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.     

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.     

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.     

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.     

Deoxyribonucleic Acid Polymerase(s) of Rous Sarcoma Virus: Effects of Virion-Associated Endonuclease on the Enzymatic Product

Purified preparations of Rous sarcoma virus (RSV) contain ribonuclease which is either a constituent of the virion surface or an adsorbed contaminant. Treatment of the virus with nonionic detergent to activate ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase renders the viral genome susceptible to hydrolysis by the external ribonuclease. The extent of this susceptibility can be substantially reduced by the use of limited amounts of detergent. At a concentration of detergent which provides a maximum initial rate of DNA synthesis, the degradation of endogenous viral RNA results in a reduced yield of high molecular weight DNA: RNA hybrid from the polymerase reaction. Attempts to detect virion-associated deoxyribonuclease, by using a variety of double helical DNA species as substrates, have been unsuccessful, but small amounts of nuclease activity directed against single-stranded DNA may be present in purified virus.     

The comparative mapping of virion and cloned DNA of the hepatitis B virus

The entire hepatitis B virus (HBV) genome and its fragments have been cloned into the BamHI site of the plasmid pBR322 vector. The identity of physical maps of cloned and authentic virion DNAs was demonstrated by restriction enzyme analysis. The location of restriction sites is suggestive of a certain similarity between the studied HBV DNA and HBV DNA, subtype ayw (Galibert et al., 1979). From the restriction enzyme analysis of virion DNA repaired and 32P-labeled by the endogenous DNA-polymerase reaction, the new information concerning the location and maximal length (approximately 1500 nucleotides) of the single-stranded region of HBV DNA has been established.     

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.     

Low-level secretion of human hepatitis B virus virions caused by two independent, naturally occurring mutations (P5T and L60V) in the capsid protein

The functional significance of naturally occurring variants of human hepatitis B virus (HBV) remains largely unknown. Previously, we reported an immature secretion phenotype caused by a highly frequent mutation at amino acid 97 of the HBV core (capsid) protein (HBcAg). This phenotype is characterized by a nonselective and excessive secretion of virions containing an immature genome of single-stranded viral DNA. To extend our study of virion secretion to other naturally occurring variants, we have characterized mutations at HBcAg codons 5, 38, and 60 via site-directed mutagenesis. Although the phenotype of the mutation at codon 38 is nearly identical to that for the wild-type virus, our study reveals that a single mutation at codon 5 or 60 exhibits a new extracellular phenotype with significantly reduced virion secretion yet maintains normal intracellular viral DNA replication. A complementation study indicates that the mutant core protein alone is sufficient for the "low-secretion" phenotype. Furthermore, the low-secretion phenotype of the codon 5 mutant appears to be induced by the loss of a parental proline residue, rather than by the gain of a new amino acid. Our study underscores the core protein as another crucial determinant in virion secretion, in addition to the known envelope proteins. Our present results suggest that a very precise structure of both alpha-helical and nonhelical loop regions of the entire HBcAg molecule is important for virion secretion. The low-secretion variants may contribute to the phenomenon of gradually decreasing viremia in chronic carriers during the late phase of persistent infection.     

Hepatitis C virus nonstructural protein 5B is involved in virus morphogenesis

The p7 protein of hepatitis C virus (HCV) is a viroporin that is dispensable for viral genome replication but plays a critical role in virus morphogenesis. In this study, we generated a JFH1-based intergenotypic chimeric genome that encoded a heterologous genotype 1b (GT1b) p7. The parental intergenotypic chimeric genome was nonviable in human hepatoma cells, and infectious chimeric virions were produced only when cells transfected with the chimeric genomes were passaged several times. Sequence analysis of the entire polyprotein-coding region of the recovered chimeric virus revealed one predominant amino acid substitution in nonstructural protein 2 (NS2), T23N, and one in NS5B, K151R. Forward genetic analysis demonstrated that each of these mutations per se restored the infectivity of the parental chimeric genome, suggesting that interactions between p7, NS2, and NS5B were required for virion assembly/maturation. p7 and NS5B colocalized in cellular compartments, and the NS5B mutation did not affect the colocalization pattern. The NS5B K151R mutation neither increased viral RNA replication in human hepatoma cells nor altered the polymerase activity of NS5B in an in vitro assay. In conclusion, this study suggests that HCV NS5B is involved in virus morphogenesis.     

The hepatitis B virus and its DNA polymerase: The prototype three-D virus

Summary The hepatitis B virus (HBV), the causal agent of serum hepatitis, has a diameter of 42 nm and is comprised of an outer surface coat and a 27 nm core. A unique DNA-dependent DNA polymerase is associated with the core of the virus. The core also houses a circular DNA that contains both double-stranded and single-stranded regions. In the endogenous reaction, the DNA polymerase repairs the single-stranded gaps of the viral DNA. The surface protein of the virus, called hepatitis B surface antigen, contains both lipid and carbohydrate, and is often present in particulate form in the blood of infected patients. In Asia and Africa HBV infection is associated with subsequent development of primary hepatocellular carcinoma. Although most patients recover completely from acute illness, the hepatitis B virus may cause chronic infection. Recently, a virus similar to human HBV was discovered in woodchucks. HBV has not yet been propagated in a cell culture system and the mode of replication of this unusual virus in hepatocytes is still moot. Although reliable therapy has not yet been provided, the problem of this world-wide infection has led to many interesting approaches to both vaccine production and anti-viral chemotherapy.     

Integration pattern of hepatitis B virus DNA sequences in human hepatoma cell lines.

Four human hepatoma cell lines established from primary hepatocellular carcinomas were examined for the presence of hepatitis B virus DNA sequences. Reassociation kinetic analysis indicated that the cell lines HEp-3B 217, HEp-3B 14, HEp-3B F1, and PLC/PRF/5 contained two, one, one, and four genome equivalents per cell, respectively. Southern blot hybridization analysis demonstrated that hepatitis B virus DNA was integrated into the cellular DNAs of these cell lines. Further liquid hybridization studies with 32P-labeled HincII restriction fragments of hepatitis B virus DNA established that DNA sequences from all regions of the HBV genome were represented in the integrated viral sequences. Although the three HEp-3B cell lines were derived from the same tumor, they differed significantly in their patterns of integration of hepatitis B virus DNA, the number of copies of viral DNA per cell, and their ability to produce the virus-coded surface antigen.     

Molecular cloning of the complete Epstein-Barr virus genome as a set of overlapping restriction endonuclease fragments.

A complete collection of fragments of Epstein-Barr virus DNA, obtained by cleavage with restriction endonuclease Eco RI, has been cloned. Fourteen different internal fragments of the virus genome, derived from linear virion DNA of the B95-8 strain, and sequences corresponding to the terminal regions of virion DNA, derived from intracellular circular EBV DNA isolated from 895-8 cells, were cloned. Sizes of fragments were determined by agarose gel electrophoresis and their sum leads to an estimated molecular weight of 110 x 10(6) for virion DNA. Large Eco RI DNA fragments of special interest were also cloned in cosmids using another source of EBV DNA, that is, to circular viral DNA derived from Raji cells. In order to provide a set of overlapping sequences, all the 29 internal Bam HI fragments of B95-8 virion DNA were cloned in pBR322. The map location within the viral genome of each cloned DNA fragment was identified by hybridizing to blots of virion DNA cleaved with several different restriction endonucleases.     

Restriction endonuclease mapping of the hepatitis B viral genome isolated from Taiwan

The Eco RI fragment of hepatitis B virus (HBV) DNA isolated from human blood plasma Dane particles were inserted into plasmid pUC8 Eco RI site and transformed into E. coli JM103 host. Two recombinants pTWL1 and pTWL2 were found to carry 3.2 kbp fragment and proved to have HBV genome by Southern hybridization method. The 1.4 kbp Bam HI fragment which carried the hepatitis B viral surface antigen (HBsAg) gene, obtained via Bam HI digestion of Dane particles DNA which was made fully double stranded by endogenous DNA polymerase reaction, was also inserted into plasmid pUC8 Bam HI site. Four recombinant clones, pTWS1, pTWS2, pTWS3, and pTWS4 were found. Only one of the clones pTWS1 carried the HBsAg gene in a correct orientation with respect to the lac promoter sequence. The physical mapping of HBV DNA was performed with several restriction endonucleases. Our results indicated that the HBV DNA insert contains unique XbaI and HpaI cleavage sites and lacks the cleavage sites for the HindIII, SmaI, KpnI, SalI, and SstI endonucleases. The locations of Bam HI, BglII, and HincII endonucleases cleavage sites within the cloned HBV DNA of the pTWL1 plasmid were similar to that HBV DNA of adw and adw2 subtypes.     

Relationship between viral DNA synthesis and virion envelopment in hepatitis B viruses.

While the intracellular pool of encapsidated hepatitis B viral DNA contains genomes in all stages of DNA replication, serum-derived virions contain predominantly mature, partially duplex, circular DNA genomes. To account for this finding, Summers and Mason proposed in 1982 that virion envelopment is somehow linked to the state of genomic maturation (J. Summers and W.S. Mason, Cell 29:403-415, 1982). Core gene mutations with phenotypes consistent with this concept have previously been identified in the duck hepatitis B virus (DHBV). Here we show that DHBV polymerase mutants with altered DNA synthesis also display defects in envelopment, and we provide quantitative estimates of the magnitude of the preference for the envelopment of mature DNA. In cells transfected with wild-type DHBV DNA, immature minus-strand DNA represents 18% of the intracellular pool but only 4% of extracellular virion DNA. A point mutation in the C-terminal domain of the polymerase strongly and selectively impairs plus-strand synthesis; in this mutant, the ratio of immature to mature DNA in the intracellular pool rises to 6:1 but is reduced to 1.5:1 in released virions. A missense mutation in the polymerase active site inactivates all viral DNA synthesis but still allows efficient RNA encapsidation; in this mutant, no detectable viral nucleic acid is enveloped and released. Thus, viral DNA synthesis is absolutely required for envelopment and export, and a strong further bias exists in favor of the export of genomes that have completed minus-strand synthesis and at least initiated plus-strand synthesis. These results imply that events within the interior of the nucleocapsid can powerfully influence its interactions with external viral envelope glycoproteins.     

Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication.

A hepatitis B virus (HBV) genome was cloned from human liver. Numerous mutations in all viral genes define this HBV DNA as a mutant, divergent from all known HBV DNA sequences. Functional analyses of this mutant demonstrated a defect blocking viral DNA synthesis. The genetic basis of this defect was identified as a single missense mutation in the 5' region of the viral polymerase gene, resulting in the inability to package pregenomic RNA into core particles. The replication defect could be trans-complemented by a full-length wild-type, but not by a full-length mutant or 3'-truncated wild-type, polymerase gene construct. Our findings indicate a critical role of the 5' polymerase gene region in the life cycle of the virus and suggest that introducing missense mutations in this region can be a strategy to terminate viral replication in vivo.     

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.