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Tat是人免疫缺陷病毒(HIV)基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制HIV复制的途径,构建了以HIV-1LTR(-158-+80)为启动子的Tat cDNA全长反义表达质粒pAS-Tat,并用已经构建的HIV LTR-158到+80为启动子,具有不同突变点的突变Tat基因表达质粒,以荧光酶基因为报告基因,共转染Jurkat细胞,结果发现无论是反义Tat表达质粒还  相似文献   

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D Harrich  C Hsu  E Race    R B Gaynor 《Journal of virology》1994,68(9):5899-5910
The human immunodeficiency virus type 1 (HIV-1) TAR element is critical for the activation of gene expression by the transactivator protein, Tat. Mutagenesis has demonstrated that a stable stem-loop RNA structure containing both loop and bulge structures transcribed from TAR is the major target for tat activation. Though transient assays have defined elements critical for TAR function, no studies have yet determined the role of TAR in viral replication because of the inability to generate viral stocks containing mutations in TAR. In the current study, we developed a strategy which enabled us to generate stable 293 cell lines which were capable of producing high titers of different viruses containing TAR mutations. Viruses generated from these cell lines were used to infect both T-lymphocyte cell lines and peripheral blood mononuclear cells. Viruses containing TAR mutations in either the upper stem, the bulge, or the loop exhibited dramatically decreased HIV-1 gene expression and replication in all cell lines tested. However, we were able to isolate lymphoid cell lines which stably expressed gene products from each of these TAR mutant viruses. Though the amounts of virus in these cell lines were roughly equivalent, cells containing TAR mutant viruses were extremely defective for gene expression compared with cell lines containing wild-type virus. The magnitude of this decrease in viral gene expression was much greater than previously seen in transient expression assays using HIV-1 long terminal repeat chloramphenicol acetyltransferase gene constructs. In contrast to the defects in viral growth found in T-lymphocyte cell lines, several of the viruses containing TAR mutations were much less defective for gene expression and replication in activated peripheral blood mononuclear cells. These results indicate that maintenance of the TAR element is critical for viral gene expression and replication in all cell lines tested, though the cell type which is infected is also a major determinant of the replication properties of TAR mutant viruses.  相似文献   

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Expression of the human immunodeficiency virus type 1 (HIV-1) is strongly activated by Tat. The proper action of Tat requires three elements: TATAA, TAR, and upstream motifs in the HIV-1 long terminal repeat. We show here that the correct spatial arrangement among Tat, Sp1, and TATAA crucially influences HIV expression. Under conditions in which basal promoter activity is unperturbed, distancing Sp1 from TATAA markedly affected Tat trans activation. An increase in the Sp1-TATAA distance from 18 to 101 nucleotides (depending on the inserted sequence) rendered HIV-1 either partially or wholly replication defective. This critical dependence on spacing suggests that Tat-, Sp1-, and TATAA-binding factors must correctly contact each other for optimal expression and replication of HIV-1.  相似文献   

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The trans-activator Tat proteins coded by human immunodeficiency virus type 1 (HIV-1) and HIV-2 appear to be similar in structure and function. However, the Tat protein of HIV-2 (Tat2) activates the HIV-1 long terminal repeat (LTR) less efficiently than Tat1 (M. Emerman, M. Guyader, L. Montagnier, D. Baltimore, and M. A. Muesing, EMBO J. 6:3755-3760, 1987). To determine the functional domain of Tat2 which contributes to this incomplete reciprocity, we have carried out domain substitution between Tat1 and Tat2 by exchanging the basic domains involved in Tat interaction with its target trans-activation-response (TAR) RNA structure. Our results indicate that Tat1 proteins containing substitutions of either 8 or 14 amino acids of the basic domain of Tat2 exhibited reduced trans activation of the HIV-1 LTR by about 1/20 or one-fourth the level induced by wt Tat1. In contrast, Tat2 containing a substitution of the 9-amino-acid basic domain of Tat1 trans activated HIV-1 LTR like native Tat1. A substitution of the highly conserved core domain of Tat2 with that of Tat1 did not have any significant effect on trans activation of the HIV-1 LTR. These results indicate that the basic domain of Tat2 contributes to its inefficient trans activation of the HIV-1 LTR. Mutation of an acidic residue (Glu) located between the core domain and the Arg-rich basic domain of Tat2 at position 77 to a Gly residue increased the activity of Tat2 substantially. These results further suggest that the presence of an acidic residue (Glu) adjacent to Arg-rich sequences may at least partially contribute to the reduced activity of the Tat2 basic domain.  相似文献   

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Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes.  相似文献   

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Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.  相似文献   

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Pathogenic JCV with rearranged regulatory regions (PML-type) causes PML, a demyelinating disease, in the brains of immunocompromised patients. On the other hand, archetype JCV persistently infecting the kidney is thought to be converted to PML-type virus during JCV replication in the infected host under immunosuppressed conditions. In addition, Tat protein, encoded by HIV-1, markedly enhances the expression of a reporter gene under control of the JCV late promoter.
In order to examine the influence of Tat on JCV propagation, we used kidney-derived COS-7 cells, which only permit archetype JCV, and established COS-tat cells, which express HIV-1 Tat stably. We found that the extent of archetype JCV propagation in COS-tat cells is significantly greater than in COS-7 cells. On the other hand, COS-7 cells express SV40 T antigen, which is a strong stimulator of archetype JCV replication. The expression of SV40 T antigen was enhanced by HIV-1 Tat slightly according to real-time RT-PCR, this was not closely related to JCV replication in COS-tat cells. The efficiency of JCV propagation depended on the extent of expression of functional Tat. To our knowledge, this is the first report of increased production of archetype JCV in a culture system using cell lines stably expressing HIV-1 Tat. We propose here that COS-tat cells are a useful tool for studying the role of Tat in archetype JCV replication in the development of PML.  相似文献   

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Tat, the trans activation protein of HIV, is produced early upon infection to promote and expand HIV replication and transmission. However, Tat appears to also have effects on target cells, which may affect Ag recognition both during infection and after vaccination. In particular, Tat targets dendritic cells and induces their maturation and Ag-presenting functions, increasing Th1 T cell responses. We show in this work that Tat modifies the catalytic subunit composition of immunoproteasomes in B and T cells either expressing Tat or treated with exogenous biological active Tat protein. In particular, Tat up-regulates latent membrane protein 7 and multicatalytic endopeptidase complex like-1 subunits and down-modulates the latent membrane protein 2 subunit. These changes correlate with the increase of all three major proteolytic activities of the proteasome and result in a more efficient generation and presentation of subdominant MHC-I-binding CTL epitopes of heterologous Ags. Thus, Tat modifies the Ag processing and modulates the generation of CTL epitopes. This may have an impact on both the control of virally infected cells during HIV-1 infection and the use of Tat for vaccination strategies.  相似文献   

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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.  相似文献   

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