Substrate specificities of SR proteins in constitutive splicing are determined by their RNA recognition motifs and composite pre-mRNA exonic elements.
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We report striking differences in the substrate specificities of two human SR proteins, SF2/ASF and SC35, in constitutive splicing. beta-Globin pre-mRNA (exons 1 and 2) is spliced indiscriminately with either SR protein. Human immunodeficiency virus tat pre-mRNA (exons 2 and 3) and immunoglobulin mu-chain (IgM) pre-mRNA (exons C3 and C4) are preferentially spliced with SF2/ASF and SC35, respectively. Using in vitro splicing with mutated or chimeric derivatives of the tat and IgM pre-mRNAs, we defined specific combinations of segments in the downstream exons, which mediate either positive or negative effects to confer SR protein specificity. A series of recombinant chimeric proteins consisting of domains of SF2/ASF and SC35 in various combinations was used to localize trans-acting domains responsible for substrate specificity. The RS domains of SF2/ASF and SC35 can be exchanged without effect on substrate specificity. The RNA recognition motifs (RRMs) of SF2/ASF are active only in the context of a two-RRM structure, and RRM2 has a dominant role in substrate specificity. In contrast, the single RRM of SC35 can function alone, but its substrate specificity can be influenced by the presence of an additional RRM. The RRMs behave as modules that, when present in different combinations, can have positive, neutral, or negative effects on splicing, depending upon the specific substrate. We conclude that SR protein-specific recognition of specific positive and negative pre-mRNA exonic elements via one or more RRMs is a crucial determinant of the substrate specificity of SR proteins in constitutive splicing. (+info)
High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector.
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Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibition of viral gene expression and replication, has been suggested for inhibition of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes of these sequences have potential as therapeutic molecular inhibitors. In the present study, we have generated several anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and TAR decoys and combinations of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA stabilization. We show that HIV replication was most strongly inhibited with the combination TAR + RRE decoy when compared with the single decoys or the tat-ribozyme. We also show that the Tat-inducible HIV promoter directs a higher level of steady-state transcription of decoys and inhibitors and that higher levels of expression directly relate to increased levels of inhibition of HIV infection. Furthermore, a stabilization of the 3' end of TAR + RRE inhibitor transcripts using a beta-globin 3' UTR sequence leads to an additional 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR + RRE, yields high level HIV inhibition for greater than 3 weeks. Taken together, both optimization for high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV. (+info)
Interactions between Tat and TAR and human immunodeficiency virus replication are facilitated by human cyclin T1 but not cyclins T2a or T2b.
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The transcriptional transactivator (Tat) from the human immunodeficiency virus (HIV) does not function efficiently in Chinese hamster ovary (CHO) cells. Only somatic cell hybrids between CHO and human cells and CHO cells containing human chromosome 12 (CHO12) support high levels of Tat transactivation. This restriction was mapped to interactions between Tat and TAR. Recently, human cyclin T1 was found to increase the binding of Tat to TAR and levels of Tat transactivation in rodent cells. By combining individually with CDK9, cyclin T1 or related cyclins T2a and T2b form distinct positive transcription elongation factor b (P-TEFb) complexes. In this report, we found that of these three cyclins, only cyclin T1 is encoded on human chromosome 12 and is responsible for its effects in CHO cells. Moreover, only human cyclin T1, not mouse cyclin T1 or human cyclins T2a or T2b, supported interactions between Tat and TAR in vitro. Finally, after introducing appropriate receptors and human cyclin T1 into CHO cells, they became permissive for infection by and replication of HIV. (+info)
Stimulation of Tat-associated kinase-independent transcriptional elongation from the human immunodeficiency virus type-1 long terminal repeat by a cellular enhancer.
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The human immunodeficiency virus type-1 (HIV-1) long terminal repeat (LTR) initiates transcription efficiently but produces only short transcripts in the absence of the trans-activator protein, Tat. To determine whether a cellular enhancer could provide the signals required to recruit an elongation-competent polymerase to the HIV-1 LTR, the B cell-specific immunoglobulin heavy chain gene enhancer (IgHE) was inserted upstream of the LTR. The enhancer increased transcription in the absence of Tat between 6- and 7-fold in transfected B cells, but the full-length transcripts remained at basal levels in HeLa cells, where the enhancer is inactive. RNase-protection studies showed that initiation levels in the presence and absence of the enhancer were constant, but the enhancer significantly increased the elongation capacity of the polymerases. Tat-stimulated elongation is strongly inhibited by the nucleoside analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), which inhibits the Tat-associated kinase, TAK (CDK9). However, polymerases initiating transcription from LTRs carrying the enhancer were able to efficiently elongate in the presence of DRB. Specific repression of TAK by expression in trans of the CDK9 kinase also inhibited Tat-stimulated elongation but did not inhibit enhancer-dependent transcription significantly. Thus, the activation of polymerase processivity by the IgHE involves a unique mechanism which is independent of TAK. (+info)
Tat-associated kinase (P-TEFb): a component of transcription preinitiation and elongation complexes.
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Human immunodeficiency virus, type 1 (HIV-1) Tat protein activates transcription from the HIV-1 long terminal repeat. Tat interacts with TFIIH and Tat-associated kinase (a transcription elongation factor P-TEFb) and requires the carboxyl-terminal domain of the largest subunit of RNA polymerase II (pol II) for transactivation. We developed a stepwise RNA pol II walking approach and used Western blotting to determine the role of TFIIH and P-TEFb in HIV-1 transcription elongation. Our results demonstrate the new findings that P-TEFb is a component of the preinitiation complex and travels with the elongating RNA pol II, whereas TFIIH is released from the elongation complexes before the trans-activation responsive region RNA is synthesized. Our results suggest that TFIIH and P-TEFb are involved in the clearance of promoter-proximal pausing of RNA pol II on the HIV-1 long terminal repeat at different stages. (+info)
Human immunodeficiency virus replication induces monocyte chemotactic protein-1 in human macrophages and U937 promonocytic cells.
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We have recently described a significant correlation between human immunodeficiency virus-1 (HIV-1) RNA replication and monocyte chemotactic protein-1 (MCP-1) levels in the cerebrospinal fluid (CSF) of individuals with the acquired immunodeficiency syndrome (AIDS) with HIV encephalitis (E). Because local macrophages (microglia) are the cells predominantly infected in the brain, we investigated whether in vitro HIV infection affects MCP-1 production in mononuclear phagocytes (MP). MCP-1 secretion and expression were consinstently upregulated over constitutive levels in human monocyte-derived macrophages (MDM) infected with the M-tropic R5 BaL strain of HIV-1. HIV replication was required for this effect, as demonstrated by the absence of chemokine upregulation after infection in the presence of 3'-azido-3'-deoxythimidine (AZT) or cell-exposure to heat-inactivated (triangle up degrees ) virus. MCP-1 induction was not restricted to HIV-1 BaL, but was also observed during productive infection of MDM with two primary isolates differing for entry coreceptor usage and of U937 cells with the X4 HIV-1 MN strain. Based on the observation that exogenous HIV-1 Tat induced MCP-1 expression in astrocytes, we also investigated its role in MDM and U937 cells. Exogenous Tat induced MCP-1 production from MDM in a concentration-dependent manner, however, it was not effective on uninfected U937 cells or on the chronically infected U937-derived cell line U1. Transfection of Tat-expressing plasmids moderately activated HIV expression in U1 cells, but failed to induce MCP-1 expression in this cell line or in uninfected U937 cells. HIV replication-dependent expression of MCP-1 in MP may be of particular relevance for the pathogenesis of HIV infection in nonlymphoid organs such as the brain. (+info)
Intercellular traffic of human immunodeficiency virus type 1 transactivator protein defined by monoclonal antibodies.
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Monoclonal antibodies (mAbs) directed against the amino-terminal region (N-terminal sequence 2-19) of transactivator protein (tat) of HIV-1 have been shown to inhibit intercellular transactivation mediated by the extracellular tat protein. The intracellular transactivation was not significantly affected by anti-tat mAbs. The specificity of anti-tat mAbs in abolishing the transactivating potential of extracellular tat is documented by studies with mAbs to HIV-1 reverse transcriptase, or to a human mammary cancer protein. None of these antibodies showed any inhibitory effect on intercellular transactivation. Specific interaction of anti-tat IgG with tat protein expressed in Jurkat cells is further supported by experiments on immunoblotting. Extracellular tat is responsible for signals which induce a variety of biological responses in HIV-infected cells, as well as in uninfected cells. The fact that anti-tat mAbs can abolish the intercellular traffic of tat protein offers a unique strategy in the development of vaccines against AIDS. (+info)
Human immunodeficiency virus type 1 Tat-dependent activation of an arrested RNA polymerase II elongation complex.
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The human immunodeficiency virus type 1 (HIV-1) Tat protein is a transcriptional activator that is essential for efficient viral gene expression and replication. Tat increases the level of full-length transcripts from the HIV-1 promoter by dramatically enhancing the elongation efficiency of the RNA polymerase II complexes assembled on this promoter. Tat could potentially activate the transcription machinery during initiation, elongation, or both. We used an immobilized HIV-1 promoter template with a reversible lac repressor (LacR) elongation block inserted downstream to dissect the stages in transcription affected by Tat. Transcription complexes assembled in the absence of Tat and blocked by LacR cannot be activated by incubation with Tat alone. These complexes can, however, be activated if Tat is added in combination with cellular factors. In this system, Tat also promoted the assembly of preinitiation complexes capable of elongating efficiently, suggesting that Tat can associate with transcription complex at an early stage. These data indicate that Tat can activate elongation of RNA polymerase by modifying an already elongating transcription complex. The data also suggest the possibility that Tat can interact with initiation complexes. (+info)