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PubMed1997

Increased length of long terminal repeats inhibits Ty1 transposition and leads to the formation of tandem multimers.

The Ty1 retrotransposon of Saccharomyces cerevisiae is bounded by long-terminal repeats (LTRs). We have constructed a variety of Ty1 elements in which the LTR length has been increased from the normal length of 334 bp to > 2 kb. Although small insertions in the LTR have minimal effects on transposition frequency, larger insertions dramatically reduce it. Nevertheless, elements with long LTRs are incorporated into the genome at a low frequency. Most of these rare insertion events represent Ty1 tandem (head to tail) multimers.

DNA, Fungal↗

PubMed1993

Replication of type 1 human immunodeficiency viruses containing linker substitution mutations in the -201 to -130 region of the long terminal repeat.

In previous transfection analyses using the chloramphenicol acetyltransferase reporter gene system, we determined that linker substitution (LS) mutations between -201 and -130 (relative to the transcription start site) of the human immunodeficiency virus type 1 long terminal repeat (LTR) caused moderate decreases in LTR transcriptional activity in a T-cell line (S. L. Zeichner, J. Y. H. Kim, and J. C. Alwine, J. Virol. 65:2436-2444, 1991). In order to confirm the significance of this region in the context of viral replication, we constructed several of these LS mutations (-201 to -184, -183 to -166, -165 to -148, and -148 to -130) in proviruses and prepared viral stocks by cocultivation of transfected RD cells with CEMx174 cells. In addition, two mutations between -93 and -76 and between -75 and -58 were utilized, since they affect the nuclear factor kappa B (NF-kappa B)- and Sp1-binding sites and were expected to diminish viral replication. Our results suggest that while transfection analyses offer an adequate approximation of the effects of the LS mutations, the analysis of viral replication using a mutant viral stock presents a more accurate picture, which is sometimes at variance with the transfection results. Three mutants (-201/-184 NXS, -165/-148 NXS, and -147/-130 NXS) had effects on viral replication that were much more severe than the effects predicted from their performance in transfection analyses, and the effects of two LS mutations (-201/-184 NXS and -183/-166 NXS) were not predicted by their effects in transfection. In addition, we observed cell type-specific permissiveness to replication of some mutant viruses. In the cell types tested, the LS mutations indicated an apparent requirement not only for the intact NF-kappa B and SP1-binding sites but also for several regions between -201 and -130 not previously associated with viral infectivity.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Kinetics of HIV-1 long terminal repeat trans-activation. Use of intragenic ribozyme to assess rate-limiting steps.

We have examined, using self-cleaving ribozymes, the intracellular trans-activation kinetics of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) by viral protein Tat. Experiments were designed to effect a competition (during RNA chain elongation) between cleavage of a nascent RNA containing the Tat-responsive target sequence (TAR) and Tat interaction with the same TAR in the process of LTR-trans-activation. We found that fast self-cleavage of nascent TAR-containing RNA abolished Tat trans-activation. Slowing the cleavage reaction kinetically rescued trans-activation. Based on our results, we conclude that the rate-limiting step in HIV-1 LTR trans-activation is the initial contact made between Tat/TAR/LTR rather than the promoter proximal pausing of RNA polymerases that are tethered to functional TAR.

Base Sequence↗

PubMed1994

Phylogenetic relationship between human immunodeficiency virus type 1 (HIV-1) long terminal repeat natural variants present in the lymph node and peripheral blood of three HIV-1-infected individuals.

PCR has been used to amplify a 540 base pair fragment of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat region encompassing the U5/R/U3 regions from proviral HIV DNA that was present in the lymph nodes and peripheral blood mononuclear cells of three HIV-infected individuals. The resulting PCR products were cloned and the DNA sequence of multiple clones from each reaction was determined to enable the distribution and phylogenetic relationship between the variants in each body site to be assessed. The results of bootstrapped parsimony phylogenetic analyses showed that a distinct polarization was evident between peripheral blood and lymph node variants in the patient who possessed a histologically intact lymph node. In contrast, similar analyses conducted on samples from two patients with extensive lymph node disruption showed similar variants in lymph node and peripheral blood. In the patient with an intact lymph node, lymph node variants were significantly more heterogeneous than those present in blood samples taken either simultaneously or 11 months later. No differences in heterogeneity between lymph node and peripheral blood were observed in patients with disrupted lymph nodes. The significance of these results for our understanding of HIV pathogenesis is discussed.

Base Sequence↗

PubMed1993

Early replication steps but not cell type-specific signalling of the viral long terminal repeat determine HIV-1 monocytotropism.

The expression of human immunodeficiency virus type 1 (HIV-1) is enhanced after cell activation because of the interaction of cell-encoded nuclear factors that interact with binding sites in the long terminal repeats (LTRs). Here we studied the contribution of cell type-specific activation signals to differences in cytotropism of HIV-1 variants. Four closely related molecular HIV-1 clones with distinct biological phenotypes and different capacities to replicate in primary monocyte-derived macrophages (MDMs) or T cell lines were used. Sequence analysis of these LTRs revealed variation in functionally important regions. Adaptation of virus variants to particular host cells by differences in LTR responsiveness was analyzed. LTR-CAT constructs were transiently transfected in T cells that were stimulated with T cell-specific activation signals such as combinations of anti-CD3 or anti-CD28 MoAB or in primary monocytes that were stimulated with IL-3, IL-4, or GM-CSF. No differences in responsiveness to cell type-specific signals were demonstrated. To further elucidate the level of restriction in cell tropism, transfection of four full-length infectious molecular HIV-1 clones into 5-day cultured MDMs was performed. From all clones, competent virus could be rescued from MDMs by coculture with PHA-stimulated PBLs. However, following cell-free inoculation, proviral DNA could be detected by PCR analysis only in monocytes exposed to HIV-1 clones that previously were shown to establish productive infection.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1991

In vivo activation of human immunodeficiency virus type 1 long terminal repeat by UV type A (UV-A) light plus psoralen and UV-B light in the skin of transgenic mice.

UV irradiation has been shown to activate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in cell culture; however, only limited studies have been described in vivo. UV light has been categorized as UV-A (400 to 315 nm), -B (315 to 280 nm), or -C (less than 280 nm); the longer wavelengths are less harmful but more penetrative. Highly penetrative UV-A radiation constitutes the vast majority of UV sunlight reaching the earth's surface but is normally harmless. UV-B irradiation is more harmful but less prevalent than UV-A. In this report, the HIV-1 LTR-luciferase gene in the skin of transgenic mice was markedly activated when exposed to UV-B irradiation. The LTR in the skin of transgenic mice pretreated topically with a photosensitizing agent (psoralen) was also activated to similar levels when exposed to UV-A light. A 2-h exposure to sunlight activated the LTR in skin treated with psoralen, whereas the LTR in skin not treated with psoralen was activated after 7 h of sunlight exposure. The HIV-1 LTR-beta-galactosidase reporter gene was preferentially activated by UV-B irradiation in a small population of epidermal cells. The transgenic mouse models carrying HIV-1 LTR-luciferase and LTR-beta-galactosidase reporter genes have been used to demonstrate the in vivo UV-induced activation of the LTR and might be used to evaluate other environmental factors or pharmacologic substances that might potentially activate the HIV-1 LTR in vivo.

Animals↗

PubMed1983

Endogenous murine leukemia proviral long terminal repeats contain a unique 190-base-pair insert.

We have determined the nucleotide sequence in the U3-R regions of the long terminal repeat (LTR) associated with NFS-Th-1 xenotropic murine leukemia virus (MuLV) DNA and the LTR components of five endogenous proviruses cloned from BALB/c mouse chromosomal DNA. The five endogenous MuLV LTRs contained the regulatory signals thought to be important in viral transcription, such as "TATAA" and CCAAT-like boxes. A unique feature in four of the endogenous LTRs was the presence of a highly conserved 190-base-pair (bp) insert bounded by 6-bp direct repeats located 48 bp upstream from the C-C-A-A-T sequence. This segment was absent from LTRs associated with ecotropic, xenotropic, or mink cell focus-forming (MCF) MuLV proviruses. All five endogenous LTR segments also contained a 14-bp duplication of a sequence located near the 5' end of the first component of the long (greater than 72-bp) direct repeat of ecotropic and MCF MuLV LTRs. An evolutionary scheme relating LTRs associated with endogenous MuLV proviral DNAs to those found in ecotropic or xenotropic proviruses is presented. Nucleotide sequence analysis also suggested that the U3 region of the MCF247 MuLV LTR is derived from an NFS xenotropic related MuLV.

Animals↗

PubMed1991

Evolution of human immunodeficiency virus type 1 nef and long terminal repeat sequences over 4 years in vivo and in vitro.

The evolution of an 851-bp segment of the human immunodeficiency virus type 1 (HIV-1) genome encoding the nef open reading frame and U3/R elements of the long terminal repeat has been followed over a 4-year period in vivo and in vitro. The population of viral sequences at any given time was established by sequencing cloned polymerase chain reaction products. The samples studied were derived from the same man for whom a detailed analysis of the tat gene was previously described (A. Meyerhans, R. Cheynier, J. Albert, M. Seth, S. Kwok, J. Sninsky, L. Morfeldt-Manson, B. Asjö, and S. Wain-Hobson, Cell 58:901-910, 1989). Once again in vitro culture resulted in the selection of minor forms. Over a 4-year period in vivo, there was no obvious selection for, or outgrowth of, any particular nef or U3/R sequence. Few defective nef protein sequences were observed, which argues against nef acting as a negative regulatory factor. Although no functionally defective promoter/trans-activation-responsive elements were identified, the transactivation efficiencies varied between 0.2 and 2 times that of the control. The sequence encoding the most efficient trans-activation-responsive region did not outgrow others. The extreme genetic heterogeneity of the different samples of the locus, either in vivo or in vitro, indicates that there is no such thing as a single, distinct HIV sequence. It is suggested that different HIV-1 loci evolve independently, recombination being responsible for their uncoupling.

Base Sequence↗

PubMed2005

Decay of human immunodeficiency virus type 1 unintegrated DNA containing two long terminal repeats in infected individuals after 3 to 8 years of sustained control of viremia.

Covert human immunodeficiency virus (HIV) replication was ongoing during the first 3 years of aviremia in 22 patients, as determined by detection of DNA containing two long terminal repeats (2 LTR DNA). Although total HIV DNA was detected in 60 2 LTR DNA-negative samples, the absence of 2 LTR DNA in 90% of patients following 7 to 8 years of highly active antiretroviral therapy suggests suppression of cryptic viral replication.

Antiretroviral Therapy, Highly Active↗

PubMed1993

Sequence similarity between the long terminal repeat coding regions of mammary-tumorigenic BALB/cV and renal-tumorigenic C3H-K strains of mouse mammary tumor virus.

The long terminal repeat (LTR) of mouse mammary tumor virus (MMTV) encodes a protein which functions as a superantigen. The BALB/cV strain differs from other exogenous MMTVs antigenically, biochemically, on the basis of restriction fragment analysis, and by the specificity of its superantigen for V beta 2+ T cells. In order to elucidate the origin of the BALB/cV virus and to better understand the interaction of its superantigen with the T cell receptor, we have determined the nucleotide sequence of the BALB/cV LTR open reading frame, including 93 bases downstream of the translation termination site. The encoded protein's C-terminal portion, thought to control superantigenic specificity, is identical to the C3H-K strain of MMTV, isolated from a rare kidney adenocarcinoma. The remainder of the coding sequence is highly related to many MMTV strains. Like other MMTV strains, the BALB/cV LTR maintains intact an 18 base pair sequence, located downstream of the translational termination site, which is lacking in the C3H-K LTR. Sequence comparison between the BALB/cV LTR and other MMTV strains indicates that the most likely origin for the BALB/cV open reading frame sequence is a recombination event involving the endogenous provirus mtv-6.

Adenocarcinoma↗

PubMed1992

A human endogenous long terminal repeat provides a polyadenylation signal to a novel, alternatively spliced transcript in normal placenta.

We have been investigating the impact that the long terminal repeats (LTRs) of the RTVL-H family of human endogenous retroviral-like elements may have on the expression of adjacent cellular genes. Using a differential hybridization strategy, we have screened a cDNA library from a normal full-term human placenta and have identified two clones containing non-RTVL-H-related cellular sequences that have been polyadenylated within an RTVL-H LTR. One of these clones, cPj-LTR, contains an open reading frame (ORF) of 223 amino acids. Southern analysis indicated that the corresponding gene, termed PLT, is most probably a single multi-exon locus and that related sequences are present in the mouse genome, suggesting that this gene has been evolutionarily conserved. Database searches detected no significant homology to previously published sequences, indicating that PLT is a novel gene. Northern analysis identified several PLT-related transcripts in placental RNA samples, one of which is associated with the LTR. The presence of this PLT-LTR fusion transcript in normal placenta was also confirmed by PCR. Additional hybridization studies with RNAs from various cell lines suggested that the PLT locus is differentially expressed in different cell types. To investigate the structure of the non-LTR-associated PLT-related transcripts, additional clones were isolated from the placental cDNA library. Analysis of these clones suggests that the PLT mRNA undergoes alternative splicing at its 3' end, with polyadenylation within an RTVL-H LTR occurring in one of the resulting transcripts.

Alternative Splicing↗

PubMedDate not supplied

[The tissue-specific methylation of human-specific endogenous retroviral long terminal repeats].

A possible involvement of retroelements in the epigenetic regulation of human gene expression was considered by the example of methylation of long terminal repeats (LTRs) of the human endogenous retrovirus family K (HERV-K). The methylation status of six HERV-K LTRs was determined in various gene-enriched regions of the human genome. The methylation of four LTRs was shown to be tissue-specific. Our results correlated with published data on the tissue-specific changes in the expression level of human genes adjacent to the LTRs under study. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 5; see also http: // www.maik.ru.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1988

Heat-shock induction of the human immunodeficiency virus long terminal repeat.

Rat cell lines were established in which the bacterial chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus (HIV) long terminal repeat (LTR) was stably integrated. The cell lines showed a repressed phenotype for CAT expression, but could be induced for it by inhibition of protein synthesis, as well as by heat-shock and chemical inducers of the cellular stress response, such as sodium arsenite, 8-hydroxyquinoline and the heavy metals cadmium and copper. A decameric sequence present in the NF-kB binding sites in the HIV LTR (GGGACTTTCC) resembles the cellular heat-shock core sequence and may therefore be involved in the heat-shock response.

Animals↗

PubMed1991

Comparison of the transcriptional activity of the long terminal repeats of simian immunodeficiency viruses SIVmac251 and SIVmac239 in T-cell lines and macrophage cell lines.

The U3 regions of the long terminal repeats (LTRs) of simian immunodeficiency viruses SIVmac251 and SIVmac239 were analyzed for basal transcriptional activity and for interaction with cellular factors in the T-cell line HUT-78 and the monocyte/macrophage cell line U937. A number of 5' deletions and mutations were made in the U3 regions of the two LTRs, and these constructs were placed upstream of a plasmid containing the bacterial chloramphenicol acetyltransferase reporter gene. The nucleotide sequences between -225 and +18 were sufficient to maintain full transcriptional activity of both LTRs in HUT-78 and U937 cells. Nucleotide sequence analysis revealed several differences between SIVmac251 and SIVmac239 within this region. Analysis of deletion mutants revealed that an additional removal of bases, from -124 to -225, had little effect on the transcriptional activity of the clone 239 LTR, whereas this deletion resulted in a significant reduction of activity in the clone 251 LTR. DNase protection assays using nuclear extracts from HUT-78 and U937 cells showed that bases within this region bound cellular factors. In addition, the NF-kappa B site was protected in DNase assays with HUT-78 cells and 12-O-tetradecanoylphorbol-13-acetate-treated U937 cells. An additional DNase footprint was detected in SIVmac239, at -52 to -38, just upstream of the TATA box. This site overlaps the 3' half of the 3'-most Sp-1 site and is downstream of 11 bases that are found in SIVmac239 but not SIVmac251. Thus, differences in the sequences in the U3 region of the LTRs of SIVmac251 and SIVmac239 have been identified which appear to alter the transcriptional activity of these promoters as well as changing the interaction of cellular proteins with sequences in the LTRs.

Base Sequence↗

PubMed1993

A protein-binding site with dyad symmetry in the long terminal repeat of the MCF13 murine leukemia virus that contributes to transcriptional activity in T lymphocytes.

We have previously identified regions in the long terminal repeat (LTR) of the MCF13 murine leukemia virus (MLV) that contribute to transcriptional activity in different cell types. We have observed that enhancer sequences and a region that resides 3' of the enhancer make significant contributions to transcriptional activity in T lymphocytes (T. Hollon and F. K. Yoshimura, J. Virol. 63:3353-3361, 1989). In this report, we have focused on the region of the MCF13 LTR that is 3' of the enhancer to identify binding sites for proteins that may play a role in the regulation of transcription in T cells. By gel shift and DNA footprint analyses, we have identified a single protein-binding site (MLPal) that includes a nucleotide sequence with dyad symmetry. A synthetic double-stranded oligonucleotide corresponding to this protein-binding site formed a specific protein-DNA complex. Deletion of this protein-binding site from the wild-type LTR decreased transcriptional activity in T lymphocytes but not in fibroblasts as determined by a transient expression assay. The MLPal sequence by itself cannot augment transcription in T cells but is able to do so in conjunction with enhancer sequences.

Animals↗

PubMed2002

The solitary long terminal repeats of ERV-9 endogenous retrovirus are conserved during primate evolution and possess enhancer activities in embryonic and hematopoietic cells.

The solitary long terminal repeats (LTRs) of ERV-9 endogenous retrovirus contain the U3, R, and U5 regions but no internal viral genes. They are middle repetitive DNAs present at 2,000 to 4,000 copies in primate genomes. Sequence analyses of the 5" boundary area of the erythroid beta-globin locus control region (beta-LCR) and the intron of the embryonic axin gene show that a solitary ERV-9 LTR has been stably integrated in the respective loci for at least 15 million years in the higher primates from orangutan to human. Functional studies utilizing the green fluorescent protein (GFP) gene as the reporter in transfection experiments show that the U3 region of the LTRs possesses strong enhancer activity in embryonic cells of widely different tissue origins and in adult cells of blood lineages. In both the genomic LTRs of embryonic placental cells and erythroid K562 cells and transfected LTRs of recombinant GFP plasmids in K562 cells, the U3 enhancer activates synthesis of RNAs that are initiated from a specific site 25 bases downstream of the AATAAA (TATA) motif in the U3 promoter. A second AATAAA motif in the R region does not serve as the TATA box or as the polyadenylation signal. The LTR-initiated RNAs extend through the R and U5 regions into the downstream genomic DNA. The results suggest that the ERV-9 LTR-initiated transcription process may modulate transcription of the associated gene loci in embryonic and hematopoietic cells.

Animals↗

PubMed1994

Comparative functional analysis of the various lentivirus long terminal repeats in human colon carcinoma cell line (SW480 cells) and feline renal cell line (CRFK cells).

Basal promoter activities of various lentiviral long terminal repeats (LTRs) in a human colon carcinoma cell line (SW480 cells) and a feline renal cell line (CRFK cells) were examined by the chloramphenicol acetyltransferase (CAT) assay using the LTR-CAT reporter plasmids. In SW480 cells, the basal promoter activities induced by LTRs of visna virus, caprine arthritis-encephalitis virus (CAEV), and simian immunodeficiency virus (SIVAGM) were moderate, and those induced by LTRs of human immunodeficiency virus (HIV) type 1 (HIV-1) and HIV type 2 (HIV-2) were low. However, the activity induced by the LTR of feline immunodeficiency virus (FIV) was extremely low. In CRFK cells, the basal promoter activities induced by LTRs of visna virus, CAEV and SIVAGM were relatively high, and those induced by LTRs of HIV-1, HIV-2 and FIV were moderate. From these data, although the structure of the LTR of FIV is reported to be similar to that of visna virus and CAEV, the function of the LTR of FIV is rather quite different from that of the LTR of these viruses.

Animals↗

PubMed2003

Evolutionary course of CsRn1 long-terminal-repeat retrotransposon and its heterogeneous integrations into the genome of the liver fluke, Clonorchis sinensis.

The evolutionary course of the CsRn1 long-terminal-repeat (LTR) retrotransposon was predicted by conducting a phylogenetic analysis with its paralog LTR sequences. Based on the clustering patterns in the phylogenetic tree, multiple CsRn1 copies could be grouped into four subsets, which were shown to have different integration times. Their differential sequence divergences and heterogeneous integration patterns strongly suggested that these subsets appeared sequentially in the genome of C. sinensis. Members of recently expanding subset showed the lowest level of divergence in their LTR and reverse transcriptase gene sequences. They were also shown to be highly polymorphic among individual genomes of the trematode. The CsRn1 element exhibited a preference for repetitive, agenic chromosomal regions in terms of selecting integration targets. Our results suggested that CsRn1 might induce a considerable degree of intergenomic variation and, thereby, have influenced the evolution of the C. sinensis genome.

Animals↗