Endogenous Genome Research Articles

Insulin Dominantly Suppresses Hepatitis B Virus Gene Expression in Cultured Human Hepatoma Cells.

We have shown previously that insulin suppresses the expression of hepatitis B surface antigen (HBsAg) gene from an endogenous integrated viral genome in cultured human hepatoma Hep3B cells. In this study, we demonstrated that insulin suppresses the viral mRNA transcribed from transiently transfected tandem repeat hepatitis B virus (HBV) dimer DNA or DNA fragment that contains only the major HBsAg gene. Insulin treatment also resulted in a decrease in HBV viral particles produced by the HBV-DNA-transfected cells in a dose-dependent manner. Furthermore, when insulin was simultaneously added with glucocorticoid, which stimulates HBV gene expression, the stimulatory effect of glucocorticoid was completely abolished. Our results suggest that insulin has a dominant negative effect on the HBV gene expression in cultured human liver cells.

Transformation of human breast epithelial cells by 7, 12, dimethylbenz(a)anthracene, but not by N-methyl-N-nitrosourea, is accompanied by up-regulation of basic fibroblast growth factor

Normal human breast epithelial cells (HBL100) are immortalized by endogenous SV40 genome and are not tumorigenic in nude mice if injected in the first 50 passages in culture. This cell line also depends on the expression of basic fibroblast growth factor (bFGF) for its viability in culture. The present study was designed to transform these cells with chemical carcinogens to establish malignant HBL100 cell lines in order to evaluate the eventual modulation in the expression of bFGF. In the first experiment, HBL100 cells were treated with 2 mu g/ml of 7, 12, dimethylbenz(a)anthracene (DMBA) for two 24 h periods. In a second experiment, HBL100 cells were transformed by exposing them to 20 mu g/ml of N-methyl-N-nitrosourea (MNU) twice a day for two days. Unlike the HBL100 parental cell line, the two newly derived cell lines (named respectively HBLTDMBA and HBLTMNU) were tumorigenic when injected in nude mice. The expression of bFGF, as measured by immunocytochemistry and Western blot analysis, was higher in the DMBA-transformed cell line than in the parental HBL100 and MNU-transformed lines. These results differentiate between transformation selectivity by two different carcinogens. The direct-acting carcinogen MNU, which induces point mutation, does not require enhanced expression of bFGF, whereas bFGF may be necessary for early events leading towards transformation by carcinogens requiring metabolism for their action, such as DMBA.

Suppressive effects of destruxin B on hepatitis B virus surface antigen gene expression in human hepatoma cells

Destruxin B, a cyclodepsipeptide was originally identified as a plant pathogen from the fungus, Alternaria brassicae. We examined the antiviral activity of destruxin B and found that it suppresses the expression of the hepatitis B viral surface antigen (HBsAg) gene in human hepatoma Hep3B cells which carry an integrated viral gene in its chromosome. In contrast, destruxin B shows no cytotoxic effect on the viability of the cells. Furthermore, it can be shown that destruxin B can reversibly suppress HBsAg production by Hep3B cells in a concentration-dependent manner with EC 50 of 0.5 μM. Northern blot analysis indicates that the suppression of HBsAg gene expression by destruxin B is mainly at the mRNA level. Destruxin B not only suppresses the endogenously expressed HBsAg in the Hep3B cells but also suppresses the HBsAg produced either from the stable transfected HBV DNA in another human hepatoma HuH-7 cell line which carry no endogenous HBV genome. These results suggest that destruxin B may have future potential for development as a specific anti-HBV drug.

A replication function associated with the activation domain of the Epstein-Barr virus Zta transactivator.

The Zta transactivator is crucial for both Epstein-Barr virus (EBV) lytic gene expression and lytic DNA replication. We have used a cotransfection-replication assay to examine the effect of mutations in the Zta activation domain (amino acids [aa] 1 to 167) on Zta replication activity. Deletion of Zta aa 25 to 86, which are critical for transcriptional activation of ori-Lyt, or aa 93 to 141 did not adversely affect replication of an ori-Lyt-containing target plasmid. However, removal of aa 2 to 25 (delta2-25) abolished replication activity. Within this subdomain, deletion of aa 2 to 10 (delta2-10) or mutation of codons 18 and 19 (m18/19) or 22 and 26 (m22/26) did not affect replication competency, while deletion of codons 13 to 19 (delta13-19) or mutation at codons 12 and 13 (m12/13) impaired Zta replication function. Each of the replication-negative Zta variants was capable of transactivating expression from both BHLF1 promoter-chloramphenicol acetyltransferase constructions and the BMRF1 promoter on endogenous EBV genomes in Raji cells with efficiency comparable to that of the wild-type polypeptide. Thus, a replication contribution of Zta was functionally separable from its transactivation activity and was supplied by the N-terminal region encompassing aa 11 to 25. Replication by a subset of the impaired Zta mutants was partially rescued upon the addition of Rta to the replication assay. The contribution of Rta mapped to domain II of the Rta activation domain and was specific for this region. A chimeric Rta-EBNA-2 transactivation domain fusion, which retains the DNA-binding and transactivation properties associated with wild-type Rta, failed to rescue replication-deficient Zta. Our data suggest that Rta may act as an ancillary replication factor in EBV ori-Lyt DNA synthesis by stabilizing Zta-replisome interactions.

The latent membrane protein 2 gene of Epstein-Barr virus is important for efficient B cell immortalization.

The viral latent membrane proteins 2 (LMP2) of Epstein-Barr virus (EBV) were analysed genetically to evaluate their role in B cell immortalization. LMP2 is transcribed as two differently spliced mRNAs which code for the LMP2A and -B proteins, also called terminal protein-1 and -2. LMP2A and -B are found in latently infected, growth-transformed B lymphocytes in vitro, in different human tumours, and in latently infected B cells in vivo. Two different approaches were used to generate EBV mutants in which the second, third and part of the fourth exon of the LMP2 gene were deleted by insertion of a marker gene. Initially, conventional homologous recombination in a Burkitt's lymphoma cell line (P3HR1) between the endogenous EBV genome and an introduced plasmid was used to generate EBV mutants. This experiment identified LMP2 as dispensable for B cell immortalization as has been reported. In a second approach, the same LMP2 mutant gene was analysed in the context of a mini-EBV plasmid. These are E. coli constructs that are sufficient when packaged into an EBV coat both to initiate and to maintain proliferation of infected B cells. In comparison with a fully competent mini-EBV, LMP2- mini-EBVs were found to be greatly reduced in their capacity to yield immortalized B cell clones. This finding confirmed the initially observed bias against LMP2- B cell clones, most of which were found to be coinfected with complementing P3HR1 virus. These results indicate that LMP2 contributes to the efficiency of B cell immortalization and that the LMP2s phenotype is auxiliary in nature.

Genetic predisposition to lymphomas in mice.

The spontaneous mouse lymphoma is a model of multifactorial genetic disease. It is induced by the endogenous murine leukemia virus (MuLV), whose genome is inherited as a Mendelian dominant trait. Lymphoma development takes place in multiple stages affected by many host genetic and epigenetic factors. An inbred strain SL/Kh with a high incidence of pre-B lymphomas has been established and the genetic predisposition of SL/Kh mice to lymphomas is being studied in the crosses with other inbred strains of mice. In the cross to the NFS/N lacking endogenous MuLV genome, it has been shown that lymphomas are induced by the expression of Emv-11 provirus (Chr. 7), and the types of B-lineage lymphomas are determined by combinations of the host genes, Esl-1 (Chr. 17) and Foc-1 (Chr. 4). Another gene, Tlsm-1 (Chr. 7) that determines the type of lymphomas to be T-lineage, is identified in the cross with AKR/Ms, with a high incidence of T-lymphomas. The role of the thymus in the development of T-lymphomas in the mouse, and the possible relevance of Tlsm-1 in this step, is discussed. The length of the latent period is determined by a gene Lia-1 (Chr. 17). A maternal resistance factor that is a maternal antibody to MuLV transmitted via milk and that epigenetically inhibits MuLV expression in SL/Ni-Eco-, one of subline of SL/NI mice, has been shown. Weak but definitive maternal resistance also operates in SL/Ni-Eco+, a subline lacking the maternal antibody to MuLV. In the latter, there is a recessive resistance gene Nir-1 (Chr. 4). In the cross with MSM/Ms, a wild mice-derived inbred strain, two resistance genes, Msmr-1 (Chr. 17) and Msmr-2 (Chr. 18), have been identified. In SL/Kh, all of these host genetic and epigenetic factors are favorable for lymphoma development. This model offers not only an understanding of the pathogenesis of virus-induced lymphomas but also may provide starting material for the comparative approach to homologous human diseases.

Functional and physical interactions between the Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1: Effects on EBV transcription and lytic replication.

The Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1 are both essential for lytic EBV replication. BZLF1 is a transcriptional activator which binds directly to the lytic origin of replication (oriLyt) and plays a critical role in the disruption of viral latency. The BMRF1 protein is required for viral polymerase processivity. Here we demonstrate that the BMRF1 gene product functions as a transcriptional activator and has direct (as well as indirect) interactions with the BZLF1 gene product. The BMRF1 gene product activates an essential oriLyt promoter, BHLF1, but does not activate two other early EBV promoters (BMRF1 and BHRF1). Direct interaction between the BMRF1 and BZLF1 gene products requires the first 45 amino acids of BMRF1 and the bZip domain of BZLF1. The effect of the BZLF1-BMRF1 interaction on early EBV transcription is complex and is promoter specific. The oriLyt BHLF1 promoter is activated by either the BZLF1 or BMRF1 gene product alone and is further activated by the combination of the BZLF1 and BMRF1 gene products. Enhanced activation of BHLF1 transcription by the BMRF1-BZLF1 combination does not require direct interaction between these proteins. In contrast, BZLF1-induced activation of the BMRF1 promoter is inhibited in the presence of the BMRF1 gene product. A point mutation in the BZLF1 protein (amino acid 200), which prevents in vitro interaction with the BMRF1 protein but which does not reduce BZLF1 transactivator function, allows the BZLF1 protein to activate the BMRF1 promoter equally well in the presence or absence of the BMRF1 gene product. Therefore, direct interaction between the BZLF1 and BMRF1 proteins may inhibit BZLF1-induced transcription of the BMRF1 promoter. BZLF1 mutated at amino acid 200 is as efficient as wild-type BZLF1 in promoting replication of an oriLyt plasmid. However, this mutation reduces the ability of BZLF1 to induce lytic replication of the endogenous viral genome in D98/HE-R-1 cells. Our results indicate that functional and physical interactions between the BMRF1 and BZLF1 proteins may modulate the efficiency of lytic EBV infection. The BMRF1 gene product clearly has a transcriptional, as well as replicative, role during lytic EBV infection.

Distribution of human endogenous retrovirus HERV-K genomes in humans and different primates

The distribution of the human endogenous retrovirus (HERV)-K genome was investigated by Southern-blot analyses using a HERV-K-env DNA probe. With the exception of one DNA-sample, obtained from a Chinese individual in whom an amplification of HERV-K was detected, Southern-blot analyses yielded identical hybridization patterns with DNA from peripheral blood lymphocytes of 37 normal healthy blood donors, with DNA from six tumor cell lines, or with 23 DNA samples prepared from various carcinoma tissues. To elucidate whether the integration of HERV-K genomes into the primate lineage occurred as a single event or as an integration with later expansion, we further examined the evolutionary history of HERV-K by Southern blot analyses with DNA samples from different primate species. We detected HERV-K genomes in Macaca mulatta and Macaca silenus, which represent Old World monkeys, but not in prosimians (Galago demidovii) and New World monkeys, represented by Saguinus fuscicollis, Saguinus oedipus, and Callithrix iacchus. Thus, we assume that the infection of the primate lineage with HERV-K had occurred after the divergence of New World and Old World monkeys, but before the evolutionary expansion of large hominoids. In contrast to the apparent lack of HERV-K-env sequences in DNA from tissue of the New World monkey Saguinus oedipus (cotton-top marmoset), we found HERV-K-DNA in the B95-8 cell-line, which is a Saguinus oedipus leukocyte cell-line, immortalized in vitro by Epstein-Barr virus (EBV) and cultivated in human cells. It may be speculated that HERV-K-DNA or HERV-K-particles were introduced into these cells during in vitro transformation with EBV.

Efficient EBV Superinfection of Group I Burkitt's Lymphoma Cells Distinguishes Requirements for Expression of the Cp Viral Promoter and Can Activate the EBV Productive Cycle

Group I Burkitt's lymphoma cell lines conditionally expressing the CD21 receptor for EBV infection were superinfected with EBV. The incoming EBV entered its normal program of gene expression, producing EBNA-2 and LMP-1 and activating the Cp EBNA promoter, but the endogenous virus in the BL lines was not induced to express EBNA-2 or transcribe RNA from Cp. LMP-1 was, however, expressed from the endogenous genome in response to superinfection. In a proportion of the superinfected Akata cells, the productive cycle antigan BZLF1 was induced and the ability of infecting virus to cause this was sensitive to inactivation by uv light. The results show that the restricted latent pattern of EBV gene expression observed in Group I BL cells is not a consequence of lack of appropriate transcription factor activity but results from inactivation of part of the viral genome, probably by methylation. Induction of BZLF1 in some of the cells also indicates a novel pathway for activation of the virus productive cycle.

Characterization of human endogenous retroviral genomes isolated from T47-D particles

Characterization of human endogenous retroviral genomes isolated from T47-D particles

Distribution of human endogenous retrovirus HERV-K genomes in normal and malignant human tissues and in different primates

Distribution of human endogenous retrovirus HERV-K genomes in normal and malignant human tissues and in different primates

Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution.

Human oocytes express murine retroviral equivalents.

Unfertilized human oocytes expressed a gp70-related epitope as observed when staining section immunocytochemically with a panel of monoclonal antibodies against gp70 of murine leukemia virus. Some oocytes also expressed virus-like particles at the cell membrane. Follicular fluids, corresponding to these oocytes, contained p30- and gp70-related antigens, reverse transcriptase, and an increased titer of interferon. The three- to four-cell human cleavage stages did not contain the gp70-related epitope. It is concluded that human oocytes, but not early cleavage stages, express products that suggest the presence of an active endogenous retrovirus genome.

Banding profiles of LTR of human endogenous retrovirus HERV-A in 24 chromosomes in somatic cell hybrids

The human genome carries multiple copies of sequences related to endogenous retroviral genomes. We investigated the distribution of one of these sequences, HERV-A, in 24 human chromosomes by Southern analyses using DNAs from flowsorted chromosomes or rodent cells carrying a single human chromosome. The results showed that HERV-A is distributed among all human chromosomes and that each chromosome has a specific Southern blot profile. The chromosome-specific pattern did not show significant polymorphism, except in a few cases, when the same chromosome obtained from different individuals was compared. These chromosome-specific Southern hybridization profiles may be useful for chromosome karyotyping. This would allow the integrity of human chromosomes in human-rodent somatic cell hybrids to be monitored without using conventional cytogenetic methods.

Endogenous retroviral LTR DNA sequences as markers for individual human chromosomes

The human genome carries multiple copies of sequences related to endogenous retroviral genomes that include long terminal repeat (LTR) sequences. We used the LTR of one such viral genome, called HERV-A, as a probe in Southern analysis to examine the distribution profiles of the hybridizing DNA in the genomes of twelve human x rodent hybrid cell lines carrying one or a few human chromosomes, and in the DNA samples prepared from six sorted, individual chromosomes. The HERV-A sequence was found to be widely distributed among different chromosomes and the Southern patterns for chromosomes 5, the X, and the Y, each obtained in duplicate from independently prepared cell lines or sorted chromosomes, were matched. Chromosome-specific Southern profiles can be used to monitor chromosomes in hybrid cells or to characterize chromosome aberrations, such as deletions.

Promoter switching in Epstein-Barr virus during the initial stages of infection of B lymphocytes.

Transcription of the genes encoding the six viral nuclear antigens present in Epstein-Barr virus latently infected lymphocytes can be initiated from one of two promoters (Cp and Wp) mapping near the left end of the viral genome. These promoters are used in a mutually exclusive manner in clonal cell lines established from either Burkitt lymphoma tumors or in vitro infection of peripheral B lymphocytes. In this paper the role of Cp and Wp during viral latency is investigated. Cp appears to be the promoter normally employed during established latent infection. Analysis of two cell lines that use Wp revealed a deletion spanning Cp in the endogenous viral genomes, suggesting that cell lines exhibiting Wp activity harbor mutated viral genomes with a nonfunctional Cp. However, in contrast to the preferred usage of Cp exhibited by established Epstein-Barr virus-infected cell lines, Wp was shown to be exclusively utilized during the initial stages of viral infection. In addition to Wp activity, Cp usage was apparent by 6 days post-infection. A model is proposed involving B-lymphocyte differentiation-driven promoter switching during the establishment of viral latency.

Endogenous virus genomes become hypomethylated tissue—Specifically during aging process of C57BL mice

In an attempt to find out a cause for age-dependent derepression of endogenous viruses, extents of DNA methylation at the endogenous B- and C-type ecotropic viruses in brain, liver and spleen of C57BL/6NJcl were examined at three ages, newborn, young adult and old. Both endogenous viruses showed a slight but significant tissue-specific either hypo- or hypermethylation during post-natal developmental phase in three tissues. After maturation, however, no such change was detectable at most of the sites examined. The exceptions were C-type ecotropic virus in brain and B-type virus in spleen, where the age-dependent decreases of methylation were observed. The changes seemed to be continuations of preceding developmental hypomethylation. They indicated that the hypomethylation could be one of the causes for the age-dependent derepression of endogenous virus. It was further suggested that a mechanism to stop the developmental changes of DNA methylation at the maturation of individuals would be important in considering the reasons for the changes in senescent phase.

A mouse linkage testing stock possessing multiple copies of the endogenous ecotropic murine leukemia virus genome

A new linkage testing stock of the laboratory mouse has been constructed. The stock, designated MEV ( multiple ecotropic pro vvirus), was developed by inbreeding and selection beginning with the cross of strains C58 J and AKXD-14. Eleven different murine leukemia virus (MuLV) proviruses have been fixed in the MEV 1Ty strain. Nine of these can be uniquely identified by Southern blotting of PvuII-digested DNA and probing with a cloned fragment of the ecotropic viral genome. Two proviruses had been mapped previously to chromosome 7, while single proviruses had been mapped to chromosomes 2, 9, and 11. The mapping of six additional proviruses, derived from C58 J , to chromosomes 1, 3, 5, 10, 18, and 19 is described. Another C58 J provirus was mapped to chromosome 8 and proved to be identical to the previously mapped C58v-1 virus inducibility gene of strain C58 Lw . Three dominant visible markers, hammer-toe ( Hm), steel ( Sl), and caracul-J ( Ca J ), located on chromosomes 5, 10, and 15, respectively, have been introduced onto the MEV genetic background by repeated backcrosses to provide additional linkage markers. It is estimated that approximately 50% of the genome can be screened by scoring 50 fully informative gametes from a linkage cross of the MEV- Hm, -Sl, -Ca J stock for the combination of viral and visible markers. A strategy for efficiently mapping new recessive visible mutations by pooling tissues for DNA extraction from mutant homozygotes among F 2 progeny is described. Ways of further improving the MEV stock are discussed. The location of the Myb proto-oncogene is defined relative to Sl and one of the C58 J proviruses on chromosome 10.

Transduction of the cellular src gene and 3' adjacent sequences in avian sarcoma virus PR2257

When injected into chickens, a transformation-defective mutant of the Prague C strain of Rous sarcoma virus induced tumors at low incidence and after a long latency. One such tumor released a replication-defective virus designated PR2257. We molecularly cloned and sequenced the proviral DNA from quail fibroblasts transformed by PR2257. Comparison of PR2257 sequence with that of Prague C, cellular src, and 3' adjacent cellular DNA showed that the spliced version of the c-src gene and about 950 base pairs (bp) of 3'-flanking cellular DNA were transduced into PR2257. This transduction eliminated nearly all replicative genes, since the gag gene splice donor site was linked to the splice acceptor site of the src gene and, on the 3' side, recombination occurred in the end of env gene. Insertion of two extra cytosines 23 bp before and 19 bp after the c-src stop codon resulted in an extension of the coding portion up to 587 amino acids, divergence of sequences after Pro-525 and replacement of Tyr-527 by a valine residue. In addition, it appears that the 5' and 3' untranslated regions of PR2257 result from multiple recombinations between exogenous and endogenous virus genomes. Limited digestion of p66src encoded by PR2257 with Staphylococcus aureus V8 protease yielded a V2 peptide (C-terminal moiety) with an apparent molecular mass of 31 kilodaltons, consistent with the 5.7-kilodalton increase expected from the DNA sequence. The structure of PR2257 suggests that the first step in the capture of c-src gene by avian lymphomatosis viruses is the trans splicing of the viral leader mRNA to exon 1 of c-src.

Human endogenous retroviruses.

AbstractSeveral studies have revealed the presence in human DNA of thousands of endogenous retrovirus genomes, or HERVs. Many HERVs are related to extant retroviruses that infect other vertebrates and some have been present in the germ line of primates for millions of years. Although the HERVs that have been isolated are defective and thus do not encode infectious retroviruses, there may be HERVs that are capable of infection. In addition, because HERVs are so ancient in the human lineage, evolution of the human genome may have included the acquisition of some HERV genes for strictly cellular functions.