26 research outputs found
Sp1 and Sp3 regulate basal transcription of the human APOBEC3G gene
APOBEC3G (A3G), a member of the recently discovered family of human cytidine deaminases, is expressed in peripheral blood lymphocytes and has been shown to be active against HIV-1 and other retroviruses. To gain new insights into the transcriptional regulation of this restriction factor, we cloned and characterized the promoter region of A3G. Transcriptional start sites were identified by 5′-rapid amplification of cDNA ends analysis. Luciferase reporter assays demonstrated that a 1025 bp A3G promoter sequence (from −959 to +66 relative to the major transcriptional start site) displayed constitutive promoter activity. In T cells, the A3G promoter was not inducible by mitogenic stimulation, interferon treatment or expression of HIV-1 proteins. Using a series of 5′ deletion promoter constructs in luciferase reporter assays, we identified a 180 bp region that was sufficient for full promoter activity. Transcriptional activity of this A3G core promoter was dependent on a GC-box (located at position −87/−78 relative to the major transcriptional start site) and was abolished after mutation of this DNA element. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that the identified GC-box represented a binding site for the ubiquitous transcription factors specificity protein (Sp) 1 and Sp3
Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease
Advanced Therapy Medicinal Products: How to Bring Cell-Based Medicinal Products Successfully to the Market - Report from the CAT-DGTI-GSCN Workshop at the DGTI Annual Meeting 2014
Regulatory evaluation of Glybera in Europe-two committees, one mission
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SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutières Syndrome Are Highly Susceptible to HIV-1 Infection
Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor
Transcriptional Regulation of Porcine Endogenous Retroviruses Released from Porcine and Infected Human Cells by Heterotrimeric Protein Complex NF-Y and Impact of Immunosuppressive Drugs
Recent studies revealed a significant promoter activity of porcine endogenous retrovirus (PERV) long terminal repeats (LTRs) in different human and mammalian cell lines, which is mediated by a 39-bp repeat located in the U3 region in different numbers, representing an enhancer (G. Scheef, N. Fischer, U. Krach, and R. R. Tönjes, J. Virol. 75:6933-6940, 2001). A statistical transcription factor analysis revealed putative binding sites for the CCAAT-binding transcription factor NF-Y inside the 39-bp repeat. Specific binding of NF-Y to the repeat sequence was demonstrated by electrophoretic mobility shift assays and supershift assays with specific antibodies directed against the three subunits of NF-Y. To identify further transcription-regulating elements, genetically modified LTRs lacking the repeat box, U3, R, or U5 were investigated. The results indicated a strong inhibitory element in the R region, as the deletion of R caused a significantly increased promoter activity. Since PERV might play a potential role in the application of xenogeneic cell therapy and xenotransplantation techniques, we have investigated whether immunosuppressive drugs that are routinely used in transplantation medicine have an impact on the promoter activity. Neither cyclosporine nor prednisolone had any influence on the promoter strength of the PERV LTRs. By performing a real-time PCR we were able to compare the proviral loads of porcine and infected human cells as well as the amount of released virions, which revealed a direct link between LTR activity and the number of released retroviruses
High level expression of the anti-retroviral protein APOBEC3G is induced by influenza A virus but does not confer antiviral activity
<p>Abstract</p> <p>Human APOBEC3G is an antiretroviral protein that was described to act via deamination of retroviral cDNA. However, it was suggested that APOBEC proteins might act with antiviral activity by yet other mechanisms and may also possess RNA deamination activity. As a consequence there is an ongoing debate whether APOBEC proteins might also act with antiviral activity on other RNA viruses. Influenza A viruses are single-stranded RNA viruses, capable of inducing a variety of antiviral gene products. In searching for novel antiviral genes against these pathogens, we detected a strong induction of APOBEC3G but not APOBEC3F gene transcription in infected cells. This upregulation appeared to be induced by the accumulation of viral RNA species within the infected cell and occurred in an NF-κB dependent, but MAP kinase independent manner. It further turned out that APOBEC expression is part of a general IFNβ response to infection. However, although strongly induced, APOBEC3G does not negatively affect influenza A virus propagation.</p
Quantitative assays reveal cell fusion at minimal levels of SARS-CoV-2 spike protein and fusion from without
Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies, as well as sera from convalescent patients, inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during coronavirus disease 2019 (COVID-19) can proceed at low levels of S protein and may not be effectively prevented by antibodies