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PubMed2000

Modular retro-vectors for transgenic and therapeutic use.

Retroviruses have been used for many years as vectors for human gene therapy as well as for making transgenic animals. However, the efficient insertion of genes by retroviruses is often complicated by transcriptional inactivation of the retroviral long terminal repeats (LTRs) and by the production of replication-competent retroviruses (RCR). Solutions to these and other difficulties are being found in modular vectors, in which the desirable features of different vector systems are combined. Examples of synergistic vectors include virosomes (liposome/virus delivery), adeno-retro vectors, and MLV/VL30 chimeras. As gene delivery systems become increasingly complex, methodology is also needed for precise assembly of modular vectors. Gene self-assembly (GENSA) technology permits seamless vector construction and simultaneous, multifragment assembly.

Animals↗
PubMed2000

Retroviruses and primate evolution.

Human endogenous retroviruses (HERVs), probably representing footprints of ancient germ-cell retroviral infections, occupy about 1% of the human genome. HERVs can influence genome regulation through expression of retroviral genes, either via genomic rearrangements following HERV integrations or through the involvement of HERV LTRs in the regulation of gene expression. Some HERVs emerged in the genome over 30 MYr ago, while others have appeared rather recently, at about the time of hominid and ape lineages divergence. HERVs might have conferred antiviral resistance on early human ancestors, thus helping them to survive. Furthermore, newly integrated HERVs could have changed the pattern of gene expression and therefore played a significant role in the evolution and divergence of Hominoidea superfamily. Comparative analysis of HERVs, HERV LTRs, neighboring genes, and their regulatory interplay in the human and ape genomes will help us to understand the possible impact of HERVs on evolution and genome regulation in the primates. BioEssays 22:161-171, 2000.

Animals↗
PubMed1992

Epstein-Barr virus integration in human lymphomas and lymphoid cell lines.

BACKGROUND: Epstein-Barr virus (EBV) is maintained as an episome in most infected cells. The presence of fused terminal restriction enzyme fragments distinguishes the circular DNA form from the linear virion form. METHODS: EBV genomic structure was analyzed in 8 lymphoid cell lines and 21 human lymphoma specimens by the Southern blot technique. RESULTS: Evidence of viral integration into host chromosomal DNA was identified in four cell lines. In the Namalwa and BL30-B95.8 cell lines, integration occurred through the terminal repeat (TR) sequences. In the BL41-P3HR1 and BL41-B95.8 cell lines, there was loss of left-end viral genomic sequences, including ori-P sequences required for episome maintenance, implying that integration was required for viral genome persistence. Integration was not detected in four other cell lines (Raji, Daudi, B95.8, and BL30-P3HR1). In 21 EBV-containing human lymphomas, including 18 immunodeficiency-related lymphomas, fused TR sequences were identified without evidence of viral genomic integration. CONCLUSIONS: These findings suggest that, although viral integration is common in Burkitt lymphoma cell lines infected in vitro, integration is not common in human lymphomas that develop in vivo in normal or immunodeficient people.

Adolescent↗
PubMed2003

Growth hormone prevents human immunodeficiency virus-induced neuronal p53 expression.

Growth hormone (GH) is neuroprotective, presumably through its actions on GH receptor-mediated pathways. Here, we examined the effects of GH using in vitro and in vivo assays of human immunodeficiency virus (HIV)-induced neuronal injury. Neuronal cultures were in assays of neurotoxicity induced by supernatants from HIV-1 tat-transfected monocytoid cells (Tat supernatant). GH treatment reduced neuronal death compared with untreated cultures (p < 0.001), which was blocked by a GH receptor antagonist, B2036. Tat supernatant-induced p53 expression in neurons was also reduced by GH treatment. Expression of both p53 and GH receptor were increased in brain tissue from HIV-infected persons compared with controls (p < 0.05). Mice receiving intrastriatal implants of Tat supernatant and treated with GH showed less neurobehavioral abnormalities together with reduced neuroinflammation and neuronal injury compared with untreated animals (p < 0.01). Three acquired immunodeficiency syndrome-defined patients with neurocognitive impairment were serially evaluated during daily GH treatment showing a sustained improvement in neuropsychological performance (p < 0.01). GH prevents neuronal death through its actions on neurons involving a p53-mediated pathway and also improved in vivo neurological function, indicating that GH may have a role in the treatment of HIV-induced neurodegeneration.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Preliminary molecular characterization of a human immunodeficiency virus (HIV-I) associated with neuropathology.

A human immunodeficiency virus (HIV-I) was isolated from the brain of a patient with progressive dementia but no obvious immunosuppression. This isolate, designated as HIV-IBR, was molecularly cloned and sequenced, and its long terminal repeat (LTR) and envelope sequences were compared with those of other HIV isolates not uniquely associated with dementia. The HIV-IBR LTR showed marked homology with the LTR sequences of the other HIV-I isolates. The predicted amino acid sequence of the external glycoprotein (gp120) of HIV-IBR revealed a pattern of conserved and variable regions similar to that of other HIV isolates. The sequence of the transmembrane portion of envelope, gp 41, was highly homologous to the counterpart region of other isolates. Further analysis is required to determine whether specific sequence variation can account for neurological manifestations of HIV-IBR infection.

Acquired Immunodeficiency Syndrome↗
PubMed2003

Quantitative characterization of specific genomic promoters using agarose gel electrophoresis.

Over the past decade a large number of studies have focused attention on the role of nucleosomes as negative and positive regulators of specific nuclear functions. Due to the lack of an analytical method to determine the higher order conformation of the nucleosomal arrays that encompass specific genetic loci (e.g., promoters, enhancers), research emphasis has mostly been centered on chromatin remodeling and histone posttranslational modifications. We have recently developed an agarose gel electrophoresis method that permits us to analyze the higher order structure of specific in vivo assembled chromatin fragments. After calibration using a well-defined in vitro system, we have been able to experimentally determine the size, shape, and conformational flexibility of the Mouse Mammary Tumor Virus long-terminal repeat promoter region in its repressed and activated states. These studies pave the way for widespread analyses of the higher order structure of specific, functionally important chromosomal loci, and in so doing enhance our understanding of the roles that the higher order structure of chromatin play in genome regulations.

Animals↗
PubMed2005

N-cadherin is not essential for limb mesenchymal chondrogenesis.

The cell adhesion molecule N-cadherin is implicated in many morphogenetic processes, including mesenchyme condensation during limb development. To further understand N-cadherin function, we characterized a new N-cadherin allele containing the lacZ reporter gene under the regulation of the mouse N-cadherin promoter. The reporter gene recapitulates the expression pattern of the N-cadherin gene, including expression in heart, neural tube, and somites. In addition, strong expression was observed in areas of active cellular condensation, a prerequisite for chondrogenic differentiation, including the developing mandible, vertebrae, and limbs. Previous studies from our laboratory have shown that limb buds can form in N-cadherin-null embryos expressing a cardiac-specific cadherin transgene, however, these partially rescued embryos do not survive long enough to observe limb development. To overcome the embryonic lethality, we used an organ culture system to examine limb development ex vivo. We demonstrate that N-cadherin-deficient limb buds were capable of mesenchymal condensation and chondrogenesis, resulting in skeletal structures. In contrast to previous studies in chicken using N-cadherin-perturbing antibodies, our organ culture studies with mouse tissue demonstrate that N-cadherin is not essential for limb mesenchymal chondrogenesis. We postulate that another cell adhesion molecule, possibly cadherin-11, is responsible for chondrogenesis in the N-cadherin-deficient limb.

Alcian Blue↗
PubMed2004

Genetic and epigenetic inactivation of tax gene in adult T-cell leukemia cells.

To clarify the status of tax gene, we analyzed human T-cell leukemia virus type-I (HTLV-I) associated cell lines and fresh adult T-cell leukemia (ATL) cells. We compared 2 types of HTLV-I associated cell lines: one was derived from leukemic cells (leukemic cell line) and the other from nonleukemic cells (nonleukemic cell line). Although all nonleukemic cell lines expressed Tax, it could not be detected in 3 of 5 leukemic cell lines, in which nonsense mutation or deletion (60 bp) of tax genes, and DNA methylation in 5'-LTR were identified as the responsible changes. We found such genetic changes of the tax gene in 5 of 47 fresh ATL cases (11%). The tax gene transcripts could be detected in 14 of 41 fresh ATL cases (34%) by RT-PCR. In ATL cases with genetic changes that could not produce Tax protein, the tax gene was frequently transcribed, suggesting that such cells do not need the transcriptional silencing. Although DNA methylation of 5'-LTR was detected in the fresh ATL cases (19 of 28 cases; 68%), the complete methylation associated with transcriptional silencing was observed only in 4 cases. Since partial methylation could not silence the transcription, and the tax gene transcription was not detected in 27 of 41 cases (66%), the epigenetic change(s) other than DNA methylation is considered to play an important role in the silencing.

Acetylation↗
PubMed1989

Complete foldback transposable elements encode a novel protein found in Drosophila melanogaster.

An apparently complete foldback (FB) transposable element homologous to FB white-crimson (FBwc) was analyzed. A complete FB element could encode one or more proteins required for regulation of FB transposition. The central DNA region (the loop) and the junctions between the loop and the inverted terminal repeats were sequenced. Three open reading frames (ORFs) are present in the loop, and a novel 308 bp inverted repeat is present at the junctions. No significant homologies were found when the DNA sequences of the loop region and the novel inverted repeat were screened against the Gene data bank. Antibodies were prepared in guinea-pigs against a peptide present near the amino terminus of ORF1, the longest ORF. A 71,000 dalton protein was isolated from an extract of Drosophila melanogaster early-stage embryos on an anti-ORF1 peptide-affinity column. Immunohistochemical studies of adult flies demonstrate localization of this protein in egg chambers.

Amino Acid Sequence↗
PubMed1994

Transactivation of the minus-strand DNA transfer by nucleocapsid protein during reverse transcription of the retroviral genome.

Two DNA strand transfers are required during reverse transcription of the RNA genome of retroviruses to complete provirus synthesis. To understand more about the first strand transfer reaction, that of the minus-strand DNA from the 5' to the 3' end of the retroviral genome, we devised an in vitro system mimicking the Moloney murine leukemia virus reverse transcription process. Two RNAs corresponding to the 5' and 3' regions of the genome were used to perform reverse transcription assays. The role of the nucleocapsid protein NCp10, which is tightly bound to the genome in the virus, was investigated in this system as well as the requirement of the 5' and 3' terminal repeats (R sequences) and the poly(A) tail. The results show that NCp10 drastically enhances the strand transfer reaction and that interactions between reverse transcriptase, nucleocapsid protein and viral RNA may be important. Both R sequences are required for an efficient and accurate DNA strand transfer and the poly(A) tail facilitates this reaction. Furthermore, it is probable that both intra- and intermolecular DNA strand transfers occur when the 5' and 3' ends of the genome are present on the same molecule.

Base Sequence↗
PubMed2003

A DNA vaccine containing inverted terminal repeats from adeno-associated virus increases immunity to HIV.

BACKGROUND: DNA vaccines have been used to induce both humoral and cellular immune responses against infectious microorganisms. This study explores whether DNA vaccine immunogenicity can be improved by introducing inverted terminal repeats (ITRs) from adeno-associated virus (AAV) into the regulatory region of the DNA plasmid. METHODS: CMV promoter-driven HIV Env expressing plasmid (pCMV-HIV) and the pCMV-HIV plasmid introduced ITRs (pITR/CMV-HIV) were transfected in HEK293 cells with LipofectAmine. The HIV Env expression was quantified with Western blot. Fifty micro g of pCMV-HIV or pITR/CMV-HIV plasmid with RIBI adjuvant were immunized to BALB/c mice on days 0, 14 and 28 by intramuscular route, and HIV-specific serum IgG titer was detected 2, 6, 10, 14 and 18 weeks after the first immunization. HIV-specific tetramer assay and HIV-specific IFN-gamma ELIspot assay were performed 1 week after the last immunization. The immune mice were intravenously challenged with a vaccinia virus expressing the HIV env gene 1 week after the last immunization. RESULTS: Significantly higher level of HIV Env expression was achieved by pITR/CMV-HIV plasmid. BALB/c mice immunized with pITR/CMV-HIV plasmid generated significantly higher HIV-specific antibody, higher cellular immune responses and lower viral loading than animals immunized with pCMV-HIV plasmid. CONCLUSIONS: AAV ITRs enhance CMV-dependent up-regulation of transgene expression and immunogenicity of DNA vaccine.

AIDS Vaccines↗
PubMed2002

Transforming growth factor-beta enhances human T-cell leukemia virus type I infection.

Human T-cell leukemia virus type I (HTLV-I), the causative agent of adult T-cell leukemia/lymphoma, is transmitted vertically by breast milk and sexually by semen. The transforming growth factor-beta (TGF-beta), a pleiotropic cytokine that is abundant in breast milk and semen, facilitates replication of HTLV-I in lymphocytes derived from asymptomatic HTLV-I carriers and transmission to cord blood lymphocytes in vitro. Transient expression assays revealed that TGF-beta can transactivate HTLV-I long terminal repeat promoter. These results suggest that TGF-beta may play a role in replication and transmission of HTLV-I.

Cells, Cultured↗
PubMed2001

On the problem of establishing the subcellular localization of Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of nuclear extracts.

At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein in vivo. Although this assumption will hold true for most of the analyzed proteins, it could also lead to false interpretations. We analyzed the subcellular distribution of endogenous and plasmid-borne proteins derived from the retrotransposon TRE5-A of Dictyostelium discoideum. In biochemical fractionation experiments the proteins encoded by TRE5-A open reading frame 1 (ORF1p) and the putative endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble material containing the nuclei. However, salt extraction of isolated nuclei did not considerably release the TRE5-A proteins. Instead, the TRE5-A proteins were strongly enriched in a fraction that contained the chromosomal DNA after removal of most cytoskeletal and histone proteins. These observations implied that ORF1p and ENp were both attached to chromatin in vivo, but this conclusion was disproved by the expression of genetic fusions of green fluorescent protein with either ORF1p or ENp. We show conclusive evidence that both fusion proteins were located as large aggregates of native protein in the cytoplasm of D. discoideum cells.

Animals↗
PubMed1999

Bystander macrophages silence transgene expression driven by the retroviral long terminal repeat.

The Moloney murine leukemia virus (MLV)-based retroviral vector has been widely used for transfer of exogenous genes to various organs and tissues. Although the long terminal repeat (LTR) of MLV allows for transgene expression in a wide range of cell type, its activity is often silenced in vivo. In reporter macrophages transduced with a MLV-based retroviral vector, activity of the LTR was transiently and reversibly suppressed following stimulation by lipopolysaccharide (LPS). When unstimulated reporter macrophages were co-cultured with LPS-stimulated, untransduced macrophages, the LTR activity was similarly depressed. Activity of the LTR in retrovirus-transduced, mesangial cells was also down-regulated when co-cultured with activated macrophages. This suppressive effect was reproduced by cross-feeding with culture media conditioned by activated macrophages. LPS-stimulated macrophages abundantly expressed cytokines including IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta1 (TGF-beta1). When externally added, TNF-alpha and/or TGF-beta1, but not IL-1beta, depressed activity of the LTR in reporter macrophages and reporter mesangial cells. These results raise a possibility that expression of transgenes driven by the MLV-LTR may be silenced in vivo when the retrovirally-transduced cells are co-localized with activated macrophages.

Animals↗
PubMed2001

Activation of HTLV-I long terminal repeat by stress-inducing agents and protection of HTLV-I-infected T-cells from apoptosis by the viral tax protein.

HTLV-I is etiologically implicated with tropical spastic paraparesis/HTLV-I associated myelopathy, adult T-cell leukemia and certain other diseases. However, after infection the virus enters into a dormant state, whereas the characteristics of the HTLV-I related diseases indicate that their genesis requires activation of the dormant virus by a Tax-independent mechanism. In the present study we demonstrate that a variety of stress-inducing agents (TPA, cisplatin, etoposide, taxol, and 3-methylcholanthrene) are capable of Tax-independent activation of HTLV-I LTR and that this activation is detected mainly in cells that are undergoing through the apoptotic process. Furthermore, it is demonstrated that both apoptosis induction and HTLV-I LTR activation are inhibited by Bcl-2 and by PKC, indicating that these two processes are mechanistically cross-linked. In addition, using an HTLV-I producing human T-cell line which permanently express the negatively transdominant tax mutant, Delta58tax, under the Tet-Off control system, we prove that the virally encoded Tax protein protects the host cells from apoptosis. Together, these data suggest that activation of the dormant virus in the carriers' infected T-cells by certain stress-inducing conditions and protecting these cells from the consequent apoptotic death by the viral Tax protein emerging after this activation, might be the basis for switching the virus from latency to a pathogenic phase.

Antineoplastic Agents↗
PubMed2000

Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.

Second- and third-generation three-plasmid vector systems, termed FELIX, were constructed from feline immunodeficiency virus (FIV). To enhance vector production, the weak FIV long terminal repeat promoter was replaced with the human cytomegalovirus enhancer/promoter. To construct a minimal system in which Gag-Pol was the only viral protein present, the cytoplasmic transport element was used in place of the FIV Rev-RRE system to facilitate nuclear export of Gag-Pol and the transfer vector. Unconcentrated vector titers routinely exceeded 1 x 10(6) IU/mL for most constructs tested. Second- and optimized third-generation vectors were capable of efficiently infecting G1/S- and G2/M-arrested cells. FIV-based FELIX vectors transduced human dendritic cells, hepatocytes, and aortic smooth muscle with efficiencies similar to that of a control 3T3 cell line. All three of these primary cell types were transducible by both the second- and third-generation FELIX vectors, demonstrating that FIV Gag-Pol alone contains the determinants necessary for transduction of primary cells. In cross-packaging tests, we observed that HIV Gag-Pol does not substantially package FIV vectors; consequently, use of such vectors in human immunodeficiency virus-infected cells should not lead to efficient mobilization of the inserted gene. Thus, this FIV-based vector system offers high efficiency and stable delivery of genes to numerous nondividing and primary cell types, opening new avenues for biological inquiry into normal human cells.

3T3 Cells↗
PubMed2001

Glucocorticoid-regulated expression of exogenous human growth hormone gene in rats.

The aim of this study was to control in vitro and in vivo expression of the growth hormone (GH) gene using a glucocorticoid-sensitive promoter, the mouse mammary tumor virus long terminal repeat (MMTV LTR). We inserted the cDNA encoding the 20-kDa form of human GH (20K-GH) downstream of the MMTV LTR of plasmid pMSG, and used lipofection to transfer it to 3Y1 cells together with plasmid pMX, which contains a puromycin-resistant element. The secretion of GH from the selected transformants was dose-dependently augmented by the application of hydrocortisone, corticosterone, or dexamethasone, among which dexamethasone was the most potent. Analysis of the time course showed that 20K-GH secretion began to increase within 2 hours after the addition of glucocorticoid and reached a maximal level of about threefold over the unstimulated control at 3 hours; secretion then gradually declined and returned to near basal levels at 19 hours. Repeated glucocorticoid application led to repeated increases in GH secretion. When GH-producing cells were microcapsulated and transplanted into the abdominal cavities of rats, 20K-GH was detected in the plasma under control conditions and increased about 3.3-fold after administration of dexamethasone. We suggest that GH expression driven by the MMTV LTR promoter may be under the control of an endogenous glucocorticoid in vivo.

Animals↗