A novel Vpr peptide interactor fused to integrase (IN) restores integration activity to IN-defective HIV-1 virions.
(17/8163)
A novel approach to complement human immunodeficiency virus type I (HIV-1) integrase (IN)-defective virions has been identified. The approach involves fusion of a 23-amino-acid stretch to the N-terminus of wild-type IN and coexpression of this chimera with the IN-defective proviral template in virus producing cells. The 23-amino-acid peptide represents a Vpr "interactor," referred to as the the WxxF or WF domain, which apparently leads to docking of the domain along with the fusion partner onto HIV-1 Vpr, thus permitting virion incorporation of the chimeric protein when expressed, in trans, with other viral products. Transfection of the WF-IN expression plasmid along with HIV-1 viral clones that produce Vpr, but bear an IN mutation, results in the release of a proportion of viral particles that are competent for integration. The extent of complementation was assessed using the MAGI cell assay, where integration of viral DNA results in the eventual appearance of easily visible multinucleated blue syncytia. The efficiency of dWF-IN (double copy of WF domain) complementation is not improved markedly by incorporation of a HIV-1 protease cleavage site (PR) between the dWF domain and IN (dWF-PR-IN), unlike that observed with Vpr fusions to IN. Furthermore, the ability of Vpr-PR-IN and dWF-PR-IN to complement IN-defective proviral clones, both of which bear an intervening protease cleavage site, appear comparable. Western blotting analyses using virions isolated through sucrose cushions demonstrate clearly the incorporation of the dWF-IN fusion protein into Vpr containing HIV-1 particles but not in Vpr-deficient virions. Additional Western blotting analyses indicate that all Vpr-IN and dWF-IN chimeras, with or without a PR site, are packaged into virions. The efficiency of virion incorporation of Vpr-IN and dWF-IN chimeras appears approximately comparable by Western blotting analysis. The ability of dWF-IN to complement IN-defective proviruses with efficiency similar to that of Vpr-PR-IN and dWF-PR-IN indicates that dWF-IN retains the full complement of functions necessary for integration of proviral DNA and is likely due to the benign nature of this small domain at the amino-terminus of IN. (+info)
Interactions between Tat and TAR and human immunodeficiency virus replication are facilitated by human cyclin T1 but not cyclins T2a or T2b.
(18/8163)
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)
The introduction of dominant-negative p53 mutants suppresses temperature shift-induced senescence in immortal human fibroblasts expressing a thermolabile SV40 large T antigen.
(19/8163)
Immortal human fibroblasts, SVts8 cells, which express a heat-labile SV40 large T antigen, induces a senescence-like phenomenon in response to upward shift in temperature. Cells with arrested division show strong induction of senescence-associated beta-galactosidase. We examined how p53 and pRB are involved in this phenomenon since they are major targets of the T antigen. Transfection of cells with plasmids encoding the wild-type T antigen or human papilloma virus type 16 E6/E7 proteins completely abolished the arrest in cell division, a plasmid encoding the E6 protein suppressed it markedly, while a plasmid encoding E7 had no effect. Plasmids encoding dominant-negative p53 mutants also suppressed the arrest in cell division to various degrees. Upon temperature shift, p21 mRNA was upregulated 10-fold in SVts8 cells, but only slightly in clones expressing the wild-type T antigen or dominant-negative p53 mutants. These data demonstrate that p53 plays a major role in this senescence-like phenomenon. (+info)
Serum response elements activate and cAMP responsive elements inhibit expression of transcription factor Egr-1 in synovial fibroblasts of rheumatoid arthritis patients.
(20/8163)
Analyzing the induction kinetics and promoter elements regulating the expression of the transcription factor Egr-1, we found elevated levels of Egr-1-encoding mRNA in synovial fibroblasts of rheumatoid arthritis (RA) patients when compared to controls. By contrast, synovial lymphocytes and macrophages do not show an elevated Egr-1 transcription. Therefore, the overexpression of Egr-1 may serve as a diagnostic marker to characterize synovial fibroblasts of RA patients. To study the regulatory mechanisms controlling Egr-1 expression we analyzed the function of transcription factor binding sites located in the Egr-1 promoter. Individual transcription factor binding sites within the Egr-1 promoter were specifically mutated and Egr-1 promoter activity was tested using reporter gene constructs. Our experiments demonstrate that serum response elements are the main positive regulators and binding to a cAMP responsive element represents the major negative regulator for Egr-1 expression in synovial fibroblasts. In addition, we functionally defined a new element, which was not yet described in the human Egr-1 promoter and which serves as a second negative regulatory element for Egr-1 expression. Therefore increased serum response factor activity or failure of Egr-1 repressing signals may account for Egr-1 overexpression in RA synovial fibroblasts. (+info)
Cot protooncoprotein activates the dual specificity kinases MEK-1 and SEK-1 and induces differentiation of PC12 cells.
(21/8163)
Mitogenic signals initiated at the plasma membrane are transmitted to the nucleus through an intricate signalling network. We identified the protooncoprotein Cot as a new component of mitogenic signalling cascades, which activates both the classic cytoplasmic cascade and the SAPK stress pathway. Wildtype and activated Cot phosphorylate and activate MEK-1 and SEK-1 in vitro. These findings are consistent with the sequence homology between Cot and the rat gene Tpl-2. Expression of oncogenic Cot in 293, NIH3T3 and PC12 cells leads to in vivo phosphorylation of endogenous c-Jun and Erk-1/2 suggesting that the serine/threonine kinase Cot functions beside c-Raf-1 and Mos as a direct activator of MEK-1. Furthermore, we have examined the biological effects of Cot on the phenotype of fibroblastic and neuronal cells. In order to test a potential c-Raf-1 dependency of Cot transformation, the effect of oncogenic Cot on Raf revertant CHP25 cells was determined. Cot could restore the transformed phenotype indicating that Cot transformation is not dependent on active c-Raf-1 and that Cot is not a target for the putative Raf inhibitor, which is presumably active in the revertant cell line. Expression of oncogenic versions of Raf as well as v-Mos leads to differentiation of PC12 cells. Cot also induces neurite outgrowth of PC12 cells. These data are consistent with the role of Cot in the classic mitogenic cascade and suggest that the simultaneously activated JNK/SAPK stress pathway has no antagonistic effects in this context. (+info)
Binding of c-Rel to STAT5 target sequences in HTLV-I-transformed T cells.
(22/8163)
The type I human T-cell leukemia virus (HTLV-I) induces abnormal growth and subsequent transformation of T cells, which is associated with the development of an acute T-cell malignancy termed adult T-cell leukemia. A characteristic of HTLV-I-transformed T cells is the constitutive nuclear expression of NF-kappaB/Rel family of transcription factors, which appears to be essential for the growth of these transformed cells. Although NF-kappaB/Rel factors are known to induce the expression of T-cell growth factor interleukin (IL)-2, it is unclear how they participate in the IL-2-independent growth of HTLV-I-transformed cells. In this study, we show that certain NF-kappaB/Rel members, predominantly c-Rel, interact with enhancer sequences for STAT5, a key transcription factor mediating IL-2-induced T-cell proliferation. Reporter gene assays reveal that the binding of c-Rel to the STAT5 site present in the Fc gammaR1 gene leads to potent transactivation of this enhancer. Binding of c-Rel to the Fc gammaR1 STAT site also occurs in human peripheral blood T cells immortalized with HTLV-I in vitro and is correlated with enhanced levels of proliferation of these cells. These results raise the possibility that NF-kappaB/Rel may participate in the growth control of HTLV-I-transformed T cells by regulating genes driven by both kappaB and certain STAT enhancers. (+info)
Search for oncogenic regulators in an autocrine tumor model using differential display PCR: identification of novel candidate genes including the calcium channel mtrp6.
(23/8163)
A hemopoietic multistep tumor model, in which IL-3 dependent PB-3c mast cells, following expression of v-H-ras progress in vivo to IL-3 producing autocrine tumors has previously been established. Central for this oncogenic progression is a recessive step, which is reversible by cell fusion and leads to stabilization of IL-3 mRNA with concomitant activation of the autocrine loop. Comparing the IL-3 dependent PB-3c and the IL-3 autocrine V2D1 tumor cells with differential display PCR revealed 12 differentially expressed genes of which eight were upregulated and four downregulated in the tumor. They included four proteases (mouse mast cell protease 2, granzyme B, pepsinogen F and serine protease 1) and two metabolic enzymes (adenine phosphoribosyltransferase and fructose1,6-bisphosphatase). For validation, expression of the identified genes was tested in independent PB-3c precursor clones and their tumor derivatives. Expression of an endogenous retroviral IAP element and three unknown transcripts were consistently upregulated in all tumor lines. In somatic cell hybrids, two of these unknown cDNAs showed a dominant and one a recessive expression pattern. One transcript, expressed in the precursor but downregulated in the tumor cells, was cloned and identified as the murine calcium channel mtrp6. (+info)
Nuclear and nucleolar targeting of human ribosomal protein S25: common features shared with HIV-1 regulatory proteins.
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The nuclear and nucleolar targeting properties of human ribosomal protein S25 (RPS25) were analysed by the expression of epitope-tagged RPS25 cDNAs in Cos-1 cells. The tagged RPS25 was localized to the cell nucleus, with a strong predominance in the nucleolus. At the amino terminus of RPS25, two stretches of highly basic residues juxtapose. This configuration shares common features with the nucleolar targeting signals (NOS) of lentiviral RNA-binding transactivators, including human immunodeficiency viruses' (HIV) Rev proteins. Deletion and site-directed mutational analyses demonstrated that the first NOS-like stretch is dispensable for both nuclear and nucleolar localization of RPS25, and that the nuclear targeting signal is located within the second NOS-like stretch. It has also been suggested that a set of continuous basic residues and the total number of basic residues should be required for nucleolar targeting. Signal-mediated nuclear/nucleolar targeting was further characterized by the construction and expression of a variety of chimeric constructs, utilizing three different backbones with RPS25 cDNA fragments. Immunofluorescence analyses demonstrated a 17 residue peptide of RPS25 as a potential nuclear/nucleolar targeting signal. The identified peptide signal may belong to a putative subclass of NOS, characterized by compact structure, together with lentiviral RNA-binding transactivators. (+info)