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PubMed2006

The varying microsporidian genome: existence of long-terminal repeat retrotransposon in domesticated silkworm parasite Nosema bombycis.

Microsporidia are a group of intracellular parasites with an extremely compact genome and there is no confirmed evidence that retroelements are parasitised in these organisms. Using the dataset of 200,000 genomic shotgun reads of the silkworm pebrine Nosema bombycis, we have identified the eight complete N. bombycis long-terminal repeat retrotransposon (Nbr) elements. All of the Nbr elements are Ty3/gypsy members and have close relationships to Saccharomycetes long-terminal repeat retrotransposons identified previously, providing further evidence of their relationship to fungi. To explore the effect of retrotransposons in microsporidian genome evolution, their distribution was characterised by comparisons between two N. bombycis contigs containing the Nbr elements with the completed genome of the human parasite Encephalitozoon cuniculi, which is closely related to N. bombycis. The Nbr elements locate between or beside syntenic blocks, which are often clustered with other transposable-like sequences, indicating that they are associated with genome size variation and syntenic discontinuities. The ratios of the number of non-synonymous substitutions per non-synonymous site to the number of synonymous substitutions per synonymous site of the open reading frames among members of each of the eight Nbr families were estimated, which reveal the purifying selection acted on the N. bombycis long-terminal repeat retrotransposons. These results strongly suggest that retrotransposons play a major role in reorganization of the microsporidian genome and they might be active. The present study presents an initial characterization of some transposable elements in the N. bombycis genome and provides some insight into the evolutionary mechanism of microsporidian genomes.

Amino Acid Sequence↗
PubMed1990

DNA-binding proteins that interact with the long terminal repeat of radiation leukemia virus.

We used the electrophoretic mobility shift assay to identify the interactions of nuclear proteins with the long terminal repeat of leukemogenic, thymotropic BL/VL3 radiation leukemia virus (RadLV). In the promoter region, we identified a CCAAT box-binding protein (CBP) that has the same binding characteristics as the CCAAT box-binding protein that binds to the Moloney murine sarcoma virus promoter and most likely represents the CP1 factor. In the upstream enhancer region unique to BL/VL3, we detected several sequence-specific complexes, one with T-lymphocyte extracts but not with fibroblast extracts. This U3 region, UEB, may be important for the T-cell specificity of BL/VL3 RadLV. In the enhancer, which has been uniquely rearranged in this virus, we identified three specific protein-binding sites. Two of them showed characteristics of the LVb and core binding sites previously described for other murine retroviruses. But one binding site, identified as Rad-1, is unique to BL/VL3 RadLV and was found downstream, only 1 nucleotide from the core sequence. Rad-1 has a corelike motif on the minus strand, and the factor that binds to it could be competed by a BL/VL3 core-containing fragment. Moreover, the protein-DNA contacts involve the typical three core Gs separated by one T. These results suggest that Rad-1 binds a factor identical or similar to the core-binding factor. Our data suggest that the LVb, core, and Rad-1 motifs may be sufficient for this enhancer, most likely in association with other U3 long terminal repeat sequences, to promote a high rate of transcription of BL/VL3 RadLV in its specific target cells (thymocytes).

Animals↗
PubMedDate not supplied

Response of human immunodeficiency virus long terminal repeat to growth factors and hormones.

We have employed a recombinant plasmid, pBHIV1, carrying the long terminal repeat (LTR) of the human immunodeficiency virus-1 (HIV-1) linked to the reporter chloramphenicol acetyl transferase (cat) gene and to the aminoglycoside phosphotransferase (aph) gene as a selectable marker. We have introduced pBHIV1 in rat 208F and human MRCSV40TGR fibroblasts and obtained stable geneticin resistant RFBHIV1-1 and SVTGHIV-1 transfectant cells respectively. Both RFBHIV1-1 and SVTGHIV1-1 cells express CAT activity from the HIV LTR promoter. The response to insulin, epidermal growth factor, hydrocortisone and dexamethasone was studied on the LTR regulated CAT activity in both cell lines. It was found that, at optimal concentrations, insulin, epidermal growth factor and hydrocortisone regulate positively the expression of CAT from the HIV LTR in rat RFBHIV1-1 but not in human SVTGHIV1-1 cells. On the other hand dexamethasone at 10(-5) M stimulated CAT activity in both types of cells.

Chloramphenicol O-Acetyltransferase↗
PubMed1996

The effects of 5-fluorouracil and doxorubicin on expression of human immunodeficiency virus type 1 long terminal repeat.

Previous work by many groups has documented induction of the human immunodeficiency virus (HIV) long terminal repeat (LTR) following exposure of cells to ultraviolet light and other DNA damaging agents. Our experiments set out to determine the relative activation or repression of the HIV-LTR in response to two classes of chemotherapeutic agents: Doxorubicin is a DNA damage-inducing agent, and 5-fluorouracil has an antimetabolic mode of action. Using HeLa cells stably transfected with a construct in which HIV-LTR drives expression of the chloramphenicol acetyl transferase reporter gene, we demonstrated an up to ten-fold induction following doxorubicin treatment at 24 h post-treatment. This induction was repressed by treatment with salicylic acid, suggesting a role for prostaglandin/cyclo-oxygenase pathways and/or NF-kappa B in the inductive response. Induction by 5-fluorouracil, in contrast, was more modest (two-fold at most) though it was consistently elevated over controls.

Antibiotics, Antineoplastic↗
PubMed1999

Activation of the human immunodeficiency virus-1 long terminal repeat by respiratory burst oxidants of neutrophils.

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) introduced in association with the luciferase reporter gene into Jurkat T cells was strongly activated by a combination of human neutrophils and phorbol myristate acetate (PMA). Activation was not observed when normal neutrophils were replaced by neutrophils which lack a respiratory burst, ie, from a patient with chronic granulomatous disease (CGD), was strongly inhibited by catalase, was potentiated by vanadate, was stimulated by relatively low concentrations of azide, and was inhibited by selective inhibitors of protein kinase C (PKC). The PMA affected activation in three ways: (1) by directly activating the LTR in Jurkat LTRluc; (2) by inducing a respiratory burst in neutrophils with the formation of H2O2; and (3) by increasing the sensitivity of Jurkat LTRluc to the activating effect of H2O2. When PMA was replaced by opsonized zymosan as the neutrophil stimulus, activation of the LTR was low unless azide was added. Activation in the presence of azide was not seen when CGD neutrophils were used or when catalase was added, suggesting that azide acts by inhibiting the degradation of H2O2. These findings indicate that activation of the HIV-1 LTR in Jurkat T cells can be induced by H2O2 released by neutrophils, particularly when PKC is concomitantly activated.

Gene Expression Regulation↗
PubMed1995

Sequence and repeat structure variants in the long terminal repeat of maedi-visna virus EV1.

Diversity in the LTR of maedi-visna virus strain EV1 has been examined by PCR-based gene amplification using DNA from infected cells both in vitro and in experimentally infected animals. In vitro, several variant structures were found in the U3 regions of the LTR which contained repeats of sequences including presumed AP-1 and AP-4 binding sites. Although these repeat variants formed a minor fraction of the LTRs present in the proviral population, they were neither produced nor lost at a significant rate when PCR was performed on cloned viral DNA and so were unlikely to be artefacts of the isolation procedure. When LTRs were isolated from two experimentally EV1 infected sheep, repeat variant structures were found to be present in efferent lymph by 14 days postinfection (p.i.) (although not seen at 9 days p.i.). They were also present at later times and in blood. Overall sequence diversity at 9 days p.i. was reduced compared both with the infecting virus and with later times of infection. When a number of the variant LTR structures were used to drive CAT reporter gene constructs in chondrocytes, all were found to be active, although consistent differences of up to fourfold in activity were seen. However, there is no evidence from these data for strong selective pressure operating on the LTR in vivo.

Animals↗
PubMed1992

Structure of DRE, a retrotransposable element which integrates with position specificity upstream of Dictyostelium discoideum tRNA genes.

Different Dictyostelium discoideum strains contain between 2 and 200 copies of a retrotransposable element termed DRE (Dictyostelium repetitive element). From the analysis of more than 50 elements, it can be concluded that DRE elements always occur 50 +/- 3 nucleotides upstream of tRNA genes. All analyzed clones contain DRE in a constant orientation relative to the tRNA gene, implying orientation specificity as well as position specificity. DRE contains two open reading frames which are flanked by nonidentical terminal repeats. Long terminal repeats (LTRs) are composed of three distinct modules, called A, B, and C. The tRNA gene-proximal LTR is characterized by one or multiple A modules followed by a single B module (AnB). With respect to the distal LTR, two different subforms of DRE have been isolated. The majority of isolated clones contains a distal LTR composed of a B module followed by a C module (BC), whereas the distal LTR of the other subform contains a consecutive array of a B module, a C module, a slightly altered A module, another B module, and another C module (BC.ABC). Full-length as well as smaller transcripts from DRE elements have been detected, but in comparison with the high copy number in D. discoideum strains derived from the wild-type strain NC4, transcription is rather poor.

Amino Acid Sequence↗
PubMed2006

Genetic analysis of the long terminal repeat (LTR) promoter region in HIV-1-infected individuals with different rates of disease progression.

The long terminal repeat (LTR) region of HIV-1 promotes and modulates proviral transcription. LTR genetic variability might influence viral replication and disease progression. Proviral LTR sequences from 32 HIV-1-infected individuals showing different rates of disease progression were examined. Non-progressors (NP, n = 11) were individuals with high and stable CD4 counts and persistently low or undetectable plasma HIV-RNA. Slow progressors (SP, n = 6) were subjects with minimal CD4 decays over time and low plasma HIV-RNA. Typical progressors (TP, n = 15) were individuals with chronic infection showing CD4 counts repeatedly below 500 cells/mul. The mutation frequency within distinct LTR functional regions involved in HIV-1 transcription were compared in these three groups of patients. No significant differences were observed in the mutation frequency in most LTR regulatory sites when comparing the three groups. However, changes in USF regulatory binding sites were more frequent in TP than in SP/NP, while changes in Sp1 binding sites were less common in the former. This is the first study examining the genetic variability of the HIV-1 LTR region in long-term non-progressors showing further divergent outcomes.

Disease Progression↗
PubMed1983

Nucleotide sequences of feline sarcoma virus long terminal repeats and 5' leaders show extensive homology to those of other mammalian retroviruses.

The nucleotide sequences of the Gardner-Arnstein feline sarcoma virus (FeSV) long terminal repeat and the adjacent leader sequences 5' to the viral gag gene were determined. These were compared with homologous portions of Synder-Theilen FeSV and with previously published sequences for Moloney murine sarcoma virus and simian sarcoma virus proviral DNA. More than 75% of the residues in the FeSV R and U5 regions were homologous to sequences within the same regions of the other viral long terminal repeats. Unexpectedly, alignment of the FeSV sequences with those of the Moloney murine sarcoma and simian sarcoma viruses showed similar extents of homology within U3. The homologous U3 regions included the inverted repeats, a single set of putative enhancer sequences, corresponding to a "72-base-pair" repeat, and sequences, including the CAT and TATA boxes, characteristic of eucaryotic promotors. The 5' leader sequences of both FeSV strains included a binding site for prolyl tRNA and a putative splice donor sequence. In addition, the FeSV leader contained a long open reading frame which was adjacent to and in phase with the ATG codon at the 5' end of the FeSV gag gene. The open reading frame could code for a signal peptide of about 7.4 kilodaltons. Our results support the concept that the virogenic portions of both FeSV and simian sarcoma virus were ancestrally derived from viruses of rodent origin, with conservation of regulatory sequences as well as the viral structural genes.

Base Sequence↗
PubMed1997

Activation of the HIV-1 long terminal repeat by nerve growth factor.

The brain is an important target for the human immunodeficiency virus type 1 (HIV-1). We show here that nerve growth factor (NGF), which induces neuronal differentiation and survival, causes a strong activation of the HIV-1 long terminal repeat by a Ras/Raf-dependent mechanism in PC12 cells. Mutation of the kappaB sequences contained whithin the long terminal repeat reduces NGF-mediated stimulation. NGF does not activate NF-kappaB in PC12 cells, but rather increases binding of other nuclear factors to the kappaB sequences. Furthermore, a nuclear receptor response element contributes to the stimulatory effect of NGF. The retinoids receptors have been identified as components of the nuclear binding to the nuclear receptor response element in NGF-treated PC12 cells. These results reveal the importance of neurotrophins and nuclear receptor signaling pathways as specific activators of HIV-1 gene expression in neural cells.

Animals↗
PubMed2000

HERV-K-T47D-Related long terminal repeats mediate polyadenylation of cellular transcripts.

The human genome harbors thousands of long terminal repeats (LTRs) that are derived from endogenous retroviruses and contain elements able to regulate the expression of neighboring cellular genes. We have investigated the ability of human endogenous retroviral (HERV)-K LTRs to provide transcriptional processing signals for nonviral sequences. Four chimeric cDNA clones isolated from a cDNA library derived from the human cell line T47D were found to be polyadenylated within an HERV-K-T47D-related LTR. Two transcripts containing an as yet unknown cellular sequence were probably derived from the same genomic locus but their 3' ends were processed at different positions of the LTR. Structural analysis of the polyadenylation site suggests RNA stem-loop structures similar to the HTLV-1 Rex responsive element that bring the two remote AAUAAA and GU-rich elements into the spatial juxtaposition necessary for correct 3' end processing. The cellular part of the third chimeric clone shows significant homology to an exon of the human tyrosine phosphatase 1 gene, although oriented in the antisense direction compared to the adjacent LTR. Furthermore, we found that the 3' untranslated region of the human transmembrane tyrosine kinase gene FLT4 is probably derived from a partial HERV-K-T47D LTR sequence. Taken together, our data suggest that LTRs of the HERV-K-T47D family display biological function by mediating polyadenylation of cellular sequences.

3' Untranslated Regions↗
PubMed1997

Long terminal repeat U3 length polymorphism of human foamy virus.

Size determination of the long terminal repeat (LTR) of an early (1985) and a more recent (1993) passage of wild-type human foamy virus (HFV) revealed that the virus has undergone substantial deletions in the U3 region upon replication in tissue culture. Two LTR deletion variants (HSRV1 and 2) have been characterized in the past and used to construct molecular clones which are replication competent in cell culture. We now report the molecular cloning, sequencing, and biological characterization of an HFV genome with full-length LTR (pHFV2). Sequence analysis revealed that the deletions in HSRV1 and 2 are nonrandom and probably occurred by misalignment during reverse transcription. The comparative analysis of HFV2 and the variant with the largest U3 deletion, HSRV2, revealed a differential ability to replicate in human cell cultures. While HSRV2 replicated faster in diploid human fibroblasts, cells which have been used extensively for amplification of HFV in the past, replication of HFV2 was faster in a lymphoblastoid cell line. Reporter gene assays indicated that the cell-type specific ability of the LTRs to respond to the viral transcriptional transactivator may be a likely, reason for the different growth properties of both viruses and for the occurrence of the HFV U3 deletions. In foamy virus-infected chimpanzees only the full-length type of LTR was observed; however, the HSRV1 deletion variant was detected as the dominating virus in an accidentally HFV-infected human.

Animals↗
PubMed1986

Nucleotide sequence analysis of the long terminal repeat of integrated caprine arthritis encephalitis virus.

The nucleotide sequence of the long terminal repeat (LTR) of caprine arthritis encephalitis virus (CAEV), a prototype lentivirus was determined. 6-bp directly repeated host cell sequences flank the 376-bp proviral LTRs. By comparison with other retroviral sequences, the CAEV LTR likely contains U3, R and U5 regions 207, 86 and 83 base-pairs in length, respectively. Sequences conforming to consensus transcriptional promoter sites were identified in the U3 region upstream of a potential transcription initiation site. A consensus polyadenylation signal is present 20 bases upstream of the putative R-U5 border and a potential poly(A) addition site. Sequence comparisons of the CAEV LTR with those of other retroviruses uncovered significant similarities with that of visna virus. No other global homologies with other retrovirus LTRs could be detected. CAEV utilizes a primer binding site complementary to lysine tRNA as does visna, AIDS associated retroviruses, and mouse mammary tumor virus. The putative primer for positive-strand DNA synthesis identified in the CAEV sequence is identical to that of visna virus and very similar to those of AIDS retroviruses and MMTV. In addition, a stretch that includes the TATA box of the CAEV LTR resembles closely the corresponding region in the AIDS retrovirus. These and other findings further strengthen the classification of AIDS retrovirus as a lentivirus.

Animals↗
PubMed1982

Retrovirus-related sequences in human DNA: detection and cloning of sequences which hybridize with the long terminal repeat of baboon endogenous virus.

Human DNA sequences which hybridized with the long terminal repeats (LTR) of baboon type C virus M7 were detected by non-stringent blot hybridization. About 7 to 10 discrete bands of the LTR-related sequences were commonly observed in the DNAs from four independent human cell lines after digestion with either Eco RI, Hind III or Bam HI. The amounts of these sequences were more abundant in tumor cell lines than in a non-malignant cell line. The human sequences related to the M7 LTR seemed to be located at relatively specific sites on the cell DNA. The human DNA clones which hybridized with M7 LTR were detected in the human DNA library described by Lawn et al. (Cell 15, 1157-1174, 1978), at a frequency of about 300 per haploid genome. Five clones were isolated which shared different extent of homology with M7 LTR and whose restriction maps were totally different one another. The DNA structures of two of them resembled the genome of retroviruses. These results suggest the presence of various types of the LTR-related sequences in human DNA: some of them might represent endogenous virus genomes of human cells.

Animals↗
PubMed1985

Generation of glucocorticoid-responsive Moloney murine leukemia virus by insertion of regulatory sequences from murine mammary tumor virus into the long terminal repeat.

The glucocorticoid-regulatory sequences from the murine mammary tumor virus long terminal repeat (MMTV LTR) were introduced into the LTR of Moloney murine leukemia virus (M-MuLV) by recombinant DNA techniques. The site of insertion was in the M-MuLV LTR U3 region at -150 base pairs with respect to the RNA cap site. Infectious M-MuLVs carrying the altered LTRs (Mo + MMTV M-MuLVs) were recovered by transfection of proviral clones into NIH-3T3 cells. The Mo + MMTV M-MuLVs were hormonally responsive in that infection was 3 logs more efficient when performed in the presence of dexamethasone, irrespective of the orientation of the inserted MMTV sequences. However, even in the presence of hormone, the Mo + MMTV M-MuLVs were less infectious than wild-type M-MuLV. In contrast to the large effect on infectivity, dexamethasone induced virus-specific RNA levels in chronically Mo + MMTV M-MuLV-infected cells only two- to fourfold. Fusion plasmids between the altered LTRs and the bacterial chloramphenicol acetyltransferase gene allowed the investigation of LTR promoter strength by the transient chloramphenicol acetyltransferase expression assay. The chloramphenicol acetyltransferase assays indicated that the insertion of MMTV sequences into the M-MuLV LTR reduced promoter activity in the absence of glucocorticoids but that promoter activity could be induced two- to fivefold by dexamethasone. The Mo + MMTV M-MuLVs were also tested for the possibility that viral DNA synthesis or integration during initial infection was enhanced by dexamethasone. However, no significant difference was detected between cultures infected in the presence or absence of hormone. The insertion of MMTV sequences into an M-MuLV LTR deleted of its enhancer sequences did not yield infectious virus or active promoters, even in the presence of dexamethasone.

DNA, Recombinant↗
PubMed1989

Role of SP1-binding domains in in vivo transcriptional regulation of the human immunodeficiency virus type 1 long terminal repeat.

Five regions of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) have been shown to be important in the transcriptional regulation of HIV in HeLa cells. These include the negative regulatory, enhancer, SP1, TATA, and TAR regions. Previous studies in which purified SP1 was used showed that the three SP1-binding sites in the HIV LTR were important in the in vitro transcription of this promoter. However, no studies to ascertain the role of each of these SP1-binding sites in basal and tat-induced transcriptional activation in vivo have been reported. To determine the role of SP1 sites in transcriptional regulation of the HIV LTR in vivo, these sites were subjected to oligonucleotide mutagenesis both individually and in groups. The constructs were tested by DNase I footprinting with both oligonucleotide affinity column-purified SP1 and partially purified HeLa extract and by chloramphenicol acetyltransferase assays in both the presence and absence of the tat gene. Mutagenesis of each SP1-binding site resulted in minimal changes in basal and tat-induced transcriptional activation. Mutations involving alterations of SP1 sites I and II, I and III, or II and III also resulted in minimal decreases in basal and tat-induced transcriptional activation. However, mutagenesis of all three SP1-binding sites resulted in a marked decrease in tat induction. The latter mutation also greatly decreased DNase I protection over the enhancer, TATA, and TAR regions when partially purified HeLa nuclear extract was used. Mutagenesis of the HIV LTR SP1 sites which converted them to consensus high-affinity SP1-binding sites with the sequence GGGGCGGGGC resulted in increased tat-induced gene expression compared with the wild-type HIV LTR template. These results suggest that SP1, through its interaction with other DNA-binding proteins, is critical for in vivo transcriptional regulation of HIV.

Base Sequence↗
PubMed1991

Transcriptional initiation and postinitiation effects of murine leukemia virus long terminal repeat R-region sequences.

Sequences within the R components of the long terminal repeats (LTRs) of several retroviruses are known to be involved at various steps in expression of the viral genomes. A series of experiments was performed to test whether sequences within the R regions of the murine leukemia viruses Akv and SL3-3 affect viral expression. By using plasmid clones of the viral LTRs linked to a reporter gene, deletion of the R region was found to decrease expression to variable extents in a series of mammalian cell lines, with the largest effects being detected in murine fibroblasts. R-region sequences from the human immunodeficiency virus type 1 LTR or a random sequence were unable to substitute for the murine leukemia virus sequences. Transcripts from the R-region-deleted templates were initiated at the proper site in the LTR, but their levels were decreased at least 10-fold. Nuclear run-on assays showed that the decrease caused by the R-region deletions was due, in part, to an effect on RNA polymerase loading, suggesting an effect on transcriptional initiation. The remainder of the activity was presumably due to a posttranscriptional effect. Analysis of the R-region sequences of murine leukemia viruses and related retroviruses led to the prediction of a conserved secondary structure in the transcribed RNA that might have a role in activity. We conclude that R-region sequences are of importance for the expression of a variety of retroviruses.

3T3 Cells↗
PubMed2006

Role of RNA polymerase III transcription factors in the selection of integration sites by the dictyostelium non-long terminal repeat retrotransposon TRE5-A.

In the compact Dictyostelium discoideum genome, non-long terminal repeat (non-LTR) retrotransposons known as TREs avoid accidental integration-mediated gene disruption by targeting the vicinity of tRNA genes. In this study we provide the first evidence that proteins of a non-LTR retrotransposon interact with a target-specific transcription factor to direct its integration. We applied an in vivo selection system that allows for the isolation of natural TRE5-A integrations into a known genomic location upstream of tRNA genes. TRE5-A frequently modified the integration site in a way characteristic of other non-LTR retrotransposons by adding nontemplated extra nucleotides and generating small and extended target site deletions. Mutations within the B-box promoter of the targeted tRNA genes interfered with both the in vitro binding of RNA polymerase III transcription factor TFIIIC and the ability of TRE5-A to target these genes. An isolated B box was sufficient to enhance TRE5-A integration in the absence of a surrounding tRNA gene. The RNA polymerase III-transcribed ribosomal 5S gene recruits TFIIIC in a B-box-independent manner, yet it was readily targeted by TRE5-A in our assay. These results suggest a direct role of an RNA polymerase III transcription factor in the targeting process.

Animals↗