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PubMed1985

Isolation of embryonal carcinoma cell lines that express integrated recombinant genes flanked by the Moloney murine leukemia virus long terminal repeat.

We have used a producer NIH 3T3 cell line that secretes, together with the helper Moloney murine leukemia virus (Mo-MuLV), a transducing recombinant virus containing the neomycin-resistance gene linked to the Mo-MuLV long terminal repeat (LTR). By infecting three embryonal carcinoma cell lines, PCC4.aza1R, F9tk-, and Nulli-SCC1, with this recombinant virus, we have isolated many transductant clones that stably express the integrated neomycin-resistance gene. These clonal transductant lines consist of undifferentiated embryonal carcinoma cells as judged by morphology, tumorigenicity in 129/Sv mice, and cell-surface antigenic markers. Analysis of the integrated recombinant viral genes by Southern blot hybridization revealed that some of the lines have single copies, whereas others have multiple copies, probably in multiple sites. Although these transductant lines contained many copies of helper Mo-MuLV integrated in the cellular genome, expression of these helper viruses was not detected either by reverse transcriptase activity or by X-C plaque assay. Two F9tk--derived, G418-resistant transductant lines were superinfected with a second recombinant transducing virus that contains the herpes simplex virus thymidine kinase gene flanked by the Mo-MuLV LTR. The frequency of transduction to yield clones able to grow in hypoxanthine/aminopterin/thymidine medium was similar to that of the parental F9tk- cells. These results suggest that the expression of the neomycin-resistance gene, linked to MoMuLV LTR in the transductant embryonal carcinoma cell clones, is due to a cisacting mechanism(s).

Animals↗
PubMed1986

The long terminal repeat of the intracisternal A particle as a target for transactivation by oncogene products.

It has been shown recently that the c-mos oncogene becomes activated in myeloma XRPC-24 via insertion of an intracisternal A particle (IAP) long terminal repeat (LTR). The inserted LTR serves as a promoter from which transcription of the 3' rearranged c-mos initiates. The insertion is in a head-to-head orientation such that the transcriptional orientations of the IAP and the 3' rearranged c-mos are opposite. It has already been shown that this IAP LTR has two promoters, one transcribing the IAP genome and the other transcribing the rearranged c-mos. Since the IAP genomes are actively transcribed in mouse myelomas but not in normal cells, it was interesting to test whether transcriptional activation of the IAP occurs in the presence of active oncogene products, especially nuclear ones. The 5' LTR of the IAP inserted in myeloma XRPC-24 was chosen as a convenient model to test the effect of viral and cellular oncogene products. These included simian virus 40 (SV40) large-T antigen, the adenovirus early 1A (E1A) gene product, the myc gene product, and p53. The LTR was coupled to the bacterial gene coding for chloramphenicol acetyltransferase (CAT) in two orientations, and the levels of CAT directed by the LTR promoters were assayed in either the presence or the absence of the oncogene products. The levels of CAT directed by the 5' LTR promoter transcribing the IAP were significantly elevated in the presence of SV40 large-T antigen, the adenovirus E1A and myc gene products, and p53. The promoter transcribing the rearranged c-mos was transactivated by SV40 large-T antigen and the adenovirus E1A gene product. The results indicate that oncogene products may have an important role in turning on promoters of other genes. The IAP LTR may serve as a useful model for studying the effect of various gene products on promoters which are known to be activated in the malignant state.

Acetyltransferases↗
PubMed1991

Analysis of the binding proteins and activity of the long terminal repeat of Moloney murine leukemia virus during differentiation of mouse embryonal carcinoma cells.

Mouse embryonal carcinoma (EC) cell lines were established which carry the stably integrated chloramphenicol acetyltransferase (CAT) gene under the control of the transcriptional elements of the long terminal repeat (LTR) of Moloney murine leukemia virus. The activity of three elements of the stably integrated LTR was analyzed in undifferentiated EC cells (stable CAT assay). Results of the study are summarized as follows. (i) In the stable assay, the promoter region of the LTR was inactive in undifferentiated ECA2 and F9 cells, and the level of the activity was 10(-4) of that in NIH 3T3 cells. (ii) In contrast to the results of the transient assay, the enhancer was active in undifferentiated ECA2 cells and in F9 cells. It activated CAT activity more than 60-fold and about 8-fold in ECA2 cells and F9 cells, respectively. (iii) Suppression by ELP, the embryonal LTR-binding protein, was more pronounced in the stable assay than in the transient assay. These data suggest that, when compared with NIH 3T3 cells, a major factor for the inactivity of the LTR in EC cells is the inefficiency of the promoter in this assay. Transcriptional activity of the LTR was analyzed during the differentiation of EC cells. In the case of ECA2 cells, the magnitude of activation by the enhancer did not change during differentiation. The activity of the promoter increased about 10-fold, and the suppression by ELP became negligible 4 days after the induction of differentiation. Upon differentiation of F9 cells, the activity of the enhancer increased more than 300-fold, but the promoter remained inactive. The pattern of LTR-binding proteins also varied during the differentiation of EC cells. Our present data suggest that the activity of LTR elements as assayed by the stable assay differs from the activity as assayed by the transient assay. It also indicates that the activity of these elements exhibits cell-type-specific changes during the differentiation of EC cells.

Animals↗
PubMed1998

In vivo protein binding and functional analysis of cis-acting elements in the U3 region of the bovine leukemia virus long terminal repeat.

Bovine leukemia virus (BLV) is a member of the human T-cell leukemia virus (HTLV)/BLV group of retroviruses. These viruses regulate their own transcription by producing Tax, a protein which activates the virus promoter region, the long terminal repeat (LTR). To explore the molecular mechanisms involved in the transactivation, we identified protein binding elements by in vivo footprinting and analyzed their function by site- directed mutagenesis. We used in vivo dimethyl sulfate footprinting by ligation-mediated PCR to detect constitutive in vivo protein-DNA interactions in a BLV-producing cell line, Bat2Cl6. The U3 region and part of the R region of the LTR were footprinted. In addition to the cis-acting elements (three cyclic AMP-responsive elements [CREs] and two AP4 sites) reported by others to be important for Tax-mediated activation of the BLV LTR, we found footprints in regions flanking these elements and in the core promoter region. The importance of these sites for transcriptional activation was studied by site-directed mutagenesis followed by promoter function analysis of the mutants with a chloramphenicol acetyltransferase reporter system. Our data corroborate those of others showing that the CREs are necessary for transactivation of the LTR, and they identify two new functional sites not previously reported by others. We show that the middle region of the BLV U3 contains multiple dual-functioning cis-acting elements which act as either positive or negative regulatory elements depending on the cell type tested. This is the first report of a functional mapping of the cis-acting elements of a virus of the HTLV/BLV group.

Animals↗
PubMed2004

PU.1 binding to ets motifs within the equine infectious anemia virus long terminal repeat (LTR) enhancer: regulation of LTR activity and virus replication in macrophages.

Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the role of these three sites in EIAV LTR activity. While DNase I footprinting studies indicated that all three sites within the enhancer were bound by recombinant PU.1, reporter gene assays demonstrated that the middle motif was most important for basal levels of LTR activity in macrophages and that the 5' motif had little impact. The impact of the 3' site became evident in Tat transactivation studies, in which the loss of the site reduced Tat-transactivated expression 40-fold. In contrast, elimination of the 5' site had no effect on Tat-mediated activity. Binding studies were performed to determine whether differences in PU.1 binding affinity for the three sites correlated with the relative impact of each site on LTR transcription. While small differences were observed in the binding affinities of the three sites, with the promoter-proximal site having the strongest binding affinity, these differences could not account for the dramatic differences observed in the transcriptional effects. Instead, the promoter-proximal position of the 3' motif appeared to be critical for its transcriptional impact and suggested that the PU.1 sites may serve different roles depending upon the location of the sites within the enhancer. Infectivity studies demonstrated that an LTR containing an enhancer composed of the three PU.1 sites was not sufficient to drive viral replication in macrophages. These findings indicate that while the promoter-proximal PU.1 site is the most critical site for EIAV LTR activity in the presence of Tat, other elements within the enhancer are needed for EIAV replication in macrophages.

Base Sequence↗
PubMed1999

Evolution of the human immunodeficiency virus type 1 long terminal repeat promoter by conversion of an NF-kappaB enhancer element into a GABP binding site.

Human immunodeficiency virus type 1 (HIV-1) transcription is regulated by the viral Tat protein and cellular factors, of which the concentration and activity may depend on the cell type. Viral long terminal repeat (LTR) promoter sequences are therefore optimized to suit the specific nuclear environment of the target host cell. In long-term cultures of a Tat-defective, poorly replicating HIV-1 mutant, we selected for a faster-replicating virus with a 1-nucleotide deletion in the upstream copy of two highly conserved NF-kappaB binding sites. The variant enhancer sequence demonstrated a severe loss of NF-kappaB binding in protein binding assays. Interestingly, we observed a new binding activity that is specific for the variant NF-kappaB sequence and is present in the nuclear extract of unstimulated cells that lack NF-kappaB. These results suggest that inactivation of the NF-kappaB site coincides with binding of another transcription factor. Fine mapping of the sequence requirements for binding of this factor revealed a core sequence similar to that of Ets binding sites, and supershift assays with antibodies demonstrated the involvement of the GABP transcription factor. Transient transfection experiments with LTR-chloramphenicol acetyltransferase constructs indicated that the variant LTR promoter is specifically inhibited by GABP in the absence of Tat, but this promoter was dramatically more responsive to Tat than the wild-type LTR. Introduction of this GABP site into the LAI virus yielded a specific gain of fitness in SupT1 cells, which contain little NF-kappaB protein. These results suggest that GABP potentiates Tat-mediated activation of LTR transcription and viral replication in some cell types. Conversion of an NF-kappaB into a GABP binding site is likely to have occurred also during the worldwide spread of HIV-1, as we noticed the same LTR modification in subtype E isolates from Thailand. This typical LTR promoter configuration may provide these viruses with unique biological properties.

Binding Sites↗
PubMed1992

Transcellular activation of the human immunodeficiency virus type 1 long terminal repeat in cocultured lymphocytes.

One of the unexplained aspects of the progression of AIDS is that immunological abnormalities are detectable before CD4+ T-helper cell depletion occurs (A.R. Gruters, F.G. Terpstra, R. De Jong, C.J.M. Van Noesel, R.A.W. Van Lier, and F. Miedema, Eur. J. Immunol. 20:1039-1044, 1990; F. Miedema, A.J. Chantal-Petit, F.G. Terpstra, J.K.M.E. Schattenkerk, F. de Wolf, B.J.M. Al, M. Roos, J.M.A. Lang, S.A. Danner, J. Goudsmit, and P.T.A. Schellekens, J. Clin. Invest. 82:1908-1914, 1988; G.M. Shearer, D.C. Bernstein, K.S. Tung, C.S. Via, R. Redfield, S.Z. Salahuddin, and R.C. Gallo, J. Immunol. 137:2514-2521, 1986). In this report, we describe a mechanism by which human immunodeficiency virus type 1 (HIV-1)-infected cells can influence neighboring HIV-1-infected T lymphocytes and uninfected T cells as well. We have examined the interaction of T-cell and macrophage cell lines that are transfected with HIV-1 DNA by using cocultured lymphocytes. The HIV-1 constructs we used lack a functional pol gene and therefore do not produce infectious virus. Cocultivation results in the transcellular activation of the HIV long terminal repeat in the cocultured T cells. This transcellular activation is evident in as little as 3 h of cocultivation, at ratios of HIV-expressing cells to target cells as low as 1:1,000, and is dependent on the Tat-responsive element. The demonstration that a small number of HIV-expressing cells can affect a large number of uninfected bystander cells in a short period of time suggests a mechanism by which global immune dysfunction can precede the high prevalence of infected cells.

Cell Line↗
PubMed1988

A comparison of bovine growth-hormone gene expression in mouse L cells directed by the Moloney murine-leukemia virus long terminal repeat, simian virus-40 early promoter or cytomegalovirus immediate-early promoter.

Three transcriptional regulatory regions including a human cytomegalovirus immediate-early region (CMVIE) promoter, the simian virus-40 early region (SV40E) promoter, and Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR) were ligated to the bovine growth hormone (bGH)-coding gene. Using a bGH transient expression system, the CMVIE and SV40E gene transcriptional regulatory regions were found to be approximately two-fold more efficient than the MoMLV LTR in directing expression of bGH in mouse L cells.

Animals↗
PubMed1982

Purified glucocorticoid receptor-hormone complex from rat liver cytosol binds specifically to cloned mouse mammary tumor virus long terminal repeats in vitro.

Purified glucocorticoid hormone-receptor complex (HRC) from rat liver binds to specific DNA sequences contained in cloned mouse mammary tumor virus (MMTV)DNA. The binding site of the hormone-receptor complex is located in the long terminal repeat (LTR) of MMTV DNA as shown by filter binding studies with labeled restriction fragments and by visualization of DNA-receptor complexes with the electron microscope. The DNAs from cloned MMTV lacking the LTR sequences were neither retained on nitrocellulose filters nor bound specifically to HRCs examined in the electron microscope. The HRC also failed to bind to restriction fragments from pBR322 and phage lambda. Specific binding of the HRC to LTR sequences is dependent upon occupancy of the receptor by a glucocorticoid. Previous work has demonstrated that the MMTV transcription is initiated within the LTR; additionally, MMTV transcription is known to be regulated by glucocorticoids. Our present results therefore support the hypothesis that HRC regulates hormone-induced transcription by binding to specific DNA sequences near the MMTV transcription start site.

Animals↗
PubMed1983

Selective cleavage in the avian retroviral long terminal repeat sequence by the endonuclease associated with the alpha beta form of avian reverse transcriptase.

M13 recombinant DNA clones containing a 350-base sequence derived from the EcoRI fragment of two tandemly linked Rous-associated virus 2 (RAV-2) long terminal repeat (LTR) sequences have been used to map reverse transcriptase-associated endonuclease (RT-endonuclease) cleavage sites by primer extension studies. Under appropriate conditions, the alpha beta form of RT-endonuclease (composed of both the alpha and beta subunits) purified from Avian sarcoma virus (Pr-C and B-77 strains) introduces a specific break in the inverted complementary repeat sequence found at the junction of the LTRs. The cleavage sites occur in the same nucleotide sequence in (-) and (+) DNA strands; together they have the potential of generating a 6-base-pair staggered overlap that spans the junction. This supports the notion that the enzyme is involved in viral DNA integration. Other RT-endonuclease sites were analyzed. A second site, which occurs in the lac region of the M13 vector DNA upstream from the unique EcoRI cloning site, bears no apparent sequence homology to the site at the junction of the LTRs. However, it also lies within an inverted complementary repeat and, as is the case for the site in the LTR, the break occurs to the 5' side of the axis of symmetry. Cleavage at this second site is suppressed when the vector contains the RAV-2 LTR insert. Thus, the viral LTR appears to exert a cis effect that can influence a region over 300 base pairs away.

Avian Sarcoma Viruses↗
PubMed1988

Addition of substitution of simian virus 40 enhancer sequences into the Moloney murine leukemia virus (M-MuLV) long terminal repeat yields infectious M-MuLV with altered biological properties.

Moloney murine leukemia virus (M-MuLV) is a replication-competent retrovirus which induces T-cell lymphoma in mice. The enhancer sequences present within the M-MuLV long terminal repeat (LTR) region of the proviral genome have been shown to influence the disease specificity of the virus strongly. We examined the contribution of the M-MuLV enhancers to the transcriptional activity and pathogenesis of M-MuLV by constructing LTRs containing heterologous enhancer elements. The simian virus 40 enhancer region (72- and 21-base-pair repeats) was inserted into the U3 region (at -150 base pairs) of the M-MuLV LTR (Mo + SV) and also into a deleted form of the LTR which lacks the M-MuLV enhancer sequences (delta Mo + SV). These chimeric LTRs were used to generate infectious M-MuLVs by transfection of corresponding proviral plasmids into mouse fibroblasts. The relative infectivities of Mo + SV and delta Mo + SV recombinant viruses as determined by rat XC cell plaque assay and reverse transcriptase assay were 60 to 70% of wild-type M-MuLV levels. To study the pathogenicity of these two recombinant viruses, we inoculated newborn NIH Swiss mice with either Mo + SV or delta Mo + SV M-MuLV. Both viruses induced disease more slowly than M-MuLV, which induces disease 2 to 4 months postinoculation. Mo + SV M-MuLV-inoculated animals became moribund at 3 to 13 months postinoculation, whereas delta Mo + SV M-MuLV-inoculated animals became moribund at 6 to 24 months postinoculation. The tumors induced by the two viruses were characterized histologically and molecularly. Mo + SV M-MuLV-induced tumors were primarily T-cell-derived lymphoblastic lymphomas containing extensive rearrangements of the T-cell receptor beta gene. In contrast, delta Mo + SV M-MuLV induced pre-B- and B-cell lymphoblastic lymphomas, B-cell-derived follicular-center cell lymphomas, and acute myeloid leukemia. The delta Mo + SV tumor DNAs from B-lineage tumors were typically rearranged at the immunoglobulin gene loci and contained germ line configurations of the T-cell receptor beta gene. Southern blot hybridization confirmed that the tumor DNAs contained the predicted Mo + SV M-MuLV or delta Mo + SV M-MuLV provirus.

Animals↗
PubMed1989

Identification of a region within the human immunodeficiency virus type 1 long terminal repeat that is essential for transactivation by the hepatitis B virus gene X.

Hepatitis B virus (HBV) X-gene product activates transcription of the chloramphenicol acetyltransferase (CAT) gene under control of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). To identify a cis-acting regulatory sequence within the HIV-1 LTR which is responsive to the HBV X-gene trans-activating function, we examined the effects of HBV X-gene expression in cells with a series of LTR/CAT deletion mutants. A region of the HIV-1 LTR containing the previously identified kappa B-like enhancer element was found to be responsive to HBV X-gene activation, and this effect was independent of, and additive with, the effect of the HIV-1 tat-III protein on CAT expression. Since kappa B-like enhancer sequences are known to regulate transcription of a variety of viruses and cellular genes, our results suggest that the X gene could activate such a gene during HBV infection and replication.

Cell Line↗
PubMed2004

Azido-containing diketo acid derivatives inhibit human immunodeficiency virus type 1 integrase in vivo and influence the frequency of deletions at two-long-terminal-repeat-circle junctions.

We previously found that azido-containing beta-diketo acid derivatives (DKAs) are potent inhibitors of human immunodeficiency virus type 1 (HIV-1) integrase (IN) (X. Zhang et al., Bioorg. Med. Chem. Lett., 13:1215-1219, 2003). To characterize the intracellular mechanisms of action of DKAs, we analyzed the antiviral activities of two potent azido-containing DKAs with either a monosubstitution or a disubstitution of azido groups, using single- and multiple-replication-cycle assays. Both azido-containing DKAs significantly inhibited HIV-1 infection in 293T, CEM-SS, and H9 cells (50% inhibitory concentration = 2 to 13 micro M) and exhibited low cytotoxicity (50% cytotoxic concentration = 60 to 600 micro M). Inhibition of HIV-1 IN in vivo was demonstrated by the observation that previously described L-708,906 resistance mutations in HIV-1 IN (T66I and T66I/S153Y) also conferred resistance to the azido-group-containing DKAs. In vitro assays and in vivo analysis indicated that the DKAs did not significantly inhibit the 3' processing and selectively inhibited the strand transfer reaction. In addition, quantitative PCR indicated that two-long-terminal-repeat (2-LTR) circles were elevated in the presence of the azido-containing DKAs, confirming that HIV-1 IN was the intracellular target of viral inhibition. To gain insight into the mechanism by which the DKAs increased 2-LTR-circle formation of 3'-processed viral DNAs, we performed extensive DNA sequencing analysis of 2-LTR-circle junctions. The results indicated that the frequency of deletions at the circle junctions was elevated from 19% for the untreated controls to 32 to 41% in the presence of monosubstituted (but not disubstituted) DKAs. These results indicate that the structure of the DKAs can influence the extent of degradation of viral DNA ends by host nucleases and the frequency of deletions at the 2-LTR-circle junctions. Thus, sequencing analysis of 2-LTR-circle junctions can elucidate the intracellular mechanisms of action of HIV-1 IN inhibitors.

Base Sequence↗
PubMed2002

HIV-1 Tat protein-mediated transactivation of the HIV-1 long terminal repeat promoter is potentiated by a novel nuclear Tat-interacting protein of 110 kDa, Tip110.

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication is highly dependent on and modulated by interactions between viral and host cellular factors. Tat protein, encoded by one of the HIV-1 regulatory genes, tat, is essential for HIV-1 gene expression. A number of host cellular factors have been shown to interact with Tat in this process. During our attempts to determine the molecular mechanisms of Tat interaction with brain cells, we isolated a cDNA clone that encodes a novel Tat-interacting protein of 110 kDa or Tip110 from a human fetal brain cDNA library. GenBank BLAST search revealed that Tip110 was almost identical to a previously cloned KIAA0156 gene with unknown functions. In vivo binding of Tip110 with Tat was confirmed by immunoprecipitation and Western blotting, in combination with mutagenesis. The yeast three-hybrid RNA-protein interaction assay indicated no direct interaction of Tip110 with Tat transactivating response element RNA. Nevertheless, Tip110 strongly synergized with Tat on Tat-mediated chloramphenicol acetyltransferase reporter gene expression and HIV-1 virus production, whereas down-modulation of constitutive Tip110 expression inhibited HIV-1 virus production. Northern blot analysis showed that Tip110 mRNA was expressed in a variety of human tissues and cells. Moreover, digital fluorescence microscopic imaging revealed that Tip110 was expressed exclusively in the nucleus, and within a nuclear speckle structure that has recently been described for human cyclin T and CDK9, two critical components for Tat transactivation function on HIV-1 long terminal repeat promoter. Taken together, these data demonstrate that Tip110 regulates Tat transactivation activity through direct interaction, and suggest that Tip110 is an important cellular factor for HIV-1 gene expression and viral replication.

Amino Acid Sequence↗
PubMed1995

Characterization of a non-long terminal repeat retrotransposon cDNA (L1Tc) from Trypanosoma cruzi: homology of the first ORF with the ape family of DNA repair enzymes.

In the present paper we describe the characterization of a Trypanosoma cruzi cDNA (L1Tc) corresponding to a transcript from a new long terminal repeat (LTR) retrotransposon. This element is present in a high-copy number, and is found dispersed throughout the T. cruzi genome. Northern analysis shows an abundant expression of L1Tc-related sequences with a major band of about 5 kb. The transcript has at its 3' end a fragment of a highly repetitive DNA sequence (E12A), at its 5' end a ribosomal mobile element-like sequence and three putative open reading frames (ORF) in different frames. The ORF2 codes for a protein which has significant homology with the retrotranscriptase-related sequences from non-LTR retrotransposons containing the seven domains present in all the retrotranscriptase and retrotranscriptase-related proteins. The ORF3 codes for a gag-like protein showing unusual cysteine motifs present in all non-LTR trypanosomatid elements, similar to the C2H2 zinc finger family of transcription factors. Interestingly, ORF1 codes for a protein with significant homology to the major human AP endonuclease protein, and maintains in similar positions most of the amino acid domains described for all the Ape family of proteins. The presence of Ape-related sequences, described for the first time in a non-LTR retrotransposon (L1Tc), may have functional relevance for these types of elements.

Amino Acid Sequence↗
PubMed1991

Isolation of cDNAs for DNA-binding proteins which specifically bind to a tax-responsive enhancer element in the long terminal repeat of human T-cell leukemia virus type I.

One of the gene products of human T-cell leukemia virus type I (HTLV-I), p40tax, activates its own viral transcription in trans through tax-responsive enhancers in viral long terminal repeats. Five species of cDNA clones for proteins that bind to the tax-responsive enhancer element in HTLV-I were isolated from the Jurkat cell library. The beta-galactosidase fusion protein prepared from the lysogen of a clone specifically recognized the cyclic AMP-responsive element in HTLV-I enhancer. The nucleotide sequence of a full-length cDNA clone (TAXREB67) had a coding capacity of 351 amino acids, which contained a basic motif followed by a leucine zipper structure near the carboxy terminus. Its mRNA was detected in human cell lines, including HTLV-I-infected or noninfected hematopoietic cell lines. The mRNA level in Jurkat cells was decreased temporarily by increasing cyclic AMP concentration but increased by increasing Ca2+ concentration. Polyclonal antibodies against the fusion protein specifically recognized a 52-kDa protein in Jurkat cells. Analyses of the function of this protein and its interactions with other cellular factors will be useful to help understand the regulatory mechanism through tax-responsive enhancers in HTLV-I.

Amino Acid Sequence↗
PubMed1995

Multiple modifications in cis elements of the long terminal repeat of retroviral vectors lead to increased expression and decreased DNA methylation in embryonic carcinoma cells.

Infection by murine retroviruses in embryonic carcinoma (EC) and embryonic stem cells is highly restricted. The transcriptional unit of the Moloney murine leukemic virus (MoMuLV) long terminal repeat (LTR) is inactive in EC and embryonic stem cells in association with increased proviral methylation. In this study, expression in F9 EC cells was achieved from novel retroviral vectors containing three modifications in the MoMuLV-based retroviral vector: presence of the myeloproliferative sarcoma virus LTR, substitution of the primer binding site, and either deletion of a negative control region at the 5' end of the LTR or insertion of a demethylating sequence. We conclude that inhibition of expression from the MoMuLV LTR in EC cells is mediated through the additive effects of multiple cis-acting elements affecting the state of methylation of the provirus.

3T3 Cells↗
PubMed1998

Transcription of the human immunodeficiency virus type 1 (HIV-1) promoter in central nervous system cells: effect of YB-1 on expression of the HIV-1 long terminal repeat.

Transcription of human immunodeficiency virus type 1 (HIV-1) is regulated by a series of host DNA-binding proteins which interact with the upstream viral promoter sequences, and the viral regulatory protein Tat which associates with TAR RNA sequences located in the leader of the viral transcript. Here we have examined the ability of the cellular protein YB-1 to modulate transcription of the HIV-1 promoter in a human astrocytic cell line (U-87MG), a neuronal cell line (SK-N-MC) and lymphoid cells (Jurkat) by transfection assay. Ectopic expression of YB-1 in U-87MG and SK-N-MC augments basal transcriptional activity of the viral sequence located between -80 and -43, which encompasses the GC-rich motif. In accord with the previous report, in Jurkat cells YB-1-mediated activation of the HIV-1 promoter required sequences which are located further upstream from the GC-rich motif. Combined overexpression of YB-1 and the GC-rich binding protein, SP1, in the transfected cells decreased the level of activation of the viral promoter, suggesting that YB-1 and SP1 may exert negative effects on each other's function. Results from band shift assay with purified YB-1 and SP1 indicated that SP1 and YB-1 bind to the GC-rich DNA sequence in the double-stranded and single-stranded configurations, respectively. However, efficient binding of SP1 to the double-stranded GC-rich motif corresponding to the HIV-1 long terminal repeat (LTR) is diminished in the presence of YB-1. Similarly, in the presence of SP1, YB-1 loses its ability to become associated with its target single-stranded DNA probe. No evidence for direct association of YB-1 and SP1 either in the presence or in the absence of DNA was observed. These data suggest that while YB-1 stimulates expression of the LTR in central nervous system cells, the level of activity of other cellular proteins, such as SP1, may dictate binding of YB-1 to its target sequence, and therefore affect the regulatory function of this protein.

Astrocytes↗