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Joachim Hauber - One of the best experts on this subject based on the ideXlab platform.
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Titration of cellular export factors, but not heteromultimerization, is the molecular mechanism of trans-dominant HTLV-1 Rex mutants
Oncogene, 1999Co-Authors: Peter Heger, Joachim Hauber, Olaf Rosorius, Roland H StauberAbstract:The HTLV-1 Rex Protein is an essential shuttle Protein required for nuclear export of unspliced and incompletely-spliced viral RNAs. Several trans -dominant (TD) mutant Rex Proteins have been reported, however, the mechanism of trans -dominance is not known. We compared TD Rex mutants and found that a natural occurring Rex mutant, Rexp21, lacking the RNA binding domain, was highly TD and inhibited also HIV-1 Rev function. Using fusions to the green fluorescent Protein (GFP) we observed that Rexp21-GFP displayed a cytoplasmic localization but was actively shuttling between the nucleus and the cytoplasm in live human cells. The presence of Rexp21-GFP inhibited the nuclear export of Rex and HIV-1 Rev as assayed by cotransfection and microinjection experiments. However, Rex-GFP or Rexp21-GFP did not form heteromultimers with nuclear Rex mutants in vivo . In contrast, shuttling was essential for trans -dominance. Thus, we propose that TD Rex mutants do not function by retaining WT Rex in the nucleus by Protein-Protein interactions, as demonstrated for Rev, but to titrate factors essential for Rex/Rev export. Our findings demonstrate differences between the regulatory Proteins Rex and Rev and implicate a novel strategy to generate highly TD Rex mutants also applicable to other Proteins.
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Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex Protein: implications for trans dominance.
Journal of Virology, 1992Co-Authors: I Weichselbraun, Marika Dobrovnik, Hal P. Bogerd, Ralph Grassmann, Joachim Hauber, Johannes Berger, Warner C. Greene, E BöhnleinAbstract:Abstract The 27-kDa Rex trans-acting Protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative Proteins. We generated missense mutations in a particular domain of the Rex Protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative Proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion Proteins demonstrated that all of the mutant Rex Proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev Proteins suggests that this Rex domain is important for oligomerization.
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Definition of the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex Protein activation domain by functional exchange.
Journal of virology, 1992Co-Authors: I Weichselbraun, E Böhnlein, G K Farrington, J R Rusche, Joachim HauberAbstract:The human retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are characterized by complex regulation of gene expression. Each virus encodes a posttranscriptional regulator, the 19-kDa HIV-1 Rev Protein and the 27-kDa HTLV-I Rex Protein, which is required for viral replication. Expression of these trans activators results in the cytoplasmic accumulation of unspliced or singly spliced viral mRNA which encode the gag, pol, and env gene products. The finding that the HTLV-I Rex Protein is able to functionally substitute for the Rev Protein of HIV-1 indicates that HIV-1 Rev and HTLV-I Rex may interact with the same component of a cellular pathway involved in either mRNA splicing or transport. In this study, we have generated functional Rev/Rex hybrid Proteins by domain exchange. We have defined, using in vivo and in vitro analyses, the activation domains of Rev and Rex which are the putative targets of a common host cell factor(s) required for Rev and Rex function.
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Functional analysis of human T-cell leukemia virus type I Rex-response element: direct RNA binding of Rex Protein correlates with in vivo activity.
Journal of virology, 1991Co-Authors: C Ballaun, Marika Dobrovnik, Joachim Hauber, G K Farrington, J R Rusche, E BöhnleinAbstract:The human T-cell leukemia virus type I Rex gene product plays a critical role in the expression of the retroviral structural Proteins Gag and Env from incompletely spliced mRNAs. Rex Protein acts through a cis element (Rex-response element [RxRE]) which is located in the U3/R region of the 3' long terminal repeat and is present on all human T-cell leukemia virus type I-specific mRNAs. Two domains of the predicted secondary structure of the RxRE are crucially important for Rex action in vivo as measured by two assay systems. In vitro studies using highly purified recombinant Rex Protein revealed a specific and direct interaction with radiolabeled RxRE sequences. The correlation between our in vivo results and the direct binding of Rex Protein to mutant and wild-type RxRE sequences supports both the existence of the predicted secondary structure and the importance of this direct interaction with the cis-acting RNA sequence for Rex function in vivo.
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Mutational analysis of the human T-cell leukemia virus type I trans-acting Rex gene product.
Journal of virology, 1991Co-Authors: L Hofer, I Weichselbraun, E Böhnlein, G K Farrington, S Quick, Joachim HauberAbstract:Expression of the human T-cell leukemia virus type I (HTLV-I) Rex gene is a prerequisite for the expression of the retroviral structural Proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex Protein. The phenotype of the various mutant Proteins was tested on the homologous HTLV-I Rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex Protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex Protein to the correct subcellular compartment required for Rex function.
E Böhnlein - One of the best experts on this subject based on the ideXlab platform.
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Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex Protein: implications for trans dominance.
Journal of Virology, 1992Co-Authors: I Weichselbraun, Marika Dobrovnik, Hal P. Bogerd, Ralph Grassmann, Joachim Hauber, Johannes Berger, Warner C. Greene, E BöhnleinAbstract:Abstract The 27-kDa Rex trans-acting Protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative Proteins. We generated missense mutations in a particular domain of the Rex Protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative Proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion Proteins demonstrated that all of the mutant Rex Proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev Proteins suggests that this Rex domain is important for oligomerization.
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Definition of the human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex Protein activation domain by functional exchange.
Journal of virology, 1992Co-Authors: I Weichselbraun, E Böhnlein, G K Farrington, J R Rusche, Joachim HauberAbstract:The human retroviruses human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) are characterized by complex regulation of gene expression. Each virus encodes a posttranscriptional regulator, the 19-kDa HIV-1 Rev Protein and the 27-kDa HTLV-I Rex Protein, which is required for viral replication. Expression of these trans activators results in the cytoplasmic accumulation of unspliced or singly spliced viral mRNA which encode the gag, pol, and env gene products. The finding that the HTLV-I Rex Protein is able to functionally substitute for the Rev Protein of HIV-1 indicates that HIV-1 Rev and HTLV-I Rex may interact with the same component of a cellular pathway involved in either mRNA splicing or transport. In this study, we have generated functional Rev/Rex hybrid Proteins by domain exchange. We have defined, using in vivo and in vitro analyses, the activation domains of Rev and Rex which are the putative targets of a common host cell factor(s) required for Rev and Rex function.
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Transdominant repressors for human T-cell leukemia virus type I Rex and human immunodeficiency virus type 1 rev function.
Journal of Virology, 1991Co-Authors: S Böhnlein, E Böhnlein, L Hofer, F P Pirker, Klaus Zimmermann, H Bachmayer, J HauberAbstract:: Human T-cell leukemia virus type I (HTLV-I) encodes a 27-kDa trans-acting gene product (Rex) which is involved in the regulated expression of transcripts coding for the viral structural Proteins. We used oligonucleotide-directed mutagenesis to generate a series of mutant HTLV-I Rex genes. Transient expression experiments demonstrated that 3 of 28 mutant Proteins are functionally inactive on the homologous HTLV-I Rex response element, whereas an additional 2 mutant Proteins are functionally inactive on the heterologous human immunodeficiency virus type 1 rev response element. One of these mutants is able to suppress the function of the wild-type HTLV-I Rex Protein in trans on the homologous Rex response element sequence. Furthermore, all of these mutants are able to inhibit Rex function on the heterologous rev response element sequence. Intriguingly, only three of these mutants are able to inhibit the human immunodeficiency virus type 1 Rev Protein in a dominant-negative manner.
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Functional analysis of human T-cell leukemia virus type I Rex-response element: direct RNA binding of Rex Protein correlates with in vivo activity.
Journal of virology, 1991Co-Authors: C Ballaun, Marika Dobrovnik, Joachim Hauber, G K Farrington, J R Rusche, E BöhnleinAbstract:The human T-cell leukemia virus type I Rex gene product plays a critical role in the expression of the retroviral structural Proteins Gag and Env from incompletely spliced mRNAs. Rex Protein acts through a cis element (Rex-response element [RxRE]) which is located in the U3/R region of the 3' long terminal repeat and is present on all human T-cell leukemia virus type I-specific mRNAs. Two domains of the predicted secondary structure of the RxRE are crucially important for Rex action in vivo as measured by two assay systems. In vitro studies using highly purified recombinant Rex Protein revealed a specific and direct interaction with radiolabeled RxRE sequences. The correlation between our in vivo results and the direct binding of Rex Protein to mutant and wild-type RxRE sequences supports both the existence of the predicted secondary structure and the importance of this direct interaction with the cis-acting RNA sequence for Rex function in vivo.
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Mutational analysis of the human T-cell leukemia virus type I trans-acting Rex gene product.
Journal of virology, 1991Co-Authors: L Hofer, I Weichselbraun, E Böhnlein, G K Farrington, S Quick, Joachim HauberAbstract:Expression of the human T-cell leukemia virus type I (HTLV-I) Rex gene is a prerequisite for the expression of the retroviral structural Proteins. We have generated internal deletion mutants of this 27-kDa nucleolar trans-acting gene product to define functional domains in the Rex Protein. The phenotype of the various mutant Proteins was tested on the homologous HTLV-I Rex response element sequence and the heterologous human immunodeficiency virus type 1 (HIV-1) rev response element sequence. Our results indicate that a region between amino acid residues 55 and 132 in the 189-amino-acid Rex Protein is required for Rex-mediated trans activation on both retroviral response element sequences. In addition, substitution of the Rex nuclear localization signal by a sequence of the HIV-1 rev gene product targets the Rex Protein to the correct subcellular compartment required for Rex function.
Hisatoshi Shida - One of the best experts on this subject based on the ideXlab platform.
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Interaction of human T-cell lymphotropic virus type I Rex Protein with Dicer suppresses RNAi silencing.
FEBS letters, 2010Co-Authors: Makoto Abe, Hisatoshi Shida, Hitoshi Suzuki, Hironori Nishitsuji, Hiroshi TakakuAbstract:Double-stranded RNAs suppress the expression of homologous genes through an evolutionarily conserved process called RNA interference (RNAi) or post-transcriptional gene silencing. A bidentate nuclease called Dicer has been implicated as the Protein responsible for the production of short interfering RNAs (siRNAs). In our experiments, Rex oveRexpression reduced the efficiency of short hairpin RNA (shRNA)-mediated RNAi. The interaction of Dicer with Rex inhibited the conversion of shRNA to siRNA. These results suggest that the interaction of Dicer with HTLV-I Rex inhibits Dicer activity and thereby reduces the efficiency of the conversion of shRNA to siRNA.
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a multifunctional domain in human crm1 exportin 1 mediates ranbp3 binding and multimerization of human t cell leukemia virus type 1 Rex Protein
Molecular and Cellular Biology, 2003Co-Authors: Yoshiyuki Hakata, Masami Yamada, Hisatoshi ShidaAbstract:Human CRM1 (hCRM1) functions in the Rex-mediated mRNA export of human T-cell leukemia virus type 1 (HTLV-1) as an export receptor and as an inducing factor for Rex multimerization on its cognate RNA. Although there are only 24 amino acid differences between hCRM1 and rat CRM1 (rCRM1), rCRM1 can hardly support Rex activity, suggesting a role for rCRM1 as a determinant restricting the host range of HTLV-1. Here, we used a series of mutants, which were generated by interchanging residues of these CRM1s, to examine the relationship of hCRM1 functions. The functions for Rex multimerization and binding to nuclear export signals are mapped to different amino acid residues, and these are separable, suggesting that CRM1 not only functions as an export receptor but also participates in the formation of the RNA export complex through higher-ordered interaction with Rex. The region for the interaction with RanBP3, comprising four residues (amino acids [aa] 411, 414, 474, and 481), and the region for Rex multimerization, including two residues (aa 411 and 414), form an overlapped domain. Our results provide the molecular basis underlying the species-specific ability of HTLV-1 to propagate in human cells.
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Rat CRM1 Is Responsible for the Poor Activity of Human T-Cell Leukemia Virus Type 1 Rex Protein in Rat Cells
Journal of virology, 2001Co-Authors: Yoshiyuki Hakata, Masami Yamada, Hisatoshi ShidaAbstract:Rat models of human T-cell leukemia virus type 1 (HTLV-1)-related diseases such as adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis have been reported. However, these models do not completely reproduce human diseases partly because HTLV-1 replicates poorly in rats. We investigated here the possible reason for this. We found that the activity of Rex in rat cells is quite low compared to that in human cells. As Rex function depends largely on the CRM1 Protein, whose human type (human CRM1 [hCRM1]) directly binds to Rex and exports it from the nucleus to the cytoplasm, we assessed whether rat CRM1 (rCRM1) could act as well as hCRM1 as a cofactor for Rex activity. We first cloned a cDNA encoding rCRM1 and found that both rCRM1 and hCRM1 could bind to and export Rex Protein to the cytoplasm with similar efficiencies. However, unlike hCRM1, rCRM1 could hardly support Rex function because of its poor ability in inducing the Rex-Rex interaction required for RNA export into the cytoplasm. These observations suggest that the poor ability of rCRM1 to act as a cofactor for Rex function may be responsible for the poor replication of HTLV-1 in rats.
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involvement of human crm1 exportin 1 in the export and multimerization of the Rex Protein of human t cell leukemia virus type 1
Journal of Virology, 1998Co-Authors: Yoshiyuki Hakata, Tomoe Umemoto, Shuzo Matsushita, Hisatoshi ShidaAbstract:We investigated the role of human CRM1 (hCRM1) (exportin 1) in the function of Rex Protein encoded by human T-cell leukemia virus type 1. hCRM1 promoted the export of Rex Protein from the nucleus to the cytoplasm. A Rex Protein with a mutation in the activation domain, RexM90, lost both the ability to bind to hCRM1 and the ability to multimerize. The oveRexpression of hCRM1 complemented the functional defects of RexM64, which contains a mutation in the multimerization domain of Rex. A dominant-negative mutant of Rex which sequesters cofactors of Rex abrogated multimerization as well as the activity of the wild-type Rex Protein. These two functions were simultaneously restored by the oveRexpression of hCRM1. Taken together, these results suggest that hCRM1 plays important roles in the multimerization and export of Rex Protein.
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Two distinct pathways for intronless mRNA expression: One related, the other unrelated to human immunodeficiency virus Rev and human T cell leukemia virus type I Rex functions
Biological signals, 1997Co-Authors: Takahiro Kiyokawa, Tomoe Umemoto, Shuzo Matsushita, Yoshihiko Watanabe, Hisatoshi ShidaAbstract:Intronless mRNAs were classified into two classes based on the sensitivities of their expression to the inhibitory effect of TAgRex, a dominant-negative mutant of the Rex Protein of human T cell leukemia virus type I, and their abilities to express the genes encoded in the intron of the human immunodeficiency virus (HIV) genome. Interferon-α mRNA could not induce the expression of the env gene of HIV, and its expression was resistant to TAgRex. In contrast, the posttranscriptional regulatory element (PRE), necessary for the nucleo-cytoplasmic export of mRNAs of hepatitis B virus, induced expression of the chloramphenicol acetyl transferase gene located within the intron of the HIV genome. PRE-mediated expression was inhibited by TAgRex. Thus, these results suggest that there are at least two distinct pathways for intronless mRNA expression, one related to and the other unrelated to Rev and Rex functions.
Masakazu Hatanaka - One of the best experts on this subject based on the ideXlab platform.
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Nucleo-cytoplasmic redistribution of the HTLV-I Rex Protein: alterations by coexpression of the HTLV-I p21x Protein.
Virology, 1996Co-Authors: Satoshi Kubota, Masakazu Hatanaka, Roger J. PomerantzAbstract:Abstract The function of the Rex Protein of human T-cell leukemia virus type I (HTLV-I) has been demonstrated to be very similar to the Rev Protein of human immunodeficiency virus type 1 (HIV-1). Both of these retroviral regulatory Proteins rescue unspliced viral RNAs from the nuclei of infected cells. The Rev Protein of HIV-1 has been reported to shuttle between the nucleus/nucleolus and the cytoplasm. Here, we have found that Rex also relocated out of the nucleus in the presence of actinomycin D. This effect was demonstrated in dose- and time-course-dependent manners. In comparison with previous reports on HIV-1 Rev, these effects with Rex seemed to be similar, but less distinct, which may reflect precise differences in the subcellular localization and/or shuttling pathways of Rev and Rex. Interestingly, the endogenous truncated form of the Rex Protein, p21 x , significantly interfered with the intracellular translocation of Rex, when coexpressed in trans. As expression of p21 x occurs in various HTLV-I-infected cells, p21 x may play a role in the life-cycle of HTLV-I, through regulating the dynamic subcellular distribution of the viral trans-activator, Rex.
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Nucleolar targeting signal of Rex Protein of human T-cell leukemia virus type I specifically binds to nucleolar shuttle Protein B-23.
The Journal of biological chemistry, 1993Co-Authors: Yoshifumi Adachi, Terry D. Copeland, Masakazu Hatanaka, Stephen OroszlanAbstract:Rex Protein, the post-transcriptional regulator of human T-cell leukemia virus type I, is located predominantly in the cell nucleolus and is associated with the cytoplasmic accumulation of unspliced and singly spliced viral mRNAs. The N-terminal 19-amino acid segment of Rex has been identified as the nucleolar targeting signal (NOS) and shown to be important for Rex function. To study the molecular interaction between the NOS region of Rex and its binding host Protein(s) in the nucleolus, we chemically synthesized a functional NOS peptide (wild type) and mutant NOS peptides. Fluorescein isothiocyanate-conjugated functional NOS peptide was rapidly taken up by human cells and was transported to the nucleolus. Using affinity chromatography, we identified nucleolar Protein B-23 as the major Protein that binds to NOS. We also identified two highly acidic regions of B-23 (amino acids 120-132 and 161-188) as acceptor regions for NOS. Previous experiments have suggested that B-23 functions as a shuttle Protein for the nucleolar transport of ribosomal components. Our results suggest that B-23 may also serve as a shuttle for the import of Rex from the cytoplasm to the nucleolus coupled to the export of viral mRNAs containing the Rex-responsive element.
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Phosphorylation of the Rex Protein of human T-cell leukemia virus type I.
The Journal of biological chemistry, 1992Co-Authors: Yoshifumi Adachi, Terry D. Copeland, Chiaki Takahashi, Tetsuya Nosaka, Aftab Ahmed, Stephen Oroszlan, Masakazu HatanakaAbstract:Abstract Rex Protein, the posttranscriptional regulator of human T-cell leukemia virus type I (HTLV-I), is required for the control of viral structural Protein expression and virus replication. Rex is a phosphoProtein found predominantly in the cell nucleolus, whose function is thought to be regulated by its nucleolar localization and phosphorylation. Therefore, we investigated the in vivo phosphorylation of Rex Protein in more detail. Phosphorylation of Rex occurred in all HTLV-I-infected cell lines examined in vivo, primarily at serine residues and to a very small extent at threonine residues. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) led to significant but transient enhancement of the incorporation of [32P]orthophosphate into Rex Protein. N-terminal truncation of Rex Protein abolished TPA-dependent phosphorylation. Chymotryptic digestion of phosphorylated Rex yielded two phosphopeptides. In vivo phosphorylation sites were identified as serine residues 70 and 177 and threonine residue 174. Serine 70 was a TPA-dependent phosphorylation site within a regulatory domain. We have already shown that the Protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) specifically blocked accumulation of viral unspliced gag-pol mRNA. Therefore, the phosphorylation at serine 70 may be involved in the regulation of Rex function in response to extracellular stimuli.
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The nucleolar localisation signal of the HTLV-I Protein p27Rex is important for stabilisation of IL-2 receptor α subunit mRNA by p27Rex
Biochemical and biophysical research communications, 1991Co-Authors: Kenneth N. White, Tetsuya Nosaka, Masakazu Hatanaka, Hiroshi Kanamori, Tasuku HonjoAbstract:In this study we investigated the mechanism of stabilisation of IL-2 receptor alpha subunit mRNA by the HTLV-I Protein p27Rex. We tested the role of the nucleolar targetting signal in Rex by introducing mutations. Three deletion mutants could not express Rex Protein in the nucleolus and although Protein was still expressed in the nucleoplasm none of the mutants could stabilise IL-2R alpha mRNA. A substitution mutant could be expressed in the nucleolus and could also stabilise IL-2R alpha mRNA. The data show that the nucleolar targetting signal is crucial for stabilisation of IL-2R alpha mRNA by Rex and raise the possibility that transport of mRNA from nucleus to cytoplasm can involve the nucleolus.
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Effects of chimeric mutants of human immunodeficiency virus type 1 Rev and human T-cell leukemia virus type I Rex on nucleolar targeting signals.
Journal of virology, 1991Co-Authors: Satoshi Kubota, Bryan R. Cullen, Tetsuya Nosaka, Masatoshi Maki, Masakazu HatanakaAbstract:Two chimeric mutant genes derived from rev of human immunodeficiency virus type 1 and Rex of human T-cell leukemia virus type I were constructed to investigate the functions of the nucleolar-targeting signals (NOS) in Rev and Rex Proteins. A chimeric Rex Protein whose NOS region was substituted with the NOS of Rev was located predominantly in the cell nucleolus and functioned like the wild-type Protein in the Rex assay system. However, a chimeric Rev with the NOS of Rex abolished Rev function despite its nucleolar localization. This nonfunctional nucleolar-targeting chimeric Protein inhibited the function of both Rex and Rev. In the same experimental conditions, this mutant interfered with the localization of the functional Rex in the nucleolus.
Ralph Grassmann - One of the best experts on this subject based on the ideXlab platform.
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The HTLV-1 Rex Protein Induces Nuclear Accumulation of Unspliced Viral RNA by Avoiding Intron Excision and Degradation
Virology, 1996Co-Authors: Monika Gröne, Claudia Koch, Ralph GrassmannAbstract:Abstract The human T-cell leukemia virus (HTLV-1) Rex Protein is essential for the cytoplasmic accumulation of incompletely spliced transcripts that code for the viral structural Proteins. In this study effects of Rex on the amounts of total, spliced, and unspliced RNA from HTLV-1 were determined. In transfected fibroblasts Rex production resulted in reduced amounts of spliced RNA and increased quantities of unspliced RNA in the nucleus. However, the total amount of viral RNA was not affected and the stability of spliced transcripts was not changed, thus indicating that only the rate of splicing was reduced. Rex action also reduced splicing in immortalized human cord blood T-cells. However, the total amount of viral transcripts and the stability of unspliced RNA in these cells were also increased in the presence of Rex. This indicates that Rex also prevents the degradation of unspliced transcripts in T-cells. The changes in the relative amounts of spliced and unspliced RNA induced by Rex were observed not only in the cytoplasm but also in the nucleus. Thus Rex affects the nucleocytoplasmic transport, splicing, and stability of HTLV-1 RNA in the nucleus. These observations may suggest that Rex directs the unspliced viral RNA to the cytoplasm via a nuclear compartment that is not accessible to splicing and degradation factors.
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A single stem-loop structure within the HTLV-1 Rex response element is sufficient to mediate Rex activity in vivo
Virology, 1994Co-Authors: Monika Gröne, Bryan R. Cullen, Susanne Berchtold, Erich Hoffmann, Ralph GrassmannAbstract:Abstract The human T-cell leukemia virus (HTLV-1) Rex Protein is required for the cytoplasmic expression of the incompletely spliced transcripts that encode the vital structural Proteins. This effect is mediated by a highly structured cis -acting RNA element of 254 nucleotides termed the Rex response element, or RexRE. Here we demonstrate that one of the four known RexRE stem-loop structures as well as a 43-nt segment derived from this element is sufficient to mediate Rex function in vivo. Upon duplication, this stem-loop is shown to function as efficiently as the full-length RexRE. In vitro RNA binding analyses with wildtype and mutagenized RNA show that this stem-loop contains a high affinity binding site for Rex that coincides with a predicted bulge structure in the central part of this stem-loop. These results indicate that a small region of the RexRE containing a high affinity binding site is sufficient to mediate Rex function and suggest that sequences outside of this binding site have no unique role in mediating Rex regulation.
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Dominant-negative mutants are clustered in a domain of the human T-cell leukemia virus type I Rex Protein: implications for trans dominance.
Journal of Virology, 1992Co-Authors: I Weichselbraun, Marika Dobrovnik, Hal P. Bogerd, Ralph Grassmann, Joachim Hauber, Johannes Berger, Warner C. Greene, E BöhnleinAbstract:Abstract The 27-kDa Rex trans-acting Protein appears to be essential for replication of human T-cell leukemia virus type I. Mutations introduced outside of the Rex RNA-binding domain-nucleolar localization signal display either wild-type activity or, conversely, yield dominant-negative Proteins. We generated missense mutations in a particular domain of the Rex Protein (amino acid residues 54 to 69) which is characterized by a cluster of dominant-negative mutants. Our results indicate that amino acids 57 to 67 are critically important for Rex function mediated through the RxRE cis-acting RNA sequence. Within this domain, only amino acids 61 to 63 could be mutated without loss of function. All other missense and deletion mutants yielded dominant-negative Proteins. In vitro RNA-binding studies performed with glutathione S-transferase-Rex fusion Proteins demonstrated that all of the mutant Rex Proteins interacted specifically with RxRE RNA. Analysis of chimeric Rex-Rev Proteins suggests that this Rex domain is important for oligomerization.
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role of human t cell leukemia virus type 1 x region Proteins in immortalization of primary human lymphocytes in culture
Journal of Virology, 1992Co-Authors: Ralph Grassmann, Bernhard Fleckenstein, S Berchtold, I Radant, M Alt, Joseph Sodroski, William A Haseltine, U RamstedtAbstract:Human T-cell leukemia virus type 1 (HTLV-1) immortalizes human CD4+ T lymphocytes in culture. Previous studies show that in the context of a herpesvirus saimiri vector, the sequence of the X region at the 3' end of the HTLV-1 genome is also capable of immortalizing CD4+ lymphocytes in the absence of HTLV-1 structural Proteins. The X region of HTLV-1 encodes two trans-acting viral Proteins, the 42-kDa Tax Protein and the 27-kDa Rex Protein. Infection of human cord blood cells with herpesvirus saimiri recombinants which contain HTLV-1 X region sequences defective for expression of tax, Rex, or both tax and Rex demonstrates that tax function is necessary and sufficient for immortalization of primary human CD4+ cord blood lymphocytes in culture in the context of the herpesvirus saimiri vector.
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In vitro binding of human T-cell leukemia virus Rex Proteins to the Rex-response element of viral transcripts.
Journal of virology, 1991Co-Authors: Ralph Grassmann, C Ballaun, Susanne Berchtold, C Aepinus, E Boehnlein, Bernhard FleckensteinAbstract:Abstract Human T-cell leukemia virus (HTLV-I, HTLV-II) Rex Protein function is required for the cytoplasmic expression of incompletely spliced viral transcripts encoding structural Proteins. The effect is mediated by a cis-acting Rex-response element (RRX) which is located near the 3' end of all viral mRNAs. We show that Rex polypeptides of HTLV-I and HTLV-II expressed in Escherichia coli are capable of specifically binding RRX-containing transcripts of both viruses in cell-free assays. Binding analyses with deletion variants of Rex Proteins revealed a domain with RNA-binding activity in the first 77 N-terminal amino acids. Removal of a basic peptide of 19 amino acids from the N terminus abrogated RNA binding, whereas a beta-galactosidase fusion Protein containing this peptide bound to the RRX. These results suggest that direct binding of Rex Protein to the RRX is important for Rex-mediated regulation of viral gene expression and that a short stretch of positively charged amino acids contributes to the specific binding of Rex to its target RNA.