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Gene transfer into haematopoietic stem cells (HSC) has been investigated for treatment of genetic disorders, conferral of chemotherapy resistance and insertion of genes to inhibit HIV-1 replication. Methods have been available for almost a decade to transduce murine HSC using high-titre retroviral vectors and stimulation of HSC proliferation with cytokines such as IL-3 and IL-6. Unfortunately, attempts to replicate the high efficiency of gene transfer using canine or simian gene transfer/bone marrow transplantation models have consistently shown that only a small fraction (0.1-1%) of reconstituting HSC are transduced using protocols similar to those which are successful in murine models. Initial clinical trials using retroviral-mediated gene transfer into human HSC also produced minimal transduction frequencies. The dicotomous results may reflect differences in the cell cycle kinetics of murine HSC versus those of larger mammals or the density of receptors for the retroviral vectors on the cells. Attempts to increase the fraction of HSC which are in active cell cycle, a prerequisite for retroviral-mediated transduction, have used either combinations of recombinant cytokines, culture on marrow stromal layers, or alternative sources for HSC, such as mobilized peripheral blood stem cells or umbilical cord blood. Other efforts have used retroviral vectors packaged with either the Gibbon Ape Leukemia virus envelope or the Vesicular Stomatitis Virus G protein. To date, none of these methods has produced a significantly increased frequency of long-term reconstituting HSC. Results using adeno-associated virus (AAV)-based vectors for HSC transduction have been conflicting, with the stable persistence of non-integrated virus particles making interpretation of results difficult using in vitro assays. Therefore, clinical trials may best be directed toward disorders that may benefit from a small fraction of genetically corrected HSC. These would include disorders where progeny of corrected HSC would be expected to have a selective survival advantage (e.g. SCID, WAS, HIV, chemoresistance) or where a small fraction of corrected cells can have a direct clinical benefit (e.g. CGD, MPS). Further basic research into HSC biology and gene delivery vectors must continue for wider application, such as haemoglobinopathies and some lysosomal storage diseases.
A replication-competent avian retrovirus mutant, containing a single amino acid substitution at amino acid residue 115 in the 3' endonuclease (IN) region of the polymerase (pol) gene, was characterized. DNA transfection experiments demonstrated that the mutant virus exhibited a delayed growth phenotype at 41 degrees while replicating efficiently at 35 degrees. Examination of virus-infected cells at the molecular level demonstrated that the mutant virus at either temperature was capable of synthesizing viral DNA as efficiently as wild-type Rous sarcoma virus, strain Prague A. This result suggested that the same mutation, which was also present in the IN moeity of the polymerase beta polypeptide, did not affect DNA synthesis. Further analyses demonstrated that at either temperature the mutant virus integrated its DNA at about 10-20% of wild-type level, although possibly less efficiently at 41 degrees than at 35 degrees. The mutation at residue 115 (Pro to Ser) appeared to lower the ability of IN to function in the integration of viral DNA relative to wild-type virus. No definitive conclusion could be made as to whether IN in this mutant possessed a temperature-sensitive lesion which caused the observed replication defect at 41 degrees.
Treatment of hepatocellular carcinoma (HCC) cells with butyrate can induce apoptosis irrespective of hepatitis B virus integration. No information is available, however, regarding the effect of butyrate on HCC in the presence of hepatitis C virus (HCV) because some HCV proteins can regulate cell survival. By gene transfer, we found that HCV core enhances but HCV NS5A antagonizes sodium phenylbutyrate (NaPB)-induced apoptosis in HCC cells, which is independent of p53. We then chose the p53-negative Hep3B HCC cell to investigate the mechanism of anti-apoptosis mediated by NS5A. In the NaPB-treated Hep3B cells without NS5A expression, induction of apoptosis was associated with Bax redistribution from the cytosol to the nucleus interior and subsequently, to a nuclear membrane-bound form. In the NS5A expressing Hep3B cells, NaPB treatment also triggered relocalization of both Bax and NS5A from the cytosol to the nucleus interior but Bax retained inside the nucleus and did not finally move to the nuclear membrane. Using double immunofluorescence and coimmunoprecipitation, we demonstrated that NS5A co-localizes and interacts with Bax in the nucleus. The HCV NS5A protein was further found to contain Bcl-2 homology domains (BH3, BH1 and BH2). Additional studies using deleted NS5A constructs were carried out to determine whether the BH2 domain or nuclear localization signal (NLS) in NS5A is required for interaction with Bax in the nucleus or inhibition of apoptosis. NS5A with deletion of both BH2 domain and NLS localized in the cytoplasm, dissociated with Bax, and lost anti-apoptosis activity during NaPB treatment. In contrast, NS5A with intact BH domains except NLS still bound directly to Bax in the perinuclear region or the nucleus, but showed less association with Bax in the nucleus and lower effect in apoptosis inhibition than full-length NS5A. These results suggest that HCV NS5A as a Bcl-2 homologue interacts with Bax to protect p53-negative HCC cells from NaPB-induced apoptosis.
This paper describes a method for the specific quantification of whole virions of foot-and-mouth disease (146S) in the presence of virus subunits (12S). The method involves the use of virus neutralising monoclonal antibodies directed against a linear epitope of the VP1 loop region of a type O virus. The monoclonal antibodies were used as both capture and detecting reagents (labelled with horse radish peroxidase) in a sandwich ELISA. Such monoclonal antibodies also have the advantage that they do not detect viruses containing proteolytically cleaved VP1, thus the assay system is ideal for estimation of whole particles in vaccine manufacture where the immunogenicity of the vaccine depends on virus integrity (whole virions being present) and uncleaved capsid protein. VP1. Other combinations of different anti-type O FMD virus monoclonal antibodies used as capture and detecting reagents were also examined. The system could be adapted to on-line continuous testing of virus being produced during a manufacturing run allowing maximisation of virus yield and quality control.
BHK-21/13 cells transformed with various chemical carcinogens and mutagens were tested for susceptibility to avian sarcoma virus infection. The chemically transformed BHK cells were highly tumorigenic. The treatment of these cells in vitro with avian sarcoma virus strain Schmidt-Ruppin D resulted in chemically transformed and viral infected cells with different rescuability of the integrated virus genome. The different rescuability is not due to the difference in virus penetration as was shown by vesicular stomatitis virus Schmidt-Ruppin virus pseudotype VSV(SR-D) technique. The sarcoma virus genome in the doubly transformed cells was not inducible by various virus inducers.
The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.
The intracellular structure and expression of integrated hepatitis B virus (HBV) DNA in the hepatoma tissue #1707 obtained from an 11-year-old boy were studied. It was found that most of the HBV genome was present in the #1707 DNA. Virus-specific 21S mRNA was also demonstrated by blot hybridization. This is the first direct evidence for the expression of the integrated viral genome in human hepatoma tissue.
A series of diketo derivatives was found to inhibit human immunodeficiency virus type 1 (HIV-1) integrase activity. Only L-708,906 inhibited the replication of HIV-1(III(B)) (50% effective concentration, 12 micro M), HIV-1 clinical strains, HIV-1 strains resistant to reverse transcriptase or fusion inhibitors, HIV-2 (ROD strain) and simian immunodeficiency virus (MAC(251)). The combinations of L-708,906 with zidovudine, nevirapine, or nelfinavir proved to be subsynergistic. In cell culture, addition of L-708,906 could be postponed for 7 h after infection, a moment coinciding with HIV integration. Inhibition of integration in cell culture was confirmed by quantitative Alu-PCR.
Thirty-four transgenic mouse strains, each carrying a single proviral insert, were generated by infection of preimplantation and postimplantation embryos with retroviruses. Animals homozygous for proviral integrations were derived for all strains with the exception of Mov 24, where the provirus is inserted on the Y chromosome, and Mov 34. Embryos homozygous at the Mov 34 locus develop normally to the blastocyst stage and die shortly after implantation, indicating that virus integration resulted in a recessive lethal mutation. The provirus and flanking sequences were cloned and the virus was mapped to the 5' side of an abundantly and ubiquitously transcribed gene. Similar to the previously derived Mov 13 mutation, proviral integration at the Mov 34 locus interferes with the expression of the adjacent gene. These and our previous results indicate that of a total of 48 proviral integrations in the germ line, two resulted in transgenic mouse strains with recessive lethal mutations.
Restriction mapping of polyoma virus DNA in mouse tumor cell lines gave patterns that varied with the cell line examined. These reflected differences in both the organization and the state of integration of virus genomes in the host chromosomes. (The cell lines were derived from tumors induced by polyoma virus in vivo and were propagated continuously in culture.) Two of the cell lines contained multiple copies of tandemly integrated virus genomes as well as free virus DNA molecules. Two other cell lines appeared to contain only integrated virus genomes arranged as tandem repeats. Based on restriction analysis with eleven different endonucleases, the virus DNA in one of the cell lines containing both free and integrated virus genomes was not detectably defective or hypermethylated. This is in contrast to most previously described polyoma virus transformed mouse cells. These virus genomes may, however, contain point mutations or unobserved rearrangements. The second cell line possessing free virus DNA molecules contained both nondefective and defective virus genomes. Most, if not all, defective virus genomes in this line were integrated. The two other cell lines possessing only detectable integrated virus DNA apparently contained only defective virus genomes. The defect in both cases was a small deletion (less than or equal to 0.2 kb) encompassing 0.12 map units on the physical map of polyoma virus DNA, a region coding for the proximal part of the large T antigen. Moreover, in contrast to the cell lines with free and detectably nondefective virus DNA, the virus DNA was extensively methylated in cell lines containing only integrated and defective virus genomes.
The integrated DNA provirus of the Gardner-Arnstein (GA) strain of feline sarcoma virus (FeSV) was molecularly cloned in a bacteriophage lambda vector. The cloned DNA fragment is 14.4 kilobase pairs long and contains a 6.7-kilobase provirus flanked by cellular sequences derived from nonproductively transformed mink cells. Transfection of mouse NIH/3T3 cells with the cloned DNA fragment induced foci of transformation at efficiencies of 10(4) focus-forming units/pmol of sarcoma virus DNA. Restriction endonuclease mapping and heteroduplex analyses were used to compare the GA-FeSV provirus with that of Snyder-Theilen (ST)-FeSV, a second strain that contains homologous transformation-specific sequences (v-fes). Both viruses have the general structure 5'-gag-fes-env-c region-3', each having retained portions of the feline leukemia virus (FeLV) gag and env genes. In addition to segments shared by the two sarcoma viruses, GA-FeSV contains 1.7 kilobases of extra sequences not found in ST-FeSV. Of these, at least 400-500 base pairs located near the 5' end of v-fes encode a portion of the GA-FeSV polyprotein; the remaining 1.2 kilobases are derived from the FeLV env gene but do not appear to encode any detectable product related to the FeLV envelope glycoprotein. The close homology of the v-fes sequences shows that GA- and ST-FeSV were formed by recombination of FeLV with similar portions of a cat cellular gene (c-fes).
Adeno-associated virus (AAV) integrates specifically into a site (AAVS1) on human chromosome 19q13.3-qter. Similarly, there is accumulating evidence that this site-specific integration occurs by transfection of AAV-based plasmid vectors. In order to further define the process of plasmid integration events, we constructed some AAV plasmids, introduced them into HeLa cells by lipofection, and isolated chromosomal integrants. One of such plasmids, pTH-5, contained the rep and neomycin-resistant (neo(r)) genes flanked by the 5'- and 3'-inverted terminal repeats of AAV and the hygromycin-resistant (hyg(r)) gene located in the plasmid backbone. Southern blot analysis revealed that among 36 G418-resistant (G418(r)) clones isolated, 22 (61%) showed site-specific integration into AAVS1. Further structural and functional analyses on the expression of the hyg(r) gene in the site-specific clones and the LacZ gene in clones generated with plasmid pTH-2 indicated that, together with the AAV sequence, the plasmid backbone was integrated into the AAVS1 site and thus the neo(r) and hyg(r) genes remained linked at high frequencies in the targeted integrants compared with random integrants. Sequence analysis of integration junctions between pTH-5 and AAVS1 revealed that the junctions occurred in the p5 promoter region of the plasmid while mainly in the partial cDNA coding region of the AAVS1 site. We also found that plasmid pTH-1 linearized in the backbone before lipofection gave a significantly lower frequency of site-specific integration (26%) than the circular form (60%). This finding may support the involvement of the double-stranded, circular form of infected AAV in the integration process. Our results may help to understand the process and mechanism of site-specific integration of lipofected AAV plasmid vectors.
It was discovered in the 1970s that oncogenic viruses could induce gene mutations in mammalian cells. The phenomenon seems to be widespread: it was observed with all groups of DNA-containing viruses and some retroviruses. The mutagenic effects of the tested viruses at gene level are not locus specific. The viruses induce point mutations, including base substitutions, as well as deletions and insertions. The mutagenic effect of SV40 is controlled by the activity of the early A gene, which encodes the T antigen. Presumably, the process of integration creates the possibility for occurrence of mutations early after infection. Mutagenesis seems to be induced by an integrated virus, though to a much smaller extent. Virus-induced mutagenesis may be connected with an activation of the cell error-prone repair systems. The sum total of the experimental data shows that virus-induced mutagenesis and transformation are interrelated: (A) viruses, like other carcinogenes, display mutagenic activity; (B) viruses that are far removed from each other systematically, whose only similarity lay in being oncogenic and capable of integration, simultaneously showed the ability to induce gene mutations; (C) agents changing the rate of transformation also changed the rate of gene mutations: (D) The function of mutagenicity was mapped in the oncogene of SV40 (gene A); and the DNA of (E) mouse mammary carcinoma virus (MMTV) and avian leukosis virus (ALLV) induced tumors has been found to contain nucleotide sequences that transform 3T3NIH cells but do not carry any viral genetic information. Mutagenesis induced by oncogenic viruses may play a part in the multistage process of malignant transformation, though its contribution may be different in various specific cases and for different groups of viruses. Further studies of the uncommon mutagens, which viruses seem to be, may greatly increase our knowledge of the virus-cell relationship. An understanding of the extent of genetic danger inherent in viruses and live viral vaccines is necessary for practical medicine.
Hepatitis B virus nucleotide sequences derived from a hepatocellular carcinoma with free and multiply integrated viral DNAs were determined. Based on a comparison within the X-gene region, cloned free viral DNA previously had been attributed to two distinct groups of preC minus genomes. The comparison of the complete sequence identified one of the genome equivalents as a recombinant between members of these groups. Four different integrated viral DNA elements were cloned and analysed. Similarity to either one of two DNAs representing the two groups of free viral DNA on one hand and the presence of certain mutations only on integrated DNA on the other hand, allowed to recognize distinct segments within the integrants. The data suggest a contribution of different but related genotypes to contiguous stretches of integrated viral DNA via homologous recombination. On this basis an evolutionary relationship between free and integrated DNAs of the preC and the preC minus genotype could be recognized when short sequence segments were compared. The observed coexistence on a given integrated DNA of segments homologous to free viral DNA and of segments homologous to another integrated DNA is consistent with (1) a long lasting association of individual genotypes with dividing cells and (2) multiple integration events being the result of a series of steps not separated by a long time span.
Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.
Circular Epstein-Barr virus (EBV) DNA molecules have been purified and characterized from a human lymphoid cell line derived from a case of heterophile antibody-positive, blood transfusion-induced infectious mononucleosis, 883L. The circular EBV DNA in three cell lines obtained by transformation of human umbilical cord blood leukocytes with a strain of EBV originally derived from 883L was also studied. As estimated from sedimentation velocity data and electron microscopy, the circular EBV DNA molecules are 10 to 15% smaller than either the circular EBV DNA previously found intracellularly in several other types of EBV-transformed cells or the linear EBV DNA present extracellularly in virus particles. In addition, the EBV-transformed cord blood cell lines studied here differed from other EBV-transformed cells in that integrated virus DNA sequences could not be detected.
OBJECTIVE: The gene encoding the peripheral cannabinoid receptor Cb2 is located in the common virus integration site Evi11 and is associated with hematopoietic malignancies in mice. To determine the effect of Cb2 overexpression on hematopoietic development in vivo, Cb2 transgenic mice were generated. MATERIALS AND METHODS: A Cb2 expression vector was constructed containing a Cb2 cDNA fragment cloned into the 14kb Sca-1 (Ly-6E.1) gene. Two transgenic lines in which Cb2 expression is controlled by the Sca-1 promoter were generated, and the effect on hematopoietic development was studied. Expression of Cb2 mRNA or protein was studied by RNase protection analysis and ligand binding assays, respectively. Leukemic predisposition was investigated by injecting newborn transgenic as well as control animals with Cas-Br-M murine leukemia virus (Cas-Br-M MuLV). RESULTS: Although increased expression of the Cb2 gene was observed in hematopoietic tissues, follow-up of more than 1 year did not reveal any hematologic defect. Interestingly, infection of newborn pSca-1/Cb2 transgenic mice with Cas-Br-M MuLV revealed that significantly more transgenic mice developed leukemia than virus-treated control littermates. Because these studies provide evidence for the cooperative potential of Cb2 in leukemia progression, we wished to identify genes that may collaborate with Cb2 in leukemic transformation. Our study suggests that Evi1, another common target for proviral integration in mouse leukemias, may be overexpressed in virus-induced leukemias in pSca-1/Cb2 transgenic mice. CONCLUSIONS: The data indicate that hematopoietic precursor cells that express high levels of Cb2 possess increased susceptibility for leukemia development and that Cb2 and Evi1 might collaborate in leukemogenesis.