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PubMed1984

Increased concentration of an indigenous proviral mouse mammary tumor virus long terminal repeat-containing transcript is associated with neoplastic transformation of mammary epithelium in C3H/Sm mice.

Increased amounts of mouse mammary tumor virus (MMTV) proviral transcripts were found in RNA dot blots from MMTV-negative, C3H/Sm mouse mammary tumors which arose spontaneously or were induced by hormonal or chemical carcinogens or both. Other dot blots probed with a long terminal repeat (LTR) probe showed that LTR (MMTV)-containing transcripts were disproportionately represented in these tumor RNAs. Different segments of the MMTV genome were used in sequential hybridizations to Northern blots to determine relative sequence content and size of MMTV transcripts in transformed mammary tissues, as compared with those in lactating mammary glands. Increased amounts of 4.4-kilobase env and 8.1-kilobase genomic MMTV transcripts were detected with an env probe in many of the tumor RNAs examined. Hybridization of the same Northern blots containing tumor RNAs with an LTR probe revealed a 2.2-kilobase transcript which was prominent in RNAs from chemically-induced, hormonally-induced, and spontaneous mammary tumors relative to those from lactating mammary glands. The LTR-containing transcript did not possess significant homology to either env or gag-pol probes. This distinctive, transformation-enhanced, 2.2-kilobase transcript may contain mouse cellular sequences in addition to LTR sequences or it may represent the message for a nonstructural viral protein encoded within the LTR open reading frame of one or more of the four C3H/Sm MMTV proviral genes.

Animals↗

PubMed1985

Retrovirus-induced spongiform encephalopathy: the 3'-end long terminal repeat-containing viral sequences influence the incidence of the disease and the specificity of the neurological syndrome.

Using chimeric murine leukemia viruses (MuLVs) constructed in vitro with parental viral genomes from the neurotropic Cas-BR-E MuLV and the nonneurotropic amphotropic 4070-A MuLV, we previously mapped the paralysis-inducing determinant of Cas-BR-E MuLV within a pol-env region. To assess the role of the long terminal repeats (LTRs) in influencing the neurological disease, we constructed another chimeric MuLV (pNEMO-1)m harboring the gag-pol-env from Cas-BR-E MuLV and the LTR region from the strongly T-cell tropic Moloney MuLV. Although the Cas-BR-E MuLV induced mainly nonthymic leukemia, pNEMO-1 MuLV induced a thymic form of leukemia, as the parental Moloney MuLV. The pNEMO-1 MuLV induced neurological diseases less frequently than Cas-BR-E MuLV when inoculated intraperitoneally into NIH/Swiss, SIM.S, and SWR/J mice. However, it induced neurological disorders more frequently and with a shorter latency than Cas-BR-E MuLV when inoculated intrathymically. Most mice with a neurological disorder induced with pNEMO-1 MuLV showed a new clinical syndrome not usually seen with the parental Cas-BR-E MuLV: They had no lower limb paralysis but were excessively tremulous, spastic, and immobile. The topographical distribution of the spongiform degeneration in the brain of mice with this new syndrome was different from that seen in mice with lower limb paralysis induced by Cas-BR-E MuLV. These results indicate that the 1.0-kilobase-pair Cla I-Pvu I LTR-containing fragment harbors sequences influencing the incidence and the clinical manifestation of the neurological disease and suggest a specificity of LTR sequences for a new tissue (brain).

Animals↗

PubMed1992

Characterization of unintegrated retroviral DNA with long terminal repeat-associated cell-derived inserts.

We have used a replication-competent shuttle vector based on the genome of Rous sarcoma virus to characterize genomic rearrangements that occur during retrovirus replication. The strategy involved cloning circular DNA that was generated during an acute infection. While analyzing a class of retroviral DNA clones that are greater than full length, we found several clones which had acquired nonviral inserts in positions adjacent to the long terminal repeats (LTRs). There appear to be two distinct mechanisms leading to the incorporation of cellular sequences into these clones. Three of the molecules contain a cell-derived insert at the circle junction site between two LTR units. Two of these molecules appear to be the results of abortive integration attempts, because of which, in each case, one of the LTRs is missing 2 bases at its junction with the cell-derived insert. In the third clone, pNO220, the cellular sequences are flanked by an inappropriately placed copy of the tRNA primer-binding site on one side and a partial copy of the U3 sequence as part of the LTR on the other side. A fourth molecule we characterized, pMD96, has a single LTR with a U5-bounded deletion of viral sequences spanning gag and pol, with cell-derived sequences inserted at the site of the deletion; its origin may be related mechanistically to pNO220. Sequence analysis indicates that all of the cellular inserts were derived from the cell line used for the acute infection rather than from sequences carried into the cell as part of the virus particle. Northern (RNA) analysis of cellular RNA demonstrated that the cell-derived sequences of two clones, pNO220 and pMD96, were expressed as polyadenylated RNA in uninfected cells. One mechanism for the joining of viral and cellular sequences suggested by the structures of pNO220 and pMD96 is recombination occurring during viral DNA synthesis, with cellular RNA serving as the template for the acquisition of cellular sequences.

Animals↗

PubMed1995

Distinct regions in human T-cell lymphotropic virus type I tax mediate interactions with activator protein CREB and basal transcription factors.

Human T-cell lymphotropic virus type I (HTLV-I) transactivator Tax augments transcription from three (cyclic AMP response element (CRE)-containing 21-bp repeats in the viral long terminal repeat and several other cis regulatory elements, including the NF-kappa B binding sites and the serum response element. Tax does not bind DNA directly; rather, it acts via cellular sequence-specific DNA binding proteins to stimulate transcription. We have shown recently that Tax forms multiprotein complexes with the heterodimeric and homodimeric forms of a ubiquitous cellular transcription factor, CREB (CRE binding protein). In vitro selection for preferred Tax-CREB binding sites indicates that the Tax-CREB complex exhibits greatly increased DNA recognition specificity and assembles preferentially on CRE motifs, TGACGT/C, flanked by long runs of G (5') and/or C (3') residues, as found in the HTLV-I 21-bp repeats. The indirect tethering of Tax to the 21-bp repeats via CREB is crucial for Tax transactivation. We now report the domain organization of Tax by characterizing its mutants. Tax mutants with alterations in the NH2 terminus, including three deletion mutants, Tax(6-353), Tax(21-353), and Tax(89-353), and two amino acid substitution mutants, M1 (H3S) and M7 (C29A, P30S), all failed to interact with CREB in vitro. In contrast, a short COOH-terminal deletion, Tax(1-319), and a Tax mutant with amino acid substitutions near the COOH end, M47 (L319R, L320S), were able to interact with CREB and the 21-bp repeats to assemble ternary Tax-CREB-DNA complexes. As demonstrated earlier, M1, M7, and M47 all failed to transactivate the HTLV-I long terminal repeat. Our data indicate that the defects in M1 and M7 result from an inability to interact with CREB. In contrast, the COOH-terminal mutations in M47 most likely inactivated the transactivation domain of Tax. As anticipated, a Tax mutant, M22 (G137A, L138S) which activated transcription from the 21-bp repeats with reduced capacity and was defective in trans activating the NF-kappa B binding sites, continued to interact with CREB in vitro, albeit with a lower level of efficiency. Finally, a glutathione S-transferase (GST)-Tax fusion protein with the GST moiety fused to the NH2 terminus of Tax failed to interact with CREB. Removal of the GST domain from GST-Tax by thrombin restores Tax's ability to assemble a ternary Tax-CREB-21-bp-repeat complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1984

In vitro transformation by bovine papillomavirus.

We have studied tumorigenic transformation of mouse tissue culture cells by bovine papillomavirus (BPV) as a model of papillomavirus-induced cell proliferation. When BPV or its 7.9-kb full-length viral DNA genome induces focal transformation of mouse calls, the viral DNA is maintained in the transformed cells as multiple extrachromosomal copies. The transforming capacity was initially localized to a 69% subgenomic fragment of the viral DNA genome. We have further characterized the BPV DNA sequences that can encode the transforming function by generating and analyzing the transforming activity of a series of BPV DNA deletion mutants. The results indicated that two discontinuous segments of the viral DNA are required for transformation. One segment, near the 5' end of the 69% transforming fragment, probably represents a control element of the viral DNA. The second segment, which lies within the 3' end of the 69% fragment, encodes transforming sequences of the viral DNA. A retroviral control element (the long terminal repeat DNA. A retroviral control element (the long terminal repeat DNA of Harvey murine sarcoma virus) will activate the 2.3-kb segment at the 3' end of the 69% fragment to transform the mouse cells.

Animals↗

PubMed1994

Repression of retinoic acid-induced transactivation by embryonal LTR binding protein.

The mechanism of repression of transcription by ELP, the embryonal long terminal repeat binding protein, was investigated. ELP represses the Moloney murine leukemia virus long terminal repeat by binding to a site which overlaps with a sequence element for retinoic acid receptor binding. This suggests possible competition of ELP with retinoic acid receptor for the same sequence elements. Oligonucleotides corresponding to ELP and/or retinoic acid receptor binding elements were placed upstream of the SV40 promoter and their effect on gene expression was analyzed by CAT assay. Elements which have affinity to both ELP and retinoic acid receptor were activated by retinoic acid receptor and these activations were repressed by ELP. An ELP binding element without affinity to retinoic acid receptor was insensitive to both activation by retinoic acid receptor and repression by ELP. Furthermore, cellular ELP binding elements and the Moloney leukemia virus long terminal repeat were activated by retinoic acid. These data suggest that one of the mechanism of transcriptional repression by ELP is competition for binding sites with transactivators such as retinoic acid receptors.

3T3 Cells↗

PubMed2005

The long terminal repeat-containing retrotransposon Tf1 possesses amino acids in gag that regulate nuclear localization and particle formation.

Tf1 is a long terminal repeat-containing retrotransposon of Schizosaccharomyces pombe that is studied to further our understanding of retrovirus propagation. One important application is to examine Tf1 as a model for how human immunodeficiency virus type 1 proteins enter the nucleus. The accumulation of Tf1 Gag in the nucleus requires an N-terminal nuclear localization signal (NLS) and the nuclear pore factor Nup124p. Here, we report that NLS activity is regulated by adjacent residues. Five mutant transposons were made, each with sequential tracts of four amino acids in Gag replaced by alanines. All five versions of Tf1 transposed with frequencies that were significantly lower than that of the wild type. Although all five made normal amounts of Gag, two of the mutations did not make cDNA, indicating that Gag contributed to reverse transcription. The localization of the Gag in the nucleus was significantly reduced by mutations A1, A2, and A3. These results identified residues in Gag that contribute to the function of the NLS. The Gags of A4 and A5 localized within the nucleus but exhibited severe defects in the formation of virus-like particles. Of particular interest was that the mutations in Gag-A4 and Gag-A5 caused their nuclear localization to become independent of Nup124p. These results suggested that Nup124p was only required for import of Tf1 Gag because of its extensive multimerization.

Active Transport, Cell Nucleus↗

PubMed1990

Effect of tamoxifen on regulation of viral replication and human immunodeficiency virus (HIV) long terminal repeat-directed transcription in cells chronically infected with HIV-1.

The protein kinase C (PKC) activator phorbol myristate acetate (PMA) was used to upregulate viral replication in a clone of promonocytic cells chronically infected with human immunodeficiency virus (HIV)-1. Induction of virus could be inhibited by the triphenylethylene anti-estrogen tamoxifen at concentrations that had minimal effects on cellular DNA synthetic responses and cell cycle kinetics. This effect correlated with tamoxifen's ability to block PMA-mediated enhancement of HIV-promoter-driven transactivation in cells of monocyte and CD4+ T-lymphocyte lineages. No interference with a primary infection was noted. Tamoxifen's mechanism of action may relate both to its capacity to inhibit PKC and to consensus sequences for gonadal steroid responsive elements in the HIV long terminal repeat, as it was able to partially inhibit another HIV activator, 5-azacytidine, which does not modulate PKC function. The finding that regulation of HIV in a model for low-level chronic or latent infection is amenable to a nonimmunosuppressive steroid antagonist may suggest approaches to pharmacologic intervention early in HIV infection.

Cell Cycle↗

PubMed2003

Enrichment for histone H3 lysine 9 methylation at Alu repeats in human cells.

The aim of this study was to identify in human cells common targets of histone H3 lysine 9 (H3-Lys9) methylation, a modification that is generally associated with gene silencing. After chromatin immunoprecipitation using an H3-Lys9 methylated antibody, we cloned the recovered DNA and sequenced 47 independent clones. Of these, 38 clones (81%) contained repetitive elements, either short interspersed transposable element (SINE or Alu elements), long terminal repeat (LTR), long interspersed transposable element (LINE), or satellite region (ALR/Alpha) DNA, and three additional clones were near Alu elements. Further characterization of these repetitive elements revealed that 32 clones (68%) were Alu repeats, corresponding to both old Alu (23 clones) and young Alu (9 clones) subfamilies. Association of H3-Lys9 methylation was confirmed by chromatin immunoprecipitation-PCR using conserved Alu primers. In addition, we randomly selected 5 Alu repeats from the recovered clones and confirmed association with H3-Lys9 by PCR using primer sets flanking the Alu elements. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine rapidly decreased the level of H3-Lys9 methylation in the Alu elements, suggesting that H3-Lys9 methylation may be related to the suppression of Alu elements through DNA methylation. Thus H3-Lys9 methylation is enriched at human repetitive elements, particularly Alu elements, and may play a role in the suppression of recombination by these elements.

Alu Elements↗

PubMed1982

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns.

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and characterization of an immunosuppressive and weakly oncogenic variant of Friend murine leukemia virus, FIS-2.

The FIS variant is a weakly leukemogenic, relatively strong immunosuppressive murine retrovirus which was isolated from the T helper cells of adult NMRI mice infected with Friend murine leukemia virus (F-MuLV) complex (FV). Unlike FV, it does not induce acute erythroleukemia but retains the immunosuppressive property of FV and induces suppression of the primary antibody response rapidly and persistently in adult mice. A previous study showed that the FIS variant contains two viral components, a replication-competent virus and a defective virus. In this study, we have biologically purified the FIS variant by end point dilution and we show that the replication-competent virus FIS-2 alone can induce immunosuppression as the parental FIS variant. Most newborn mice infected with FIS-2 developed erythroleukemia, but with an increased latency period compared with that of F-MuLV clone 57. In contrast, FIS-2 induced suppression of the primary antibody response and disease more rapidly than F-MuLV clone 57 in immunocompetent, adult mice. FIS-2 was further molecularly cloned and characterized. Restriction mapping and nucleotide sequence analysis of FIS-2 showed a high degree of homology between FIS-2 and F-MuLV clone 57, suggesting that FIS-2 is a variant of F-MuLV. The striking difference is the deletion of one of the tandem repeats in the FIS-2 long terminal repeat and the single point mutation in the binding sites for core-binding protein and FVa compared with the long terminal repeat of F-MuLV clone 57. Two single point mutations led to the appearance of two extra potential N glycosylation sites in the FIS-2 gag-encoded glycoprotein. Together, the results suggest that FIS-2 represents an interesting murine model to study retrovirus-induced immunosuppression on the basis of its unique combined property of low leukemogenicity and relatively strong and persistent immunosuppressive activity in adult mice.

3T3 Cells↗

PubMed1996

Repression of human immunodeficiency virus type 1 long terminal repeat-driven gene expression by binding of the virus to its primary cellular receptor, the CD4 molecule.

We have previously postulated that the binding of the human immunodeficiency virus type 1 (HIV-1) to cell surface CD4 induces signal transduction pathways that down-modulate production of progeny virions in acutely infected T cells (M. Tremblay, S. Meloche, S. Gratton, M. A. Wainberg, and R.-P. Sékaly, EMBO J. 13:774-783, 1994). To evaluate the possibility that CD4 cross-linking might indeed affect viral gene expression, we have introduced a molecular construct made of the luciferase reporter gene placed under the control of the regulatory elements of HIV-1 in several CD4-positive T-cell lines. We found that cross-linking of CD4 with defective HIV-1 particles and heat-inactivated viruses inhibits long terminal repeat-dependent luciferase expression. Experiments revealed that the gp120-CD4 interaction was necessary to repress HIV-1 long terminal repeat-dependent luciferase activity. The cytoplasmic domain of CD4 was also found to be required for this effect to occur. The virus-mediated signal transduction was shown to be mediated via p56lck-dependent and -independent pathways. These results indicate that the earliest event in the HIV-1 replicative cycle, namely, the binding of the virus to its cellular receptor, can lead to signal transduction culminating in down-modulation of viral gene expression. Thus we propose that defective viruses could regulate the pathogenesis of HIV disease as they constitute the vast majority of circulating HIV-1 particles.

CD4 Antigens↗

PubMed1990

RNA transcripts of the human immunodeficiency virus transactivation response element can inhibit action of the viral transactivator.

Tandem repeats of the transactivation response element (TAR) of the human immunodeficiency virus 1 (HIV-1) were generated using a specially constructed "tandemizing" plasmid, pGem-Tan. This plasmid exploits the rotational nonequivalence of Ava I restriction sites to generate multiple copies of an inserted sequence. Twelve tandem repeats of the TAR were then placed in sense and antisense orientations behind a strong human beta-actin gene promoter. The TAR constructs were transfected with an appropriate HIV-1-driven reporter and tat gene expression plasmids into NT2/D1 cells, a pluripotential human embryonic teratocarcinoma cell line. Twelve tandem TAR repeats in the sense orientation suppressed 85-90% of the transactivating function of the virus-encoded tat protein, whereas the antisense construct or constructs containing single copies of TAR in either sense or antisense orientations were relatively ineffective. The suppression was specific for reporter gene constructs containing an intact HIV-1 long terminal repeat: Reporter genes driven by other promoters or by an HIV-1 long terminal repeat lacking the TAR were not suppressed. Suppression of activation by tat required transcription into RNA: Similar constructs containing the TAR repeats but lacking a eukaryotic promoter failed to suppress tat activation. In the absence of tat, the TAR DNA stimulated 2- to 5-fold the expression of gene constructs driven not only by the HIV-1 long terminal repeat but also by the human beta-actin gene and the simian virus 40 promoters.

Actins↗

PubMed1990

Evidence that retroviral transduction is mediated by DNA not by RNA.

Retroviral transduction of cellular nucleic acid sequences requires illegitimate RNA or DNA recombination. To test a model that postulates transduction via efficient illegitimate recombination during reverse transcription of viral and cellular RNAs, we have measured the ability of Harvey sarcoma viruses (HaSVs) with artificial 3' termini to recover a retroviral 3' terminus from helper Moloney virus (MoV) by illegitimate and homologous recombination. For this purpose, mouse NIH 3T3 cells were transformed with Harvey proviruses and then superinfected with MoV. The proviruses lacked the 3' long terminal repeat and an untranscribed region of the 5' long terminal repeat to prevent virus regeneration from input provirus. Only 0-11 focus-forming units of HaSV were generated upon MoV superinfection of 3 x 10(6) cells transformed by Harvey proviruses with MoV-unrelated termini. This low frequency is consistent with illegitimate DNA recombination via random Moloney provirus integration 3' of the transforming viral ras gene in the 10(6)-kilobase mouse genome. When portions of murine viral envelope (env) genes were attached 3' of ras, 10(2)-10(5) focus-forming units of HaSV were generated, depending on the extent of homology with env of MoV. These recombinants all contained HaSV-specific sequences 5' and MoV-specific sequences 3' of the common env homology. They were probably generated by recombination during reverse transcription rather than by recombination among either input or secondary proviruses, since (i) the yield of recombinants was reduced by a factor of 10 when the env sequence was flanked by splice signals and (ii) HaSV RNAs without retroviral 3' termini would be inadequate templates for provirus synthesis. We conclude that there is no efficient illegitimate recombination in retroviruses. In view of known precedents of illegitimate DNA recombination, the structure of known viral onc genes, and our evidence for illegitimate DNA recombination via provirus integration, we favor the DNA model of transduction over the RNA model.

Animals↗

PubMed1988

Mineralocorticoid regulation of transcription of transfected mouse mammary tumor virus DNA in cultured kidney cells.

Renal cells contain two corticosteroid-binding entities defined on the basis of hormone-binding selectivity as type I (mineralocorticoid) and type II (glucocorticoid). The mineralocorticoid, aldosterone can bind to both type I and type II receptors. This poses problems in defining the characteristics of a true mineralocorticoid regulated expression of specific genes. We have used chimaeric constructs bearing the mouse mammary tumor virus (MMTV) promoter to study aldosterone action in the feline renal cell line CRFK. We have shown that in these cells aldosterone induces MMTV transcription through its own receptor (type I). This induction of MMTV transcription by aldosterone is a primary response to the hormone. We have shown that the DNA sequences that mediate the aldosterone response overlap the hormone response element (HRE) required for the glucocorticoid, progestin, and androgen induction of transcription at the MMTV long terminal repeat region. Thus the aldosterone regulation of MMTV long terminal repeat transcription is identical to the mode of action of the other steroid hormones at this promoter.

Aldosterone↗

PubMed1993

Herpes simplex virus type 1 latency-associated transcript (LAT) promoter deletion mutants can express a 2-kilobase transcript mapping to the LAT region.

The results of studies in several laboratories suggest that a TATA box-containing promoter located in the herpes simplex virus type 1 internal long repeat and long terminal repeat elements drives expression of the latency-associated transcripts (LATs). In the present study, we show that expression of a 2-kb LAT-related transcript can occur in the absence of this LAT TATA promoter, indicating the existence of a cryptic promoter. By Northern (RNA) blot analysis, we have examined LAT expression by herpes simplex virus type 1 variant strains KOS/29 and 1704, which contain deletions of the LAT promoter region. Our data indicate that KOS/29, despite lacking the 203-bp fragment which contains the LAT TATA box, can express a 2-kb LAT-related transcript during productive infection in tissue culture and in mouse trigeminal ganglia during acute infection and reactivation. Similarly, strain 1704, which contains a larger deletion in this promoter region, also expresses a 2-kb LAT-related transcript during tissue culture infection and reactivation of latently infected trigeminal ganglia. However, LATs are not expressed with either virus during latency. Northern blot analysis with a single-stranded, oligonucleotide probe demonstrates that the 2-kb LAT and LAT-related transcript are colinear and share a large area of sequence similarity. These findings suggest the existence of a second promoter in the LAT gene which can function during lytic infection and reactivation, at least in the absence of the LAT TATA promoter. We propose that this cryptic promoter is located either in a proximal region approximately 300 bp upstream of the start site of the 2-kb LAT or in a distal region starting over 1,226 bp upstream of this site.

Animals↗

PubMed1995

Human foamy virus Bel1 transactivator contains a bipartite nuclear localization determinant which is sensitive to protein context and triple multimerization domains.

The Bel1 protein of human foamy virus is a 300-amino-acid nuclear regulatory protein which transactivates the gene expression directed by the homologous long terminal repeat and the human immunodeficiency virus type 1 long terminal repeat. While previous reports suggested that the single basic domain of Bel1 from residues 211 to 222 and/or 209 to 226 is necessary and sufficient for efficient nuclear localization (L. K. Venkatesh, C. Yang, P. A. Theodorakis, and G. Chinnandurai, J. Virol. 67:161-169, 1993; F. He, J. D. Sun, E. D. Garrett, and B. R. Cullen, J. Virol. 67:1896-1904, 1993), our recent data showed that another basic domain, from amino acid residues 199 to 200, is also required for nuclear localization of Bel1 (C. W. Lee, C. Jun, K. J. Lee, and Y. C. Sung, J. Virol. 68:2708-2719, 1994). To clarify this discrepancy, we constructed various bel1-lacZ chimeric constructs and several linker insertion mutants and determined their subcellular localization. When the region of Bel1 containing basic domains was placed at an internal site of the lacZ gene, the nuclear localization signal (NLS) of Bel1 consisted of two discontinuous basic regions separated by an intervening sequence. Moreover, insertion of specific amino acids between two basic regions disrupted the activity of the Bel1 NLS. On the other hand, Bel1 residues 199 and 200 were not required to direct the Bel1-beta-galactosidase chimeric protein to the nucleus when the Bel1 NLS was appended to the amino terminus of beta-galactosidase. These results indicate that the function of the Bel1 NLS is sensitive to the protein context within which the sequence is present. In addition, we demonstrated that the Bel1 protein forms a multimeric complex in the nuclei of mammalian cells by using a sensitive in vivo protein-protein interaction assay. Mutational analyses revealed that the regions which mediate multimer formation map to three domains of Bel1, i.e., residues 1 to 31, 42 to 82, and 82 to 111. Furthermore, our results show that the region of Bel1 from residues 202 to 226 prevents Bel1 from forming a multimeric complex.

Amino Acid Sequence↗

PubMed2006

Inhibition of LINE-1 expression in the heart decreases ischemic damage by activation of Akt/PKB signaling.

Microarray analyses indicate that ischemic and pharmacological preconditioning suppress overexpression of the non-long terminal repeat retrotransposon long interspersed nuclear element 1 (LINE-1, L1) after ischemia-reperfusion in the rat heart. We tested whether L1 overexpression is mechanistically involved in postischemic myocardial damage. Isolated, perfused rat hearts were treated with antisense or scrambled oligonucleotides (ODNs) against L1 for 60 min and exposed to 40 min of ischemia followed by 60 min of reperfusion. Functional recovery and infarct size were measured. Effective nuclear uptake was determined by FITC-labeled ODNs, and downregulation of L1 transcription was confirmed by RT-PCR. Immunoblot analysis was used to assess changes in expression levels of the L1-encoded proteins ORF1p and ORF2p. Immunohistochemistry was performed to localize ORF1/2 proteins in cardiac tissue. Effects of ODNs on prosurvival protein kinase B (Akt/PKB) expression and activity were also determined. Antisense ODNs against L1 prevented L1 burst after ischemia-reperfusion. Inhibition of L1 increased Akt/PKBbeta expression, enhanced phosphorylation of PKB at serine 473, and markedly improved postischemic functional recovery and decreased infarct size. Antisense ODN-mediated protection was abolished by LY-294002, confirming the involvement of the Akt/PKB survival pathway. ORF1p and ORF2p were found to be expressed in rat heart. ORF1p showed a predominantly nuclear localization in cardiomyocytes, whereas ORF2p was exclusively present in endothelial cells. ORF1p levels increased in response to ischemia, which was reversed by antisense ODN treatment. No significant changes in ORF2p were noted. Our results demonstrate that L1 suppression favorably affects postischemic outcome in the heart. Modifying transcriptional activity of L1 may represent a novel anti-ischemic therapeutic strategy.

Animals↗