigenemia after a latent period of 2 to 3 months; three of these animals have remained viremic for over 9 months. Like hepatitis B virus, GSHV demonstrates marked hepatotropism, with viral DNA detected in significant quantities only in the liver, where an average of 6 X 10(2) to 6 X 10(3) viral DNA molecules per cell were found by molecular hybridization. However, histological signs of liver injury after acute infection are minimal. In contrast to infection of its natural host, parenteral administration of GSHV to rats, mice, guinea pigs, and hamsters did not result in demonstrable antigenemia, suggesting that the host range of GSHV, like that of hepatitis B virus, is narrow.     

Expression of integrated hepatitis B virus X variants in human hepatocellular carcinomas and its significance

Hepatitis B virus X protein (HBX) has been implicated in the transactivation of diverse cellular genes and possibly also the pathogenesis of human hepatocellular carcinoma (HCC). We report the characterization of HBX variants from HBV-related human hepatocellular carcinoma (HCC). These HBX variants were integrated into the host chromosomes and also expressed in the HCC tissues. In addition, we report a novel in vitro HBX activity assay based on color changes that were indicative of the beta-galactosidase enzyme activity. Conducted in wheat germ lysates, the transactivating function of either wild type or mutant HBX protein was measured through their interaction with the Early Growth Response factor 1 (Egr-1) that controls the beta-galactosidase gene. Further analysis of these HBX deletion mutants using this assay may shed new insights on the significance of various mutations occurring in HCC-associated HBX.     

Structural relationships between the surface antigens of ground squirrel hepatitis virus and human hepatitis B virus.

Several physical, chemical, and serological properties of surface antigen particles from ground squirrel hepatitis virus (GSHsAg) and human hepatitis B virus (HBsAg) were compared. GSHsAg and HBsAg particles were purified from positive sera by gel chromatography and isopycnic centrifugation. Both antigens consisted mainly of spherical particles with an average diameter of approximately 20 nm and a buoyant density in CsCl of approximately 1.19 g/ml. Their UV absorption spectra indicated the presence of more tryptophane than tyrosine and the absence of detectable nucleic acid. GSHsAg was found to contain two major polypeptides of approximately 23,000 and 27,000 daltons, with electrophoretic migration rates distinctly faster than those of the two major polypeptides of HBsAg particles. After radiolabeling of purified antigen preparations with Bolton-Hunter reagent, the two major polypeptides of GSHsAg showed almost identical tryptic peptide maps. The tryptic peptide map of the major polypeptide from GSHsAg contained 13 of 37 spots also present in the map of the major HBsAg polypeptide, and 13 of 27 spots in the map of the major HBsAg polypeptide were also present in the map of the major GSHsAg polypeptide. This suggests considerable sequence homology between the major surface antigen polypeptides of the two viruses. However, there was only a weak serological cross-reactivity between antigens of the two viruses. Using an anti-HBs-containing serum with a relatively strong cross-reactivity, GSHsAg was found to consist of at least two antigenically different subspecies. The more strongly cross-reacting from had a slightly higher buoyant density than the other antigenic form.     

Cloning and structural analysis of integrated woodchuck hepatitis virus sequences from hepatocellular carcinomas of woodchucks

Woodchuck hepatitis virus (WHV), like the related hepatitis B virus, induces in its natural host hepatocellular carcinomas that contain integrated viral sequences. As a first step in determining whether and how the integrated sequences contribute to formation of the tumors in which they are found, we have cloned two such integrations of WHV and have determined their structure by restriction mapping and heteroduplex electron microscopy. The identity of the cloned sequences was confirmed by comparison of restriction sites in the clones with those located by Southern blot analysis of tumor DNA. Viral sequences in both integrations are extensively rearranged, and in neither were all parts of the viral genome represented. In this respect, the behavior of WHV in vivo is similar to that of other DNA tumor viruses that have been studied in vitro. We discuss the implications of these results in relation to possible mechanisms for tumor induction by WHV.     

Ground squirrel hepatitis virus DNA: molecular cloning and comparison with hepatitis B virus DNA

Ground squirrel hepatitis virus (GSHV) shares many ultrastructural antigenic, molecular, and biological features with hepatitis B virus (HBV) of humans, indicating that they are members of the same virus group. Both viruses contain small circular DNA molecules which are partially single stranded. Here, we ligated an endonuclease EcoRI digest of GSHV DNA with EcoRI-cleaved plasmid vector pBR322 and cloned recombinant plasmids in Escherichia coli C600. Two cloned recombinants were characterized. One (pGS2) was found to contain only part of the GSHV genome, and the other (pGS11) was found to contain the entire viral DNA. A restriction endonuclease cleavage map of the GSHV insert in pGS11 and the locations of certain physical features of the virion DNA were determined. The relative positions of the single-stranded region, the unique 5' end of the short DNA strand, and the unique nick in the long DNA strand in GSHV DNA were found to be the same as those previously described for HBV DNA. Hybridization with an HBV [32P]DNA probe containing the apparent coding sequence for the major polypeptide of HBV surface antigen and a probe containing the putative coding sequence for the major polypeptide of the HBV core revealed specific homology with different restriction fragments of GSHV DNA. The two homologous regions had approximately the same locations relative to the single-stranded region, the 5' end of the short strand, and the nick in the long strand in the two viral DNAs. These results suggest that in both viruses the genes for the major HBV surface antigen and core polypeptides have the same locations relative to unique physical features of the viral DNAs.     

Antigenic and structural relationships of the surface antigens of hepatitis B virus, ground squirrel hepatitis virus, and woodchuck hepatitis virus.

The surface antigens of human hepatitis B (HBsAg), ground squirrel hepatitis (GSHsAg), and woodchuck hepatitis (WHsAg) viruses were compared serologically, and their major polypeptides were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping. Results showed that both GSHsAg and WHsAg are antigenically cross-reactive, that their major pairs of polypeptides have identical mobilities on sodium dodecyl sulfate gels, and that the major polypeptides of GSHsAg and WHsAg migrate faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than do the corresponding bands of HBsAg. The peptide maps of the major (P-22) surface antigen polypeptides of GSHsAg and WHsAg showed that they shared over half of their spots. Peptide mapping of HBsAg subtypes indicated a close relationship between the major polypeptides (P-24) of adw and adr and a more distal relationship to ayw. Only about 25% of the spots shared by the combined HBsAg subtypes were also found in the peptide maps of GSHsAg and WHsAg, indicating at least some structural homology among the major polypeptides of the human and animal virus surface antigen particles. This is also reflected in the serological cross-reactivity among HBsAg, GSHsAg, and WHsAg. Further, the detection of ground squirrel and woodchuck antigens by Ausria II radioimmunoassay, combined with peptide mapping data indicating the common origin of these viruses, suggests that the common a determinant is shared by each and is restricted to approximately 25% of the sequences in their major polypeptides.     

Structural rearrangement of integrated hepatitis B virus DNA as well as cellular flanking DNA is present in chronically infected hepatic tissues.

Cellular DNAs from human livers chronically infected with hepatitis B virus (HBV) were analyzed by Southern blot hybridization for the presence of integrated HBV DNA. In 15 of 16 chronically infected hepatic tissues, random HBV DNA integration was evident. By molecular cloning and structural analyses of 19 integrants from three chronically infected hepatic tissues, deletion of cellular flanking DNA in all cases and rearrangement of HBV DNA with inverted duplication or translocation of cellular flanking DNA at the virus-cell junction in some cases were noted. Thus, the rearrangement of HBV DNA or cellular flanking DNA is not a specific incident of hepatocellular carcinoma formation. Detailed analyses of various integrants bearing rearranged viral DNA failed to indicate any gross structural alteration in cellular DNA, except for a small deletion at the integration site, indicating that viral DNA rearrangement with inverted duplication possibly occurs before integration of HBV DNA. Based on nucleotide sequencing analyses of virus-virus junctions, a one- to three-nucleotide identity was found. A mechanism for this inverted duplication of HBV DNA is proposed in which illegitimate recombination between two complementary viral strands may take place by means of a nucleotide identity at the junction site in a weakly homologous region (patchy homology) on one side of adjoining viral sequences. For virus-cell junctions, the mechanism may be basically similar to that for virus-virus junctions.     

Nonoverlapping antigenic sites of woodchuck hepatitis virus surface antigen and their cross-reactivity with ground squirrel hepatitis virus and hepatitis B virus surface antigens.

Five nonoverlapping antigenic sites (sites I through V) of woodchuck hepatitis virus surface antigen were identified with competitive binding assays involving monoclonal antibodies. Site I contributed to cross-reactions among surface antigens of hepatitis B-like viruses infecting woodchucks, ground squirrels, and humans. At least three distinct sites (sites I, II, and III) are responsible for cross-reactions between woodchuck and ground squirrel hepatitis virus surface antigens. Sites IV and V of woodchuck hepatitis virus surface antigen are not major cross-reactive sites, suggesting that these elicit virus-specific antibodies. There were no cross-reactions with duck hepatitis B virus surface antigen.     

Upstream sequences and cap proximity in the regulation of polyadenylation in ground squirrel hepatitis virus.

The polyadenylation signal of mammalian hepadnaviruses is unusual in that its hexanucleotide element is the variant UAUAAA rather than AAUAAA. This signal functions inefficiently and must be augmented by multiple activator elements located in the upstream 400 nucleotides (nt) to promote efficient processing. Here we characterize one of these upstream elements, termed PS2, in the ground squirrel hepatitis virus. PS2 is located within the 107 nt 5' to the UAUAAA and raises the efficiency of polyadenylation by this signal from < 10% to 50 to 60%. It can function independently of the more 5' activator elements and conversely is not required for their function. Its action is orientation dependent, and a predicted stem-loop structure within the element is not necessary for its activity. PS2 is the sole upstream element that maps within the terminal redundancy of viral genomic RNA. Thus, it is present, together with the UAUAAA, at both the 5' and 3' ends of this RNA. During genomic RNA synthesis, the poly(A) signals in the 5' repeat are bypassed, while those in the 3' copy are used. The ability of PS2 to function independently of the other, more upstream activators suggests that the absence of the latter elements from the 5' redundancy is insufficient to account for bypass of the 5' poly(A) site, as we had earlier proposed. Rather, the short distance from the cap site to the UAUAAA at the 5' end of genomic RNA actively suppresses its use, as this suppression can be experimentally relieved by increasing this distance to 230 to 400 nt.     

Nucleotide sequence of an infectious molecularly cloned genome of ground squirrel hepatitis virus.

We have determined the complete nucleotide sequence of an infectious cloned genome of ground squirrel hepatitis virus (GSHV), a nonpathogenic member of the hepadnavirus group. The genome is 3,311 base pairs long and contains the major open reading frames described for the related human and woodchuck hepatitis B viruses (HBV and WHV, respectively). These reading frames include genes for the major structural proteins (the surface and core antigens), unassigned open reading frames (A and B), the longer of which is presumed to encode the viral DNA polymerase, and an open reading frame preceding and continuous with the surface antigen gene. The arrangement of these open reading frames is similar to that encountered in the genomes of HBV and WHV: all of the reading frames are encoded on the same strand, they are positioned in the same fashion with respect to each other, and a large portion (at least 51%) of the genome can be translated in two reading frames. Comparisons of the predicted translational products of the three mammalian hepadnaviruses reveal 78% amino acid homology between the proteins of GSHV and WHV and 43% homology between those of GSHV and HBV. In addition, a perfect direct repeat of 10 to 11 base pairs, separated by ca. 46 to 223 base pairs, is present in the three mammalian viruses and in duck hepatitis B virus; the position of the repeats near the 5' termini of the two strands of virion DNA suggests a role in viral replication.     

Closed circular viral DNA and asymmetrical heterogeneous forms in livers from animals infected with ground squirrel hepatitis virus

To identify possible intermediates in the replication of ground squirrel hepatitis virus, we characterized the major forms of intracellular virus-specific DNA in the livers of infected ground squirrels. A variety of DNA species were found: covalently closed circular molecules, relaxed circular molecules, and a heterogeneous collection of molecules that migrated ahead of closed circular DNA during agarose gel electrophoresis. The heterogeneous DNA was at least partly single stranded, consisted of minus strands in a greater than eight-fold mass excess of plus strands, and was tightly associated with protein.     

In vitro recombinants of ground squirrel and woodchuck hepatitis viral DNAs produce infectious virus in squirrels.

Hepatitis B viruses of humans, woodchucks, ground squirrels, and ducks are similar biochemically but differ with respect to host range and pathogenicity. To pursue the genetic basis of these properties in the absence of a cell culture system for virus growth, we exploited the demonstrated infectivity of cloned hepatitis B virus DNA in whole animals. We constructed several recombinant molecules in vitro between cloned infectious genomes of woodchuck hepatitis virus (WHV) and ground squirrel hepatitis virus (GSHV) and assayed the recombinants for infectivity after intrahepatic injection in ground squirrels, which support growth of GSHV but not WHV. Two of the recombinants molecules initiated productive infection; in one recombinant genome, 76% of the coding region for the major surface glycoprotein of GSHV and for the overlapping portion of the presumptive gene for DNA polymerase was replaced by WHV DNA; in the other, 29% of the same coding domain was replaced by WHV DNA. These findings demonstrate the feasibility of generating viable recombinants of hepatitis B viruses from different animal species and suggest that the major host range determinants are not encoded within the surface antigen gene of these viruses.     

Antibodies to pre-S and X determinants arise during natural infection with ground squirrel hepatitis virus.

The DNA sequence of the ground squirrel hepatitis virus (GSHV) genome predicts the existence of several proteins in addition to the major surface (S) and core antigens. These include the pre-S1 and pre-S2 proteins, initiated at sites within the open reading frame preceding and continuous with the coding region for the S gene product, and the X protein, the putative product of an independent reading frame. Using an antibody directed against a peptide predicted by codons 130 to 143 of the pre-S1 reading frame, we identified a 43-kilodalton product of the pre-S1 coding region in preparations of GSHV surface antigen purified from the sera of infected animals. In addition, by immunoprecipitation of S- and pre-S-specific in vitro translation products with ground squirrel sera obtained after GSHV infection, we determined that antibodies arise to both S and pre-S determinants. The antibody response to pre-S includes, in some cases, reactivity to pre-S1-specific domains and is not always associated with an anti-S response. Similarly, by production of the viral X gene product in vitro followed by immunoprecipitation with ground squirrel sera, we showed that antibodies to this viral gene product also arise during infection, indicating that X antigenic determinants are synthesized during viral infection and are recognized by the host immune system.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.     

Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.