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At least 217 records · Page 12Linked to original sources
PubMed1992

Probing protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 by in vivo footprinting.

We have analyzed protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1 in a productively infected T-cell line by in vivo dimethyl sulfate footprinting. Major footprints are evident at the basal promoter and enhancer elements. In particular, proteins appear to occupy the TATA box, the Sp1 sites, and the two repeats of the enhancer region. In the negative regulatory element, protections are detected over the USF/MLTF and NFAT-1 sites. Furthermore, two previously unrecognized sites, from nucleotides -260 to -275 and from nucleotides -205 to -216, respectively, appear to be involved in protein-DNA interactions. These two sites are purine rich and share a common sequence motif.

Base Sequence↗
PubMed1997

Regulation of the long terminal repeat in visna virus by a transcription factor related to the AML/PEBP2/CBF superfamily.

The long terminal repeats of maedi visna virus strain 1514 contain a consensus AP-1 binding site which has been shown to be important in controlling virus transcription. However, this consensus site is absent in strain EV-1. Here, we have compared the ability of oligonucleotides corresponding to LTR sequences from EV-1 with those from 1514 to bind transcription factors in competitive gel retardation assays and activate reporter gene expression. The experiments demonstrated no observable binding of AP-1 to the EV-1-derived sequences and significant differences in the abilities of the 1514 and EV-1 sequences to activate transcription. However, both viral sequences interacted with a second, previously undetected, transcription factor. This factor gave specific gel shifts which were competed by an oligonucleotide containing the consensus sequence for the AML/PEBP2/CBF family of transcriptional factors, but not by control AP-1 or OCT-1 oligonucleotides. The factor was therefore denoted AML (vis). A second AML (vis) site, noted upstream of the TATA box proximal AP-1 site, gave single shifts which were competed by the downstream AML (vis) oligonucleotide. Both sites were functional in transfection assays. In gel shift retardation assays, polyclonal antisera directed against known runt domain proteins were able to supershift part of the AML (vis) binding activity in nuclear extracts from physiologically relevant cell types. The results thus suggest that the AML (vis) binding factor belongs to the AML/PEBP2/CBF family of transcription factors and may be important in controlling virus replication in these and other strains of ruminant lentiviruses.

Animals↗
PubMed1987

Sequence instability in the long terminal repeats of avian spleen necrosis virus and reticuloendotheliosis virus.

Sequence divergence between the 3' long terminal repeats (LTR) of avian reticuloendotheliosis virus (REV), deletion variant proviral clone 2-20-4, and spleen necrosis virus (SNV)-proviral clones 14-44, 60, and 70-was found to involve two classes of base substitutions: low-frequency interspersed and high-frequency clustered substitutions. Clones 2-20-4 and 14-44 have diverged 4.4% owing to low-frequency substitutions. In contrast, two high-frequency substitution segments have diverged by 30% and 29%, respectively. Clustered substitutions appear to be located either within or next to tandem repeats, suggesting their introduction concomitant with sequence deletions and duplications commonly associated with such repeats. A new 19-bp tandem repeat is found in clone 2-20-4. Its sequence could have evolved from the 26-bp repeats found in the SNV clones.

Base Composition↗
PubMed2006

Long terminal repeats are not the sole determinants of virulence for equine infectious anemia virus.

The long terminal repeats (LTRs) of equine infectious anemia virus donkey leukocyte-attenuated virus (EIAV-DLA) were substituted with those of the wild-type EIAV-L (wt EIAV-L, the parent virus of EIAV-DLA). The resulting chimeric plasmid was designated pOK-LTR DLA/L. Purified pOK-LTR DLA/L was transfected into monocyte-derived macrophage (MDM) cultures prepared from EIAV-negative, heparinized whole blood from a donkey. Eighth-passage cell cultures developed the typical cytopathogenic effects (CPE) of EIAV infection, and virions with typical EIAV profiles were observed with an electron microscope. Horses were inoculated with the chimeric virus or EIAV-DLA and challenged with the wt EIAV-L strain six months later. All of the horses inoculated with either the chimeric virus or EIAV-DLA were protected from disease, whereas the control horses died with typical EIA symptoms.

Animals↗
PubMed1997

Deletion analysis of both the long terminal repeat and the internal promoters of the human foamy virus.

Deletion analyses of the long terminal repeat (LTR) and internal promoters (IP) of human foamy virus (HFV) showed that a negative acting element resides in the U5 region of the 5' LTR reducing reporter gene expression tenfold. The basal activity of the IP was higher than that obtained with LTR promoter constructs and strongly elevated in permissive BHK-21 cells whereas semi-permissive COS-7 cells showed low basal activity. Since the basal activity of the IP is critical for initiating HFV gene expression by providing Bel 1 transactivator early after infection, the basal activity of the IP may be the crucial factor that contributes to whether cells are permissive for HFV infection or not. Deletion mutagenesis allowed to define the minimal IP region. A region strongly transactivated by Bel 1 extends from -136 to +58 relative to the cap site of the IP. The major Bel 1 response element of the IP required for transactivation is located upstream of the cap site between -136 and -88 relative to the internal cap site. A DNA fragment reported to be protected by recombinant Bel 1 was deleted with marginal reduction of Bel 1 transactivation. HFV gene expression directed by the IP and LTR promoters is thus multiply regulated by positive and negative acting response elements in cis and their binding partners in trans.

Animals↗
PubMed2003

Patterns of sequence conservation at termini of long terminal repeat (LTR) retrotransposons and DNA transposons in the human genome: lessons from phage Mu.

Long terminal repeat (LTR) retrotransposons and DNA transposons are transposable elements (TEs) that perform cleavage and transfer at precise DNA positions. Here, we present statistical analyses of sequences found at the termini of precise TEs in the human genome. The results show that the terminal di- and trinucleotides of these TEs are highly conserved. 5'TG...CA3' occurs most frequently at the termini of LTR retrotransposons, while 5'CAG...CTG3' occurs most frequently in DNA transposons. Interestingly, these sequences are the most flexible base pair steps in DNA. Both the sequence preference and the degree of conservation of each position within the human LTR dinucleotide termini are remarkably similar to those experimentally demonstrated in transposable phage Mu. We discuss the significance of these observations and their implication for the function of terminal residues in the transposition of precise TEs.

Bacteriophage mu↗
PubMed1984

Proteins encoded by the long terminal repeat region of mouse mammary tumor virus: identification by hybrid-selected translation.

The long terminal repeat (LTR) region of mouse mammary tumor virus (MMTV) is known to contain an open reading frame of sufficient length to code for a protein of 36,000 Mr. The coding capacity of the 3' sequences of MMTV genomic RNA has been demonstrated by in vitro translation studies, which have reported the synthesis of four related proteins: p36, p24, p21, and p18. These proteins are overlapping translation products of the same open reading frame, with the smaller ones initiating at internal methionine codons. From the predicted amino acid sequence of the LTR protein, we have selected a region likely to be antigenic, obtained a synthetic peptide of that region, and raised antiserum to the peptide. The antipeptide serum specifically immunoprecipitated all four proteins from in vitro translated genomic 3' MMTV RNA, plus an additional one of 32,000 Mr. Published sequence data of MMRV LTRs show an internal AUG codon at a position which could initiate a protein of 32,000 Mr. The three smaller in vitro translation products (p24, p21, and p18) were consistently synthesized in much greater amounts than the p36 or p32 protein. The relative amount of each in vitro synthesized protein from genomic MMTV RNA could be predicted and was in good agreement with the postulated effect of flanking nucleotides on the efficiency of the respective AUG initiation codon. Polyadenylated RNAs, isolated from various mouse tissues, were selected by hybridization to plasmid DNA containing MMTV LTR sequences immobilized on nitrocellulose. In vitro translation of hybrid-selected mRNAs isolated from BALB/c mouse lactating mammary glands and carcinogen-induced mammary tumors, followed by immunoprecipitation with antipeptide serum, revealed that only one polypeptide was synthesized by the MMTV LTR-specific mRNA, the 36,000 Mr species.

Animals↗
PubMed1989

Differences in activities of murine retroviral long terminal repeats in cytotoxic T lymphocytes and T-lymphoma cells.

Transcriptional activities of the long terminal repeats (LTRs) of various murine leukemia viruses were tested in the cytotoxic T-cell lines CTLL-1 and CTLL-2. In contrast to T-lymphoma cells, in which the LTRs of T-lymphomagenic virus SL3-3 and Moloney murine leukemia virus are more active than those of other viruses, transcriptional activity in these mature, interleukin-2-dependent cells is not correlated with the specificity of viral leukemogenicity. Several approaches were used to investigate the molecular basis for LTR activity differences in lymphoma cells and mature cytotoxic T cells. Deletion analysis of the Moloney virus LTR showed that the direct repeats associated with enhancer activity have, at most, a slight effect on expression in CTLL-1 cells, whereas they stimulate expression six- to eightfold in T-lymphoma cells. This suggests that the mature T-cell line lacks one or more factors present in T-lymphoma cells that function to augment transcription from the Moloney murine leukemia virus LTR. We also used recombinant viral LTRs to investigate the role of the enhancer core element of SL3-3 in CTLL-1 and CTLL-2 cells. A one-base-pair difference between the core sequences of SL3-3 and nonleukemogenic Akv virus, which is important for SL3-3 activity in T-lymphoma cells, had no effect in these cells. The inability to distinguish the single-base-pair difference in expression assays was correlated with the absence of binding of a cellular factor, S-CBF, to the SL3-3 enhancer core in extracts of CTLL-1 and CTLL-2 nuclei. These studies may have implications for identification of the target cells for viral leukemogenesis, as well as for tracing of changes in the transcriptional machinery during T-lymphocyte differentiation.

Cell Line↗
PubMed1992

Bovine immunodeficiency-like virus encodes factors which trans activate the long terminal repeat.

Lentiviruses are known to encode factors which trans activate expression from the viral long terminal repeat (LTR); the primary trans activator is the tat gene product. One of the putative accessory genes (tat) of the bovine immunodeficiency-like virus (BIV) bears sequence similarity to other lentivirus tat genes. This finding suggests that BIV may encode a trans-activating protein capable of stimulating LTR-directed gene expression. To test this hypothesis in vitro, BIV LTR-chloramphenicol acetyltransferase (CAT) reporter gene plasmids were constructed and transfected into three cell lines established from canine, bovine, or lapine tissues that are susceptible to BIV infection. The level of BIV LTR-directed CAT gene expression was significantly elevated in BIV-infected cells compared with uninfected cells. The relatively high basal-level expression of BIV LTR-CAT in uninfected canine and bovine cell lines suggests that cellular factors play a role in regulating BIV LTR-directed gene expression. Additionally, by using a clonal canine cell line in which the BIV LTR-CAT plasmid is stably expressed, BIV LTR-directed CAT expression is elevated 15- to 80-fold by cocultivation with BIV-infected cells, supporting the notion that BIV encodes a trans activator. The relative specificity of this viral activation was assessed by coculturing the clonal BIV LTR-CAT cell line with bovine leukemia virus- or bovine syncytial virus-infected cells; these bovine retroviruses increased expression from the BIV LTR only two- to threefold. Thus, BIV LTR regulatory elements in infected cells, like those of human immunodeficiency virus type 1 and other lentiviruses, are trans activated, presumably through the action of a Tat-like protein and cellular factors.

Animals↗
PubMed1993

Mutation of the C/EBP binding sites in the Rous sarcoma virus long terminal repeat and gag enhancers.

Several C/EBP binding sites within the Rous sarcoma virus (RSV) long terminal repeat (LTR) and gag enhancers were mutated, and the effect of these mutations on viral gene expression was assessed. Minimal site-specific mutations in each of three adjacent C/EBP binding sites in the LTR reduced steady-state viral RNA levels. Double mutation of the two 5' proximal LTR binding sites resulted in production of 30% of wild-type levels of virus. DNase I footprinting analysis of mutant DNAs indicated that the mutations blocked C/EBP binding at the affected sites. Additional C/EBP binding sites were identified upstream of the 3' LTR and within the 5' end of the LTRs. Point mutations in the RSV gag intragenic enhancer region, which blocked binding of C/EBP at two of three adjacent C/EBP sites, also reduced virus production significantly. Nuclear extracts prepared from both chicken embryo fibroblasts (CEFs) and chicken muscle contained proteins binding to the same RSV DNA sites as did C/EBP, and mutations that prevented C/EBP binding also blocked binding of these chicken proteins. It appears that CEFs and chicken muscle contain distinct proteins binding to these RSV DNA sites; the CEF binding protein was heat stable, as is C/EBP, while the chicken muscle protein was heat sensitive.

Animals↗
PubMed1997

Structure and function of the long terminal repeats of feline leukemia viruses derived from naturally occurring acute myeloid leukemias in cats.

Long terminal repeats of feline leukemia viruses cloned from feline acute myeloid leukemias frequently contained direct repeats of 40 to 74 bp in the upstream region of the enhancer (URE). The repetitive URE conferred an enhancer function upon gene expression in myeloid cells, suggesting its association with tumorigenic potential in myeloid cells.

Acute Disease↗
PubMed1993

Research note: a rapid method for the detection of the Rous-associated endogenous solitary long terminal repeat, ev15.

A rapid method has been developed for the detection of the solitary long terminal repeat ev15, a member of the avian leukosis virus (ALV) family of endogenous viral elements (ev genes) in chickens. Detection is accomplished by a polymerase chain reaction (PCR) assay that can be performed on purified genomic DNA samples or crude preparations of partially purified whole blood lysates. The test discriminates unambiguously between birds that are homozygous ev15-, homozygous ev15+, or heterozygous ev15-/ev15+. The incorporation of a modified touchdown amplification profile significantly improved the specificity of the PCR assay. Small-scale screening of birds from a variety of chicken breeds has revealed that ev15 is present in populations of both egg-strain birds and broilers.

Animals↗
PubMed1982

Nucleotide sequences of the murine retrovirus Friend SFFVp long terminal repeats: identification of a structure with extensive dyad symmetry 5' to the TATA box.

The two long terminal repeats (LTRs) of the integrated provirus of the polycythemia strain of the Friend spleen focus forming virus have been sequenced. Each of the identical LTRs is 514 nucleotides long and together confer on the provirus the features of a transposable element. They are terminated by perfect 11 base-pair inverted repeats, and the entire provirus is flanked by an apparent duplication of host DNA four nucleotides long. The assumed transcription regulatory sequences (Hogness-Goldberg box, CAAT box, polyadenylation signal) can be identified within the LTRs, as well as a region of an imperfect inverted repeat which extends from approximately 140 to 270 nucleotides 5' from the point of transcription initiation. This sequence may form a hairpin-type structure which might have some function in the promotion of transcription.

Animals↗
PubMed1987

Significance of DNase I-hypersensitive sites in the long terminal repeats of a Moloney murine leukemia virus vector.

A Moloney murine leukemia virus-derived retroviral vector (N4) carrying the bacterial neomycin resistance gene (neo) was used to study the chromatin configuration of integrated proviral DNA in NIH 3T3-derived cell lines containing one copy of the vector DNA per cell. Three independently obtained cell lines were examined. In two of these cell lines, the vector was introduced by viral infection, while in the third the construct was introduced by DNA transfection. Such transfected cell lines (including the one examined) usually express 10- to 50-fold less virus-specific RNA than do cell lines obtained by viral infection. All three cell lines exhibited similar patterns of DNase I-hypersensitive (HS) sites. Two strong DNase I HS sites were detected in the 5' long terminal repeat, which contains signals required for proper and efficient initiation of viral transcription. One of these sites was found to overlap the viral enhancer sequences, while the other site mapped very close to the start site for viral transcription. A third HS site was detected in nearby internal viral sequences. Only one HS site was found in the 3' long terminal repeat, which contains the signal(s) required for proper addition of a poly(A) tail to viral transcripts. This HS site was located in the region of the viral enhancer. Several weak DNase I HS sites were also found in the cellular sequences adjacent to the integration sites, at different locations in each cell line analyzed. No common pattern of cellular DNase I HS sites was found. These observations suggest that the 5' and 3' long terminal repeats of integrated retroviral proviruses exhibit different chromatin conformations, possibly reflecting the different functions encoded by the otherwise identical sequences, and the DNase I HS sites detected in these studies reflect only a potential for transcription and are not a reflection of the high transcriptional activity characteristic of retroviruses.

Animals↗
PubMed1993

Activation of the human immunodeficiency virus type 1 long terminal repeat by skin-sensitizing chemicals in transgenic mice.

Topical dinitrochlorobenzene (DNCB) is often used for evaluating contact skin hypersensitivity in immunocompromised patients. We have determined, in this study, that topical application of DNCB alone, even without induction of contact skin hypersensitivity, was sufficient to observe activation of the human immunodeficiency virus promoter (long terminal repeat) in the skin of an HIV-1 long terminal repeat-luciferase transgenic mouse model. Such treatment might be contra-indicative in patients infected with the human immunodeficiency virus, because in earlier studies DNCB-exposed skin dendritic cells might migrate into draining lymph nodes which play an important role in AIDS pathogenesis.

Animals↗
PubMed1985

Construction of recombinants between molecular clones of murine retrovirus MCF 247 and Akv: determinant of an in vitro host range property that maps in the long terminal repeat.

The leukemogenic mink cell focus-forming (MCF) retroviruses such as MCF 247 have biological properties distinct from those of their ecotropic progenitors. Nucleotide sequences encoding portions of gp70, Prp15E, and the long terminal repeat differ between the two types of viruses. To investigate the role of each of these genetic elements in determining the biological properties of MCF viruses, we prepared infectious molecular clones of MCF 247 and generated a set of recombinants between these clones and a molecular clone of Akv, the ecotropic parent of MCF 247. Each molecular clone of MCF 247 was distinct. All the recombinants between Akv and MCF 247 yielded infectious virus upon transfection. Most interestingly, recombinants which contain the long terminal repeat of MCF 247 were found to have an in vitro host range property that has been correlated with high oncogenic activity and thymotropism of certain MCF isolates; namely, they plated with higher efficiency on SC-1 cells than on NFS mouse embryo cells. Nononcogenic MCF isolates showed a slight preference for NFS cells, whereas Akv virus plated with approximately equal efficiency on the two cell types.

Animals↗
PubMed1998

Inhibition of NF-kappaB and HIV-1 long terminal repeat transcriptional activation by inducible nitric oxide synthase 2 activity.

In the human lymphoblastoid T cell line JJhan-5.1, stably transfected with a human immunodeficiency virus-1 long terminal repeat luciferase vector, the level of luciferase activity is dependent on activation of the nuclear factor kappaB (NF-kappaB) transcription factor. Tumor necrosis factor-induced luciferase activity was not modified in JJhan-5.1 cells co-cultivated with murine adenocarcinoma EMT-6 cells but was strongly decreased when nitric oxide (NO) synthase 2 expression was induced in these cells. Two NO synthase inhibitors counteracted this inhibitory effect. Tumor necrosis factor-alpha binding to JJhan-5.1 cells was not modified after incubation with EMT-6 cells. Viability and protein synthesis in JJhan-5.1 cells were also unchanged. Induction of NF-kappaB DNA binding activity was inhibited when EMT-6 cells expressed NO synthase 2 activity. Aminoguanidine, which completely abolished nitrite production, prevented this inhibition. NF-kappaB activation was also strongly inhibited by S-nitrosoglutathione but was marginally affected by N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1, 2-ethylenediamine. Taken together, these results indicated that NO-related species, released by EMT-6 effector cells and probably different from NO itself, inhibited NF-kappaB activation in human lymphoblastoid target cells. Consequently, transcriptional activity of a long terminal repeat-driven luciferase gene construct was markedly diminished.

Animals↗