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PubMed1984

Genesis of Kirsten murine sarcoma virus: sequence analysis reveals recombination points and potential leukaemogenic determinant on parental leukaemia virus genome.

The genome of Kirsten murine sarcoma virus was formed by recombination between Kirsten murine leukaemia virus sequences, and rat sequences derived from a retrovirus-like '30S' (VL30) genetic element encompassing the Kras oncogene. Using cloned DNAs we have determined the nucleotide sequences of the long terminal repeats and adjacent regions, extending across the points of recombination on the sarcoma and leukaemia virus genomes. Our results suggest that discrete regions of homology and other cryptic sequence features, may have constituted recombinational hot-spots involved in the genesis of the Kirsten murine sarcoma virus genome. We have also compared the sequence of the Kirsten murine leukaemia virus p15 env and adjacent long terminal repeat with the corresponding regions of the AKV and Gross A murine leukaemia virus genomes. This comparison has identified a leukaemogenic determinant in the U3 domain of the long terminal repeat, possibly within a enhancer-like sequence element.

Amino Acid Sequence↗

PubMed1991

A transposon with an unusual LTR arrangement from Chlamydomonas reinhardtii contains an internal tandem array of 76 bp repeats.

TOC1, a transposable element from Chlamydomonas reinhardtii, is 5662 bases long. The 217 and 237 base long terminal repeat sequences of TOC1 are unusually arranged around the 4600 and 123 base unique regions: [217]-4600-[237] [217]-123-[237]. Although TOC1 contains long terminal repeats and most TOC1 elements are complete, features shared with virus-like retroposons, its unique 4600 base region is more similar to the structure of the L1 family of non-virus retroposons: first, 11 3/4 tandemly repeated copies of a 76 base repeat are found 813 bases from the left end of TOC1, and second using the universal genetic code large open reading frames were not found in TOC1. The relationship between TOC1, virus-like retroposons and the L1 family of non-virus retroposons is unclear and may be very distant since only poor similarity was found between the TOC1 encoded ORFs and retrovirus polypeptides. The length of the tandem array of 76 base repeat sequences was conserved in most TOC1 elements and solo 76 base repeat sequences were not found outside TOC1 elements in the C. reinhardtii genome. Nucleotide substitutions allow all copies of the 76 base repeat to be distinguished from one another.

Base Sequence↗

PubMed1991

The transcriptional transactivator of human foamy virus maps to the bel 1 genomic region.

The human foamy virus (HFV) genome possesses three open reading frames (bel 1, 2, and 3) located between env and the 3' long terminal repeat. By analogy to other human retroviruses this region was selected as the most likely candidate to encode the viral transactivator. Results presented here confirmed this and showed further that a deletion introduced only into the bel 1 open reading frame of a plasmid derived from an infectious molecular clone of HFV abolished transactivation. In contrast, deletions in bel 2 and bel 3 had only minor effects on the ability to transactivate. The role of the bel 1 genomic region as a transactivator was further investigated by eukaryotic expression of a genome fragment of HFV spanning the bel 1 open reading frame. A construct expressing bel 1 under control of a heterologous promoter was found to transactivate the HFV long terminal repeat in a dose-dependent fashion. Furthermore, it is shown that the U3 region of the HFV long terminal repeat is sufficient to respond to the HFV transactivator.

Animals↗

PubMed1986

Expression and characterization of the trans-activator of HTLV-III/LAV virus.

The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.

Cell Line↗

PubMed1988

Dexamethasone stimulates expression of transposable type A intracisternal retroviruslike genes in mouse (Mus musculus) cells.

Dexamethasone treatment enhances expression of transposable intracisternal type A particles (IAP) at RNA and proteins levels in a murine retrovirus-transformed cell line (Ki-BALB). This effect was ascertained by electron microscopic numeration of IAP. By sequence comparison, we located glucocorticoid-responsive elements in IAP long terminal repeats. Their regulatory potential was tested on the promoter activity of an IAP long terminal repeat construct coupled with the chloramphenicol acetyltransferase gene. Our findings suggest that the IAP activation by dexamethasone occurs at the level of transcription.

Animals↗

PubMed1989

Thymic atrophy characteristic in transgenic mice that harbor pX genes of human T-cell leukemia virus type I.

The human T-cell leukemia viruses (HTLV) are associated with T-cell malignancies in humans. The malignant transformation occurs after a long latency in some carriers, and its mechanism appears to be distinct from that of other classes of retroviruses which induce transformation through viral or cellular oncogenes. A widely postulated explanation is that the products of novel pX genes transactivate endogenous cellular genes which lead to tumor development in T cells. To directly examine the pathological effects of pX genes in vivo, we produced transgenic mice harboring the HTLV type I pX genes under several regulatory units: HTLV type I long terminal repeat, immunoglobulin enhancer-simian virus 40 promoter, and mouse mammary tumor virus long terminal repeat. Atrophy of the thymus was characteristic in these mice no matter which regulatory unit directed the expression of the genes.

Animals↗

PubMed1989

Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kappaB-like transcription factor.

We have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). We found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, we placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by R. Sen and D. Baltimore (Cell 46:705-716, 1986) as the binding site for the nuclear factor NF kappa B. In a cotransfection competition experiment, we could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF kappa B-binding sequence. The same long-terminal-repeat DNA containing mutant NF kappa B-binding sequences (G. Nabel and D. Baltimore, Nature [London] 326:711-713, 1987) did not affect CAT expression, which suggested that binding by an NF kappa B-like factor is required for increased SAA transcription.

Base Sequence↗

PubMed1990

Sequence-specific binding of DNA by the Moloney murine leukemia virus integrase protein.

Genetic studies have indicated that integration of retroviral DNA into the host genome depends on the presence of the inverted repeats at the free termini of the long terminal repeats on the unintegrated DNA and on the product of the 3' end of the pol gene (the integrase [IN] protein). While the precise function of the Moloney murine leukemia virus IN protein is uncertain, others have shown that it is a DNA-binding protein and functions in the processing of the inverted repeats prior to integration. By using site-directed mutagenesis, we cloned and expressed the IN protein in Escherichia coli. Crude extracts of total cellular protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, denatured in guanidine, renatured, and incubated with oligonucleotide probes. Single- and double-stranded oligonucleotides corresponding to the termini of unintegrated linear viral DNA were specifically bound by the IN protein in this assay. These data suggest that the role of the Moloney IN protein in the early steps of integration involves sequence-specific recognition of the DNA sequences found at the ends of the long terminal repeats.

Base Sequence↗

PubMed1991

Hormonal control of interacting promoters introduced into cells by retroviruses.

The interaction of promoters contained in a Moloney murine leukemia virus (MoMLV)-based retroviral vector was studied after infection of FTO-2B rat hepatoma and NIH 3T3 mouse fibroblast cells. Segments of the phosphoenolpyruvate carboxykinase (PEPCK) promoter-regulatory region, which are known from previous studies to confer responsiveness to hormones, were linked to the structural genes for bovine growth hormone, amino-3'-glycosyl phosphotransferase (neo), and herpes-virus thymidine kinase and inserted into a MoMLV-based retroviral vector. In vectors in which PEPCK was the only internal promoter, it was the major site of gene transcription. This dominant effect was independent of the orientation of the PEPCK promoter relative to the 5' long terminal repeat of the provirus and was noted with as little as -174 base pairs of the 5'-flanking sequence. NIH 3T3 cells, which do not express the endogenous PEPCK gene, transcribed the transduced PEPCK-chimeric genes at the same high levels as was observed in hepatoma cells. When two promoters were present in the provirus, the expression of chimeric structural genes depended on the relative position and orientation of these genes as well as the type of cell infected by the retrovirus. Differential responses of proviral promoters in infected cells were also observed in the presence of hormones. Dibutyryl cyclic AMP increased the expression of genes linked to the PEPCK promoter in FTO-2B and NIH 3T3 cells, whereas glucocorticoids stimulated transcription from both the PEPCK promoter and the long terminal repeat in FTO-2B cells. The effect of these hormones on transcription of proviral promoters depended on their position relative to the 5' long terminal repeat. In contrast, insulin uniformly inhibited transcription from the PEPCK promoter in a position-independent manner but only in hepatoma cells and not in fibroblasts. In clonally isolated FTO-2B cells infected with a retrovirus, the site of proviral integration was also a major factor determining the expression and hormonal regulation from the internal promoters. The data suggest that the hormonal regulation of the expression of genes contained in retroviral vectors depends on the type and position of the regulatory elements present in the provirus and the lineage of the infected cell.

Animals↗

PubMed1992

Isolation and characterization of cDNAs from BamHI-H gene family RNAs associated with the tumorigenicity of Marek's disease virus.

It has been reported that loss of the tumorigenic potential of attenuated Marek's disease virus (MDV) is strongly associated with amplification of the 132-bp repeat sequences found within the BamHI-D and BamHI-H fragments contained within the long terminal repeat and the long internal repeat, respectively. The expansion of this region results in loss of transcripts that are 3.8, 3.0, and 1.8 kbp long that are produced by tumorigenic strains of MDV. This evidence suggests that production of one or more of these three RNAs is strongly associated with the tumorigenic potential of the virus. In this study, we have cloned and sequenced 1.69-, 1.5-, 1.9-, and 2.2-kbp cDNAs from the BamHI-H gene family RNAs associated with tumorigenicity. The 1.69- and 2.2-kbp cDNAs are derived from nonspliced transcripts, whereas the 1.5- and 1.9-kbp cDNAs are from single spliced mRNAs spanning the BamHI-H and BamHI-I2 fragments of MDV DNA. Sequence analysis has shown two potential open reading frames in each of the cDNAs. The putative 63-amino-acid protein encoded by the first open reading frame in the 1.69-kbp cDNA and a putative 75-amino-acid protein encoded by the first open reading frame in the 1.5-kbp cDNA showed limited homology with the mouse T-cell lymphoma oncogene and the fes/fps family of kinase-related transforming proteins.

Amino Acid Sequence↗

PubMed1989

Structure of simian immunodeficiency virus regulatory genes.

Three full-length cDNA clones were obtained from cells infected with the simian immunodeficiency virus (SIV) isolated from captive macaques (SIVMAC). Nucleotide sequence analyses suggested that these represented mRNA for the SIV MAC genes tat, rev (formerly, art/trs), and nef (formerly, 3'orf). The putative tat-specific clone was active in trans-activation of the SIV MAC long terminal repeat in COS-1 and Jurkat cells. In contrast, the human immunodeficiency virus 1 long terminal repeat was significantly trans-activated only in the COS-1 cells. This suggests that trans-activation by the SIV tat gene is modulated by cell-specific factors. The structure of all of the clones suggested an mRNA splicing pattern more complex than that described for human immunodeficiency virus 1.

Amino Acid Sequence↗

PubMed1985

Nucleotide sequence of HBI, a novel recombinant MC29 derivative with altered pathogenic properties.

HBI is a recombinant avian retrovirus with novel pathogenic properties that was derived from the myc-containing virus MC29. In contrast to MC29, which causes endotheliomas in chickens, HBI induces lymphoid tumors. The results of molecular cloning and nucleotide sequencing of HBI reported here show that the virus contains sequences derived from both c-myc and ring-neck pheasant virus, in addition to MC29. The 3' half of the myc gene was largely replaced by c-myc sequences, and most of the long terminal repeat and gag regions were replaced by ring-neck pheasant virus sequences. The long terminal repeat contained a triplicate sequence which was homologous to the core enhancer sequence of the simian virus 40 72-base-pair repeat. The significance of these changes in relation to the unusual biological properties of the virus are discussed.

Amino Acid Sequence↗

PubMed1992

Multiple complex families of endogenous retroviruses are highly conserved in the genus Gallus.

We have analyzed the genome of the domestic chicken for the presence of genetic sequences related to the envelope protein-encoding genes of avian sarcoma/leukosis retroviruses to determine the organization, structure, potential functionality, and distribution of such sequences. We have previously identified in the genus Gallus an extensive group of endogenous avian retroviruses termed EAV-0. Southern blot and sequence analysis presented here of EAV-0 elements revealed that the majority of the EAV-0 elements in the domestic chicken genome have large deletions in their env genes. Screening of a line 0 chicken genomic DNA library for potential full-length env gene-containing endogenous elements yielded three provirus clones of a previously unrecognized group of endogenous retroviruses. These three clones, E13, E33, and E51, are more closely related to each other (80% or more sequence identity) than to other avian retroviruses (70% or less sequence identity). The E13 element has a large deletion in env, but the E51 element has full-length and highly divergent SU- and TM-coding domains. Complete sequence analysis of the E51 env gene region revealed a defective SU-coding domain and an intact TM-coding domain. Sequence analysis of the E51, E33, and E13 3' termini revealed highly distinctive long terminal repeats of approximately 360 bp which appear to be the products, in part, of long terminal repeat domain shuffling. Hybridization analysis with E51 and E33 env gene probes indicated that they are members of an extensive group of elements present in all Gallus species, and at least one element, E51, could be shown by polymerase chain reaction amplification and direct sequencing to have integrated prior to Gallus speciation.

Amino Acid Sequence↗

PubMed1990

Multimeric arrays of the yeast retrotransposon Ty.

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.

Bacteriophage lambda↗

PubMed1995

An autocrine loop of HIV type-1 Tat protein responsible for the improved survival/proliferation capacity of permanently Tat-transfected cells and required for optimal HIV-1 LTR transactivating activity.

Human immunodeficiency virus type 1 (HIV-1) transactivating Tat protein is pivotal to virus replication. Tat's potential effects on HIV-1 pathogenesis, however, go well beyond its role in the virus's life cycle. Current data indicate that biologically active Tat is released from HIV-1-infected cells and readily endocytosed and targeted to the nucleus of nearby, or perhaps distant, cells, where it may exert a series of pleiotropic effects. This paracrine action has been extensively investigated, and depending on the amounts of exogenously added Tat, its effects may extend from the suppression of immunocompetent cells to transactivation of heterologous genes to the promotion of growth of Kaposi's sarcoma spindle cells. We have already observed that various cell lines, either permanently transfected with an expressive HIV-1 tat gene construct or cultured in the presence of exogenously added Tat protein, are protected from programmed cell death after serum withdrawal or other apoptotic stimuli. The present article shows that various types (lymphoblastoid, epithelial, neuronal) of permanently tat-transfected cell lines actively release fully bioactive Tat protein. The addition of anti-Tat antibody to the culture medium completely abolishes their increased survival/proliferation capacity in serum-free culture. In these conditions, therefore, the enhanced survival/proliferation potential of permanently tat-transfected cells seems entirely dependent on a Tat-protein autocrine loop. The finding that anti-Tat antibody, added to culture medium, exerts a negative influence on the expression of a Tat-responsive HIV-1 long terminal repeat chloramphenicol-acetyltransferase construct, transiently transfected into permanently tat-transfected cells, suggests that the Tat autocrine loop may also be required for optimal HIV-1 long terminal repeat transactivation.

Animals↗

PubMed1987

Handicapped retroviral vectors efficiently transduce foreign genes into hematopoietic stem cells.

Retroviral vectors, designated handicapped, are described. These are genetically defective viruses that allow transfer of nonselectable genes under the transcriptional control of internal promoters. The basic handicapped vector (pHHAM) is derived from Harvey, Abelson, and Moloney murine retroviruses. It contains a 327-base-pair deletion in the 3' long terminal repeat that spans enhancer and promoter sequences. The deletion is successfully transferred to the 5' long terminal repeat after reverse transcription of viral RNA, yielding a provirus incapable of synthesizing viral transcripts. HHAM viruses containing the mouse c-myc gene under the control of immunoglobulin kappa chain gene regulatory elements, along with a selectable gene (neo) driven by a weak promoter (tk), were stably transmitted to cultured mouse B cells. The donor c-myc gene was transcribed from the kappa promoter in these cells. Helper-free virus-producing cell lines were generated at titers favorable for the efficient introduction of HHAM viruses, even without selection, into hematopoietic stem cells from mouse bone marrow. When returned to unirradiated congenic recipient mice, the cells were capable of long-term reconstitution of the myeloid and lymphoid lineages of W/Wv mutants and the lymphoid system of scid mutants.

Animals↗

PubMed1995

Sequences in the human immunodeficiency virus type 1 U3 region required for in vivo and in vitro integration.

A series of mutants with alterations in the U3 region of the human immunodeficiency virus type 1 long terminal repeat were made, and the effects of these mutations were evaluated both in vitro and in vivo. When the subterminal 6 to 8 nucleotides of the U3 long terminal repeat were mutated, the resulting provirus was unable to efficiently replicate in vivo, and a mutant oligonucleotide which mimicked the mutation could not be efficiently cleaved but could be joined to target DNA by wild-type recombinant integrase protein in vitro. These results suggest that this region is important in the specific recognition of the viral DNA by the integrase protein.

Base Sequence↗

PubMed1994

An interaction between the DNA-binding domains of RelA(p65) and Sp1 mediates human immunodeficiency virus gene activation.

Induction of human immunodeficiency virus type 1 (HIV-1) gene expression in stimulated T cells has been attributed to the activation of the transcription factor NF-kappa B. The twice-repeated kappa B sites within the HIV-1 long terminal repeat are in close proximity to three binding sites for Sp1. We have previously shown that a cooperative interaction of NF-kappa B with Sp1 is required for the efficient stimulation of HIV-1 transcription. In this report, we define the domains of each protein responsible for this effect. Although the transactivation domains seemed likely to mediate this interaction, we find, surprisingly, that this interaction occurs through the putative DNA-binding domains of both proteins. Sp1 specifically interacted with the amino-terminal region of RelA(p65). Similarly, RelA bound directly to the zinc finger region of Sp1. This interaction was specific and resulted in cooperative DNA binding to the kappa B and Sp1 sites in the HIV-1 long terminal repeat. Furthermore, the amino-terminal region of RelA did not associate with several other transcription factors, including MyoD, E12, or Kox15, another zinc finger protein. These findings suggest that the juxtaposition of DNA-binding sites promotes a specific protein interaction between the DNA-binding regions of these transcription factors. This interaction is required for HIV transcriptional activation and may provide a mechanism to allow for selective activation of kappa B-regulated genes.

Animals↗