Intrinsic protein fluorescence of reverse transcriptases from HIV-1 and HIV-2 provides a sensitive signal for monitoring the interaction of the enzymes with primer/template duplex molecules. Kd values for 18/36-mer DNA/DNA duplexes were found to be in the range of a few nanomolar (about 3 times higher for the enzyme from HIV-2 than for that from HIV-1). The quenching of protein fluorescence induced on binding primer/template, together with an increase in extrinsic fluorescence on interaction with primer/template containing a fluorescent nucleotide at the 3'-end of the primer, was used to investigate the kinetics of interaction with reverse transcriptase from HIV-1. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial association (k(ass) = ca. 5 x 10(8) M-1 s-1) followed by a slow isomerization step (k = ca. 0.5 s-1). These (forward) rate constants are increased in the presence of a non-nucleoside inhibitor (S-TIBO) of HIV-1 reverse transcriptase, while the reverse rate constant for the second step is decreased, leading to an increase in affinity between the enzyme and primer/template by a factor of at least 10 when S-TIBO is bound. The results are discussed in terms of present knowledge of the structure of reverse transcriptase.
Thjazolo-iso-indolinone derivatives with high specificity toward the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) were identified. The most potent compound, BM +51.0836, inhibited HIV-1 RT at a 50%o inhibitory concentration of 90 nM in vitro. In cell culture assays, similar 50%o inhibitory concentrations were obtained with high specificity for HIV-1. These substances were equally active against a zidovudine-resistant isolate. No antiviral effect was observed with an HIV-2 isolate. HIV-1 isolates resistant to the thiazolo-iso-indolinones were generated in cell culture, and the nucleotide sequences of the respective RT genes were analyzed subsequently. Comparison Fig. lb], a substance with 10-fold lower 50% inhibitory concentrations (IC50s) than the lead compound BM 21.1298 and higher antiviral activity than 0-TIBO and nevirapine (BI-RG-587).
MATERIALS AND METHODSCompounds. The thiazolo-iso-indolinones were synthesized by Boehringer Mannheim GmbH. A detailed description of the synthesis will be published elsewhere. Zidovudine (AZT; Wellcome), nevirapine (BI-RG-587; Boehringer Ingelheim), and 0-TIBO (Janssen) were also synthesized by the chemical department of Boehringer Mannheim GmbH. The inhibitors were dissolved in dimethyl sulfoxide, and the mixture was added to the RT assay mixture (the dimethyl sulfoxide concentration was less than 5%). For cell culture assays, stock solutions were prepared by dissolving the inhibitors in 50% dimethyl sulfoxide-50% culture medium. In the cell culture supernatant, the dimethyl sulfoxide concentration was less than 1%.Nonradioactive Bl-1 RT assay. Selective inhibitors of HIV-1 RT were identified by using a previously published high-efficiency screening system (4). The assay contains purified HIV-1 RT (11) expressed in Escherichia coli, an in vitro transcript of the HIV-1 long terminal repeat (21), and the primer-binding site as the template and as well as an 18-mer oligonucleotide as the primer. Inhibition of RT was determined by a nonradioactive RT assay (ELISA) with biotin-and digoxigenin-labeled nucleoside triphosphates (4). In brief, 1 ,ug
Torque Teno Virus (TTV) has been assigned to the floating genus Anellovirus. TTV ssDNA genomes have a size of 3.6 to 3.8 kb and display up to 30% nucleotide diversity. The pathogenic potential of TTV is under investigation. To address a putative link of pathogenicity with the observed sequence variations, the transcription profile of P/1C1 (genogroup 1) isolated from a patient diseased with a non A-G hepatitis was analysed. Four mRNAs were identified, which encoded the seven proteins ORF1, ORF1/1, ORF1/2, ORF2, ORF2/2, ORF3 and ORF4. Expression of the ORF1 protein and its splice variant ORF1/1 in cell culture was detected by an ORF1-specific antiserum. Analysis of N-terminal tagged P/1C1-encoded proteins revealed that ORF1, ORF1/1 and ORF1/2 were localised in the nucleoli, ORF3 and ORF4 resided in the nucleoplasm, ORF2/2 appeared either in the nucleoli or the whole nucleus while ORF2 was the only protein seen in the cytoplasm.
Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.
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