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A recombinant plasmid (pSVS dhfr) encoding the pre-S region and the S gene of human hepatitis B virus (HBV) and murine dihydrofolate reductase (DHFR) cDNA has been used for the transfection of Chinese hamster ovary (CHO) DHFR- cells. Selection of clones resistant to methotrexate has permitted amplification of HBV sequences and an increase in production of hepatitis B surface antigen (HBsAg). HBV-specific transcripts have been characterized. The HBsAg 22-nm particles contain a receptor for polymerized human serum albumin (pHSA) and elicit in animals the synthesis of antireceptor antibodies. This property is ascribed to a 34,000-dalton polypeptide in the particles, which is most likely encoded by the S gene and part of the pre-S region. Especially because the pHSA receptor is most abundantly present on the virion and because, in hepatitis B infection, the appearance of anti-pHSA receptor antibodies seems to be a highly reliable criterion for viral clearance, the HBsAg particles obtained may constitute a particularly efficient vaccine.

The ETn (for "early transposon") family of long repeated sequences in abundantly transcribed in early mouse embryos from retroviral-like long terminal repeats. Nucleotide sequencing of two elements does not reveal any long open reading frame nor significant homology to retroviral proteins. The genetic polymorphism, monitored by Southern blotting within and across mouse species, reflects a concerted mode of evolution for the ETn sequences.

Integrated hepatitis B virus (HBV) DNA sequences have been cloned from cellular DNA of two human liver tumors. The structure of the clones was determined by restriction mapping, and the host-viral DNA junctions were sequenced. In each clone one junction mapped to within an 11-base-pair sequence, 5' T-T-C-A-C-C-T-C-T-G-C, which is directly repeated near the extremities of the cohesive-end region of the free viral genome. The two copies of this sequence are termed DR1 and DR2. While one clone carried a host-viral junction within DR1, the second one carried a host-viral junction within DR2. The first 1 or 2 base pairs of the repeat were deleted upon recombination with the host genome, leaving at the junctions a common 9-base-pair segment of HBV DNA, 5' C-A-C-C-T-C-T-G-C. The other two host-viral junctions mapped to the pre-S region and to the core region of the viral genome, showing no peculiar feature. These results show that HBV DNA can integrate via a specific viral DNA sequence.

Lymphadenopathy-associated virus (LAV) is a human retrovirus first isolated1from a homosexual patient with lymphadenopathy syndrome, frequently a prodrome or a benign form of acquired immune deficiency syndrome (AIDS)2. Other LAV isolates have subsequently been recovered from patients with AIDS or pre-AIDS3–5and all available data are consistent with the virus being the causative agent of AIDS. The virus is propagated on activated T lymphocytes and has a tropism for the T-cell subset OKT4 (ref. 6), in which it induces a cytopathic effect. The major core protein of LAV is antigenically unrelated to other known retroviral antigens1,2,7. LAV-like viruses have more recently been independently isolated from patients with AIDS and pre-AIDS. These viruses, called human T-cell leukaemia/lymphoma virus type III (HTLV-III)8–11and AIDS-associated retrovirus (ARV)12, seem to have many characteristics in common with LAV and probably represent independent isolates of the LAV prototype. We have sought to characterize LAV by the molecular cloning of its genome. A cloned LAV complementary DNA was used to screen a library of recombinant phages constructed from the genomic DNA of LAV-infected T lymphocytes. Two families of clones were characterized which differ in a restriction site. The viral genome is longer than any other human retroviral genome (9.1–9.2 kilobases).

A restriction map of hepatitis B virion DNA was established after cloning of the whole viral genome in Escherichia coli. By use of EcoRI, Xho I, Bgl II, Xba I, BamHI, HincII, and Hae III endonucleases, a total of 28 restriction sites were mapped. The single-stranded region was localized on the restriction map and 5' end of the short strand was mapped at a fixed position.

Mouse L cells transformed with recombinant plasmids carrying hepatitis B virus (HBV) DNA fragments were used to study the transcription of the viral surface antigen gene (gene S). An HBV-specific, polyadenylated, 2.3-kilobase RNA was mapped on the HBV genome. This RNA hybridized with approximately 75% of the genome and excluded the region of the HBV core antigen gene (gene C). The 2.3-kilobase RNA species was present only in cell lines that produced hepatitis B surface antigen. An HBV-specific 2.3-kilobase RNA was also detected in human hepatoma cell line PLC/PRF/5 which produced hepatitis B surface antigen. A study of gene S expression in the transformed mouse L cells allowed us to localize the regions of initiation and termination of gene S transcription. Our results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.

Hepatocellularcarcinoma (HCC) that occur in woodchucks chronically infected with woodchuck hepatitis virus (WHV) were screened for activation of cellular oncogenes. Enhanced expression and allelic alterations of the c-myc oncogene were found in three HCC out of nine. Variations in the size of the c-myc transcripts, ranging from 2.0 kilobases (kb) to 5.6 kb, as well as in the level of c-myc gene expression, 5–50-fold higher than in adjacent liver tissues, were observed among the three HCC. Rearrangements of the c-myc locus were either upstream of the gene or within the first intron. Cloning and sequencing of the break-point region from one of the three tumours showed that the c-myc gene was truncated and joined to a unique cellular sequence of unknown function. WHV DNA was not integrated near the c-myc coding exons, excluding a direct role of the virus in c-myc activation. The novel type of rearrangement and activation of the c-myc gene, reported here in liver tumours of hepatitis virus infected animals, appears strikingly similar to those resulting from chromosomal translocations in human Burkitt's lymphomas, acute B- and T-cell leukaemias and mouse plasmacytomas.

The complete nucleotide sequence of hepatitis B virus genome (subtype ayw) cloned in Escherichia coli has been determined using the Maxam and Gilbert method and the dideoxynucleotide method. This sequence is 3,182 nucleotides long. Location of the nonsense codons shows that the coding capacity of the L chain is larger than the coding capacity of the Schain. Eight open regions, able to code for polypeptide chains larger than 100 amino acids, have been located. Region 6, which is the largest, covers more than 80% of the genome. The gene S which codes for polypeptide I of the Hbs Ag and was previously located between coordinates 95.1 and 73.6 is contained in region 7.

The domain of the hepatitis B virus (HBV) S gene specifying the HBV surface antigen (HBsAg) and comprising 25 base pairs of the 5'-transcribed noncoding region, the structural gene sequences, and the 3'-noncoding gene sequences including the polyadenylylation site was fused to the promoter-regulatory regions of the beta-thymidine kinase and of the alpha 4 gene of herpes simplex virus type 1 (HSV-1). The chimeric constructs were then inserted into the HSV-1 genome and specifically into the thymidine kinase gene by homologous recombination through flanking sequences. Cells infected with recombinants carrying the chimeric genes produced and excreted the HBsAg into the extracellular medium for at least 12 hr concurrently with the multiplication of the HSV-1 vector. The temporal patterns of expression and the observation that HBV S gene linked to the HSV-1 alpha promoter-regulatory region was regulated as an HSV-1 alpha gene indicate that the HBsAg gene chimeras inserted into the virus were regulated as viral genes. The HBsAg banded in isopycnic CsCl density gradients at a density of 1.17 g/cm3. Electron microscopic studies revealed that HBsAg harvested from the extracellular medium and banded in CsCl density gradients contained spherical particles 15-22 nm in diameter, characteristic of empty HBV envelopes. The results indicate that HSV-1 is a suitable vector for the expression of foreign genes placed under the control of HSV promoter-regulatory regions.

We have identified long-incubation viral agents that share epitopes with hepatitis B virus (HBV). During chimpanzee infectivity studies, these agents may be recognized in the liver since they possess complementary nucleic acid sequences with HBV DNA; the genomic size was found to be 3.2 kilobases, identical to that of HBV. Liver injury was produced and there was antigen expression in hepatocytes. Chimpanzees were not protected by prior immunization with hepatitis B surface antigen; conversely, they were still susceptible to HBV after recovery from infection with such agents. These findings suggest that these hepatitis B virus-related variants appear to be immunologically distinct from HBV.

ABSTRACTDirect activation of the N-myc2oncogene 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-myc2promoter in hepatoma cell lines. To better define the integrated WHV regulatory sequences responsible for N-myc2promoter activation in woodchuck liver tumors, we analyzed the structure and enhancer activity of a single viral integrant found at the winlocus 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-myc2promoter 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-myc2activation.

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. We have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possibly related to HBV integration.

Recombinant phage clones carrying integrated hepatitis B virus (HBV) DNA sequences have been isolated from two phage libraries made from human DNA of a hepatoma and a hepatoma-derived cell line. One clone from each library has been characterized both by restriction mapping and by electron microscopy. In one clone there is at least one complete and uninterrupted HBV genome, and in the other the HBV sequences are composed of two major subgenomic fragments inverted with respect to each other. The host-virus junctions are localized within the positions 1,700-2,600 base pairs on the physical map of the free viral genome. The pre-S/S (surface antigen gene) region is conserved between the two clones. The two clones do not have common cellular sequences nor do they contain cellular homologues to six retroviral oncogenes. For one clone, the hepatitis B surface antigen gene was found to be functional when introduced into mouse thymidine kinase-negative cells by transfection.

Fragments of the human immunodeficiency virus (HIV) envelope coding region have been fused with the hepatitis B virus envelope middle protein. In this system, HIV antigenic determinants are exposed at the surface of a highly antigenic structure, the hepatitis B surface antigen particle. Immunization of rabbits with these particles elicited antibodies directed against both parts of the hybrid protein. One of the rabbit antisera not only exhibited a neutralizing effect on the original HIV1 isolate but also on a divergent Zairian isolate. The HIV sequence in this recombinant is 84 amino acids long and contains conserved and variable domains and a region critical for interaction with the CD4 receptor. Such recombinant antigens could be primary elements in the design of a polyvalent vaccine.

Using the Southern blot technique and cloned hepatitis B virus (HBV) DNA as a probe, we studied the state of HBV DNA in the liver of 13 patients with hepatocellular carcinoma, 17 patients with chronic hepatitis, and 2 patients with acute hepatitis. The hybridization results were compared with the serological and immunohistological data. Integration of HBV DNA in cellular DNA of the liver from patients with hepatocellular carcinoma was demonstrated. In two patients from which tumorous and nontumorous liver tissue samples were available the integration patterns were different. In one patient with hepatitis B e antigen (HBeAg)-positive early hepatocellular carcinoma, free viral DNA was present in the liver. In some patients with HBeAg-negative chronic hepatitis, without tumor, integration of HBV DNA in cellular DNA was also demonstrated. This suggests that HBV is not the only factor involved in the development of a tumor. In patients with HBeAg-positive chronic hepatitis, free viral DNA was detected in the liver. In the two acute hepatitis patients analyzed, the restriction endonuclease patterns strongly suggested HBV DNA integration. Therefore, viral DNA integration seems to occur early in infection. Whatever the form of the disease, discrete bands were observed, suggesting the existence of limited and specific integration sites in host cellular DNA. The presence of integrated or free DNA sequences has implications for antiviral therapy. In addition, detection of HBV DNA in the liver is another sensitive viral marker that could be useful for diagnostic purposes.

A striking characteristic of the simian immunodeficiency virus (SIV) and of the human immunodeficiency virus type 2 (HIV-2) is the presence of a nonsense mutation in the env gene resulting in the synthesis of a truncated transmembrane protein lacking the cytoplasmic domain. By mutagenesis of an infectious molecular clone of SIVmac142, we investigated the function of the cytoplasmic domain and the significance of the env nonsense mutation. When the nonsense codon (TAG) was replaced by a glutamine codon (CAG), the virus infected HUT78 cells with markedly delayed kinetics. This negative effect was counterselected in vitro as reversion of the slow phenotype frequently occurred. The sequencing of one revertant revealed the presence of a new stop codon three nucleotides 5' to the original mutation. Deletions or an additional nonsense mutation introduced 3' to the original stop codon did not modify SIV infectivity. In contrast, the same deletions or nonsense mutation introduced in the clone in which the stop codon was replaced by CAG abolished infectivity. These results indicated that the envelope domain located 3' to the stop codon is not necessary for in vitro replication. However, the presence of this domain in SIV transmembrane protein leads to a reduced infectivity. This negative effect might correspond to a function controlling the rate of spread of the virus during in vivo infection.

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication.

We have previously isolated from a human hepatocellular carcinoma a hepatitis B virus integration in a 147-base-pair cellular DNA fragment, similar to steroid- and c-erb- A/thyroid-hormone receptor genes1. We have now cloned the corresponding complementary DNA from a human-liver cDNA library. Nucleotide sequence analysis revealed that the overall structure of the cellular gene, which we have named hap, is similar to that of the DNA-binding hormone receptors. That is, it displays two highly conserved regions identified as the putative DNA-binding and hormone-binding domains of the c-erb A/steroid receptors2–9. Six out of seven hepatoma and hepatoma-derived cell-lines express a 2.5-kilobase (kb) hap messenger RNA species which is undetectable in normal adult and fetal livers but present in all non-hepatic tissues analysed. The data suggest that the hap gene product may be a novel ligand-responsive regulatory protein whose inappropriate expression in liver may relate to the hepatocellular carcinogenesis.

Because of the growing incidence of AIDS (acquired immune deficiency syndrome), the need for studies on animal models is urgent. Infection of chimpanzees with the retroviral agent of human AIDS, the human immunodeficiency virus (HIV), will have only limited usefulness because chimpanzees are in short supply and do not develop the disease. Among non-human primates, both type D retroviruses and lentiviruses can be responsible for immune deficiencies. The D-type retroviruses1–3, although important pathogens in macaque monkey colonies, are not satisfactory as a model because they differ in genetic structure and pathophysiologi-cal properties from the human AIDS viruses4,5. The simian len-tivirus, previously referred to as simian T-cell lymphotropic virus type III (STLV-III), now termed simian immunodeficiency virus (SIV) is related to HIV by the antigenicity of its proteins and in its main biological properties, such as cytopathic effect and tropism for CD4-bearing cells6–9. Most importantly, SIV induces a disease with remarkable similarity to human AIDS in the common rhesus macaques, which therefore constitute the best animal model currently available10. Natural or experimental infection of other monkeys such as African green monkeys or sooty mangabeys has not yet been associated with disease8,9,11. Molecular approaches of the SIV system will be needed for biological studies and development of vaccines that could be tested in animals. We have cloned and sequenced the complete genome of SIV isolated from a naturally infected macaque that died of AIDS. This SIVMACappears genetically close to the agent of AIDS in West Africa, HIV-2 (ref. 12), but the divergence of the sequences of SIV and HIV-2 is greater than that previously observed between HIV-1 isolates13.

DNA recombinant technology has radically changed hepatitis B virus (HBV) virology. The genetic organization, transcription and replication of the virus are basically understood, structures of integrated HBV sequences in hepatocellular carcinoma have been characterized, and new vaccines produced by recombinant DNA technique are being developed.

We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), which shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison génétique in vitro" committee (D.G.R.S.T.).

We have investigated the basis for liver-specific and sex-linked expression of hepatitis B surface antigen (HBsAg) gene in transgenic mice by monitoring the level of liver HBsAg mRNA and serum HBsAg at different stages of development and in response to sex-hormone regulation. Transcription of the HBsAg gene starts at day 15 of development, together with that of the albumin gene, and reaches a comparable level at birth. HBsAg mRNA level and HBsAg production are parallel in males and females during prenatal development and until the first month of life, but HBsAg gene expression increases 5-10 times in males at puberty. After castration, the level of expression decreases dramatically in both males and females and is subsequently increased by injection of testosterone or estradiol. Glucocorticoids also regulated positively expression of the HBsAg gene. Our results suggest that sex hormones play a role in hepatitis B virus gene expression during natural infection and could explain the difference in incidence of chronic carriers between men and women.

Analysis of RNA (after 5 or 60 min of labeling with [3H]uridine) from L5178Y cells by electrophoresis in 1.7% polyacrylamide gels demonstrates that the ribosomal RNA “45S fraction“ is not homogeneous but consists of at least three molecular species, conventionally designated 47S RNA, 46S RNA, and 45S RNA. Alternatives to the classical scheme for the biosynthesis of ribosomal RNA in mammalian cells are discussed.