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PubMed1990

Differences in replication and cytopathogenicity of human immunodeficiency virus type 1 (HIV-1) are not determined by long terminal repeats (LTR).

The growth properties of molecular clones of a highly cytopathic Zairian HIV1-NDK and prototype viruses were compared to correlate genetic variations with biological changes. The cloned HIV1-NDK retained the highly replicating cytopathic phenotype and formed larger syncytia than the prototype. One of the major differences in the alignment of the nucleotide sequence of the HIV1-NDK and HIV1-BRU prototypes was localized in the negative regulatory element (NRE) of the long terminal repeat (LTR). In a chloramphenicol acetyl transferase (CAT) assay, we failed to detect a significant difference between LTR promoter activity of the prototype and HIV1-NDK, suggesting that the LTR of both phenotypes had a similar function. The complete recombinant provirus DNA molecules bearing HIV1 LTR derived from one phenotype and the rest of the genomes from the other phenotype were constructed and transfected. The high cytopathogenicity of both the original and the chimeric viruses was correlated with the high speed of virus replication. Cytopathogenicity, morphology of syncytia, and replication kinetics of the recombinant viruses were determined by the functions coded within an internal part of HIV1 genome, covering the gag to env region, which were, however, not within LTR.

Animals↗
PubMed2001

Identification of a novel retrovirus long terminal repeat (LTR) that is targeted by p51A (TAp63gamma) and selective dominant-negative activity of p73L (DeltaNp63alpha) toward p53-responsive promoter activities.

The p51/p73L/p63/p40 gene, recently identified as a p53 homolog, encodes two major isoforms, p51A and p73L, which are suggested to have similar functions synonymous with p53 and dominant-negative activity toward both p53 and p51A, respectively. We have cloned a high affinity genomic fragment bound to p51A that was assigned to be a novel retrovirus long terminal repeat. Strikingly, this fragment was found to bind to both p53 and p73L with similar affinity to p51A. Additional demonstration with known p53 response elements suggested that DNA-binding profiles of p51A and p73L were very similar but were distinct from that of p53. Consistent with this novel finding, transient cotransfection experiments in mammalian cells suggested that p73L, when it was expressed at a low level, selectively suppressed p53-dependent transactivation of p21-luc and mdm2-luc but not of cyclinG-luc and bax-luc reporters. These data raise the possibility that p73L differentially modulates the p53 function according to the distinct DNA-binding affinity between these two proteins.

Animals↗
PubMed1982

Mouse EcoRI satellite DNA contains a sequence homologous to the long terminal repeat of the intracisternal A particle gene.

EcoRI restriction endonuclease fragments from the mouse 1,350-base-pair EcoRI satellite sequence were cloned in pBR322 plasmid. One of these fragments, SAT-1, was hybridized to various restriction fragments from cloned mouse intracisternal type A particle (IAP) genes and to mouse total genomic DNA. Hybridization occurred between the SAT-1 sequence and each of the IAP clones. The common region of hybridization in each of the clones was the long terminal repeat region of the IAP genes.

Animals↗
PubMed1987

Transcription from a spleen necrosis virus 5' long terminal repeat is suppressed in mouse cells.

To determine the block(s) to spleen necrosis virus (SNV) replication in mouse cells, we studied the expression of a dominant selectable marker, neo, or a gene whose product is easily assayed, the chloramphenicol acetyltransferase (cat) gene, in SNV-derived and murine leukemia virus-derived vectors. Using transient (CAT) and stable (Neor phenotype) transfection assays, we showed that the SNV promoter was used in mouse cells only when the 3' SNV long terminal repeat (LTR) was absent. Infection of mouse cells with recombinant SNV viruses was 1% as efficient as infection of permissive dog (D17) cells. The SNV proviruses in mouse cells appeared normal by Southern blot analysis, indicating that their integration probably occurred by normal mechanisms. S1 nuclease analyses of Neor mouse cell clones, each harboring a single recombinant SNV provirus, showed that the selected (internal) promoter was active, but that the 5' SNV LTR promoter was not. However, in the rare (less than 10(-6)) Neor colonies in which expression of the 5' LTR was selected, both promoters were active. Thus, the block to SNV infection of mouse cells is at least at two levels; one is a 100-fold-decreased efficiency at some step(s) up to and including integration, and the other is at transcription.

Animals↗
PubMed1986

Sequences near the 5' long terminal repeat of avian leukosis viruses determine the ability to induce osteopetrosis.

Avian leukosis virus (ALV)-induced osteopetrosis is associated with the accumulation of unintegrated viral DNA in osteoblasts. Viruses constructed from the DNAs of an osteopetrosis-inducing ALV (Br21) and a non-osteopetrosis-inducing ALV (RAV-0) have been used to test for the role of viral genes in the induction of osteopetrosis. Our results map osteopetrotic potential to a 1,400-base-pair region near the 5' long terminal repeat. This region contains signals for the splicing, translation, and packaging of viral RNAs and coding sequences for the gag proteins p19 and p10 and the N terminus of p27.

Animals↗
PubMed1999

Germ cell expression of an isolated human endogenous retroviral long terminal repeat of the HERV-K/HTDV family in transgenic mice.

In contrast to most other human endogenous retroviral families, various HERV-K members have open reading frames that code for functional viral proteins which can form noninfectious particles in some germ cell tumors. The HERV-K viral genes are highly transcribed in germ cell tumors but are transcribed to lower or undetectable levels in most other tissue and tumor types. To further analyze the expression patterns of these proviruses, long terminal repeats (LTRs) were isolated from the human genome and used in reporter gene assays. Expression of some HERV-K LTRs was found to be high in human and murine germ cell tumors (testicular teratocarcinomas) and low in non-germ-cell tumors. Furthermore, upon differentiation of a teratocarcinoma cell line, the expression of an active LTR dropped dramatically, suggesting developmental regulation of these proviral LTRs. Transgenic mice harboring an active LTR driving lacZ expression were generated and analyzed. Adult mouse testes showed the highest levels of expression, and the transgene staining appeared to be restricted primarily to the more undifferentiated spermatocytes. Most other tissues analyzed revealed very low or undetectable levels of expression both by reverse transcription-PCR and by Northern blot analysis. Whether the restricted expression of HERV-K in germ cells and in germ cell-derived tumors is of significant importance during development or tumorigenesis remains to be elucidated. Germ line expression of these viruses would allow for their expansion and movement, while somatic repression would ensure limited insertional mutagenesis and misexpression in an individual.

Animals↗
PubMed1999

Long terminal repeat sequences of equine infectious anaemia virus are a major determinant of cell tropism.

The Wyoming strain of equine infectious anaemia virus (EIAV) is a highly virulent field strain that replicates to high titre in vitro only in primary equine monocyte-derived macrophages. In contrast, Wyoming-derived fibroblast-adapted EIAV strains (Malmquist virus) replicate in primary foetal equine kidney and equine dermis cells as well as in the cell lines FEA and Cf2Th. Wyoming and Malmquist viruses differ extensively both in long terminal repeat (LTR) and envelope region sequences. We have compared the promoter activities of the Wyoming LTR with those of LTRs derived from fibroblast-adapted viruses by examining their abilities to drive a luciferase reporter gene as well as by construction of infectious molecular clones differing only in LTR sequence. Our results indicate that LTR sequences are a major restriction for growth of the Wyoming strain of EIAV in fibroblasts.

Animals↗
PubMed2001

The rapamycin sensitivity of human T-cell leukaemia virus type I-induced T-cell proliferation is mediated independently of the polypyrimidine motifs in the 5' long terminal repeat.

The immunosuppressant rapamycin can regulate the translation of a subset of messenger RNAs, a phenotype which has been linked to the presence of a polypyrimidine motif [C(N)(4-14)] downstream of the mRNA cap structure. T-cell clones naturally infected with transcriptionally active human T-cell leukaemia virus, type I (HTLV-I) undergo autologous proliferation; this phenotype is inhibited by rapamycin but not FK506, which reverses the rapamycin effect. Within the R region of the HTLV-I 5' long terminal repeat (LTR) there are seven polypyrimidine motifs. We sought to determine if these were involved in the sensitivity of proliferation to the presence of rapamycin. Here we illustrate the generation of an in vitro model of this rapamycin-sensitivity and the analysis of LTR mutants which were created to determine the importance of the polypyrimidine motifs. Reporter gene assays suggest the effect is independent of the polypyrimidine motifs in the virus leader sequence.

Animals↗
PubMed2004

A mouse mammary tumor virus-like long terminal repeat superantigen in human breast cancer.

We previously reported a 660-bp mouse mammary tumor virus (MMTV)-like env gene sequence in approximately 38% of human breast cancer DNA, but not in normal breasts or other tumors. This MMTV-like env gene sequence was expressed in 66% of the env gene-positive human breast cancers. An entire proviral structure was identified in human breast cancer DNA with high homology to MMTV and low homology to known human endogenous retrovirus. MMTV-like long terminal repeat (LTR) sequences were also detected in 41.5% of human breast cancers. They contain hormone-responsive elements, TEF-1 family elements, and the open reading frame for the superantigen (SAg). We have now amplified and sequenced MMTV-like sag sequences from 10 human breast cancers, and we found that they are highly homologous to those of MMTV. However, deletions and insertions at the COOH-terminal of sag were observed. The immune function of the human MMTV-like LTR SAg was also investigated. The sag gene was cloned and expressed in a human B-cell line (Ramos). T-cell proliferation and cytokine releasing assays were performed after cocultivation of T cells with irradiated Ramos SAg-expressing cells. The results indicate that expression of the human SAg stimulates T-cell activation in vitro, as the mouse SAg does. Because the T-cell responses in vitro are considered similar to those in vivo, these results suggest that the human LTR SAg might also play a role in human breast carcinogenesis.

Amino Acid Sequence↗
PubMed1989

Signaling through T lymphocyte surface proteins, TCR/CD3 and CD28, activates the HIV-1 long terminal repeat.

The state of T cell activation and proliferation controls HIV-1 replication and gene expression. Previously, we demonstrated that the administration of PHA and PMA to the human T cell line Jurkat activates the HIV-1 enhancer, which is composed of two nuclear factor kappa B (NF kappa B) binding sites. Here, we show that PMA alone is sufficient for this effect. In addition, activation of T cells through the surface proteins TCR/CD3 and CD28 increased gene expression directed by the HIV-1 long terminal repeat (LTR) to the same extent as PMA. Analysis of 5' deletions in the LTR revealed that the NF kappa B binding sites and sequences in the upstream U3 region are required for this response. Whereas cyclosporin A did not inhibit the effect of PMA, it reduced the effects of agonists to TCR/CD3 and CD28 on the LTR. H7, an inhibitor of protein kinase C (PKC), blocked the effects of all stimuli. Thus, PMA activates the NF kappa B sites through a PKC-dependent pathway while ligands to TCR/CD3 and CD28 activate the LTR through a cyclosporin A-sensitive, PKC-dependent pathway of T cell activation. We conclude that mechanisms involved in the expression of IL-2 and the alpha-chain of the IL-2R alpha genes also play a role in the regulation of HIV-1. Physiologic stimuli can activate HIV-1 gene expression; agents that block T cell activation also inhibit activation of the LTR. These observations might serve as a model for the regulation of HIV-1 gene expression in peripheral blood T cells.

Animals↗
PubMed1992

SV40 large T antigen trans-activates the long terminal repeats of a large family of human endogenous retrovirus-like sequences.

The Simian Virus 40 (SV40) large T antigen (T) is required for the initiation of viral replication, the autoregulation of early gene expression, and the activation of late gene expression in productively infected cells. In addition to these roles, T has been implicated in the transcriptional activation of a variety of viral and cellular promoters. We have used the chloramphenicol acetyltransferase (CAT) reporter gene system to study the effect of T on the long terminal repeats (LTRs) of a large family of human endogenous retrovirus-like sequences, RTVL-H. Here we show that T can activate expression from certain RTVL-H LTRs 5- to 30-fold. Competition experiments in which an excess of plasmid containing only an RTVL-H LTR was cotransfected with an LTR-CAT reporter gene construct confirmed that this effect is specific for RTVL-H sequences. Restriction enzyme analysis using methylation-sensitive enzymes has shown that this activation is not due to plasmid replication. We have also observed this trans-activation effect in two CV-1 cells lines containing stably integrated LTR-CAT constructs. These results demonstrate that a known transforming protein can alter the transcriptional capabilities of RTVL-H LTRs. As there are approximately 3000 related LTRs in the genomes of humans and other primates, these findings suggest that a large number of these promoters and their associated transcripts may be transcriptionally stimulated by this and other oncogens.

Animals↗
PubMed2003

Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells.

Although DNA methylation and transcriptional repression are generally associated, a causal role for DNA methylation in silencing of retroviral vectors has not been established. The newer generation murine stem cell virus retroviral vector (MSCV) lacks many of the repressive cis-acting DNA sequences identified in Moloney murine leukemia virus but remains sensitive to transcriptional silencing in various cell types. To determine the contribution of cytosine methylation to MSCV silencing, we mutated CpG dinucleotides located in the MSCV long terminal repeat (LTR) that are clustered in the U3 region and directly spanning the transcription start site in the R region. Effects of the CpG mutations on MSCV silencing were assessed in murine embryonic stem cells. An analysis of numerous clonal proviral integrants showed that mutation of CpGs in both clusters eliminated proviral integrants that were completely silenced. Variegated expression was shown to represent a substantial component of intraclonal silencing and was independent of the presence of CpGs in the LTR. Treatment of transduced cells with 5-azadeoxycytidine delayed establishment of the silenced state but had only a modest effect on expression of some proviral integrants at late times post-transduction. These results are direct evidence for a causal contribution of DNA methylation in the LTR to MSCV silencing and define the promoter region CpGs as a repressive element in embryonic stem cells. Furthermore, distinct mechanisms are suggested for establishment and maintenance of the silenced proviral state.

Animals↗
PubMedDate not supplied

Grasshopper, a long terminal repeat (LTR) retroelement in the phytopathogenic fungus Magnaporthe grisea.

The fungal phytopathogen Magnaporthe grisea parasitizes a wide variety of gramineous hosts. In the course of investigating the genetic relationship between pathogen genotype and host specificity we identified a retroelement that is present in some strains of M. grisea that infect finger millet and goosegrass (members of the plant genus Eleusine). The element, designated grasshopper (grh), is present in multiple copies and dispersed throughout the genome. DNA sequence analysis showed that grasshopper contains 198 base pair direct, long terminal repeats (LTRs) with features characteristic of retroviral and retrotransposon LTRs. Within the element we identified an open reading frame with sequences homologous to the reverse transcriptase, RNaseH, and integrase domains of retroelement pol genes. Comparison of the open reading frame with sequences from other retroelements showed that grh is related to the gypsy family of retrotransposons. Comparisons of the distribution of the grasshopper element with other dispersed repeated DNA sequences in M. grisea indicated that grasshopper was present in a broadly dispersed subgroup of Eleusine pathogens, suggesting that the element was acquired subsequent to the evolution of this host-specific form. We present arguments that the amplification of different retroelements within populations of M. grisea is a consequence of the clonal organization of the fungal populations.

Amino Acid Sequence↗
PubMed1991

A single point mutation activates the Moloney murine leukemia virus long terminal repeat in embryonal stem cells.

The expression of Moloney murine leukemia virus (Mo-MuLV) and Mo-MuLV-derived vectors is restricted in undifferentiated mouse embryonal carcinoma and embryonal stem (ES) cells. We have previously described the isolation of retroviral mutants with host range properties expanded to embryonal cell lines. One of these mutants, the murine embryonic stem cell virus (MESV), is expressed in ES cell lines. Expression of MESV in these cells relies on DNA sequence motifs within the enhancer region of the viral long terminal repeat (LTR). Here we show that replacement of the Mo-MuLV enhancer region by sequences derived from the MESV LTR results in the activation of the Mo-MuLV LTR in ES cells. The enhancer regions of MESV and Mo-MuLV differ by seven point mutations. Of these, a single point mutation at position -166 is sufficient to activate the Mo-MuLV LTR and to confer enhancer-dependent expression to Mo-MuLV-derived retroviral vectors in ES cells. This point mutation creates a recognition site for a sequence-specific DNA-binding factor present in nuclear extracts of ES cells. This factor was found by functional assays to be the murine equivalent to human Sp1.

Base Sequence↗
PubMed1983

Molecular cloning of viral DNA from leukemogenic Gross passage A murine leukemia virus and nucleotide sequence of its long terminal repeat.

The viral DNA genome of the leukemogenic Gross passage A virus was cloned in phage Charon 21A as an infectious molecule. The virus recovered by transfection with this infectious DNA was ecotropic, N-tropic, fibrotropic, and XC+. It was leukemogenic when reinjected into newborn SIM mice, indicating that ecotropic murine leukemia virus (MuLV) from an AKR mouse thymoma can harbor leukemogenic sequences. Its restriction map was similar to that of nonleukemogenic AKR MuLV, its putative parent, but differed at the 3' end and in the long terminal repeat (LTR). The nucleotide sequence of the Gross A virus LTR was identical to the AKR MuLV LTR sequence (Van Beveren et al., J. Virol. 41:542-556, 1982) in U5, R, and part of U3. All differences between both LTRs were found in U3. Only one copy of the U3 tandem direct repeat was conserved in the Gross A virus LTR, and it was rearranged by the insertion of a 36-base-pair sequence and by five point mutations. Only one additional point mutation common to several oncogenic MuLVs was present in U3. These structural changes in the U3 LTR and at the 3' end of the genome may be related to the leukemogenicity of this virus.

Animals↗
PubMed1984

The long terminal repeat of an endogenous intracisternal A-particle gene functions as a promoter when introduced into eucaryotic cells by transfection.

We describe experiments designed to determine whether an endogenous intracisternal A-particle (IAP) gene randomly selected from a mouse embryo library has the potential to be transcriptionally active. Assays for IAP gene transcription were done with permanently transformed rat cells and transiently transfected monkey and mouse cells. The rat cells, which had integrated IAP gene copies, contained IAP RNA. A start site within the IAP 5' long terminal repeat (LTR) was localized by S1 mapping. The promoter activity of the IAP LTR was also measured in cells 48 h after the introduction of recombinant plasmids in which bacterial chloramphenicol acetyl transferase (CAT) encoding sequences were under the control of the LTR. The IAP LTR promoted CAT activity in mouse and monkey cells. In mouse L-cells, the levels of CAT activity were 10 to 25% of those promoted by an analogous recombinant containing the Moloney murine sarcoma virus LTR as the promoter. In contrast to the Moloney murine sarcoma virus LTR, the IAP LTR was five- to eightfold more active in monkey cells than in mouse cells. The 5' and 3' LTRs were equally active, and promoter activity was dependent on having the orientation of the LTRs with respect to the CAT gene the same as their orientation with respect to the IAP gene. A 5'-flanking sequence containing a member of the highly repetitive R-sequence family increased CAT activity in COS cells 11-fold when present along with the LTR. Our results indicate that the LTR of an endogenous mouse IAP gene can function as an efficient promoter in heterologous as well as homologous cells.

Acetyltransferases↗
PubMed1995

Formation of simian immunodeficiency virus long terminal repeat circles in resting T cells requires both T cell receptor- and IL-2-dependent activation.

Although immunodeficiency viruses can enter resting CD4+ T lymphocytes, activation of T cells is required for complete viral cDNA synthesis and transport of double-stranded viral DNA to the nucleus. Cross-linking T cell receptors (TCRs) on resting CD4+ T cells induces reverse transcription of full-length simian immunodeficiency virus (SIV) genomes, but TCR engagement alone is insufficient to stimulate SIV DNA to move to the nucleus and form long terminal repeat (LTR) circles. Neither ligation of TCR or CD28 receptors nor interleukin 2 (IL-2) alone induces formation of LTR circles; however, the combination of TCR ligation with either CD28 ligation or IL-2 doses. Anti-IL-2 serum inhibits the formation of LTR circles induced by cross-linking CD3 and CD28, but has no effect on the induction of increased viral reverse transcription. Thus, two signals appear to be required for immunodeficiency viruses to move to the T cell nucleus, one from the TCR to promote reverse transcription of the viral genome, the other through an IL-2-dependent process leading to formation of LTR circles.

Animals↗