Common and unique transcription factor requirements of human U1 and U6 snRNA genes

13 pages, 7 figures.-- PMID: 8253082 [PubMed].-- PMCID: PMC413633.The human U1 and U6 genes have similar basal promoter structures. A first analysis of the factor requirements for the transcription of a human U1 gene by RNA polymerase II in vitro has been undertaken, and these requirements compared with those of human U6 gene transcription by RNA polymerase III in the same extracts. Fractions containing PSE-binding protein (PBP) are shown to be essential for transcription of both genes, and further evidence that PBP itself is required for U1 as well as U6 transcription is presented. On the other hand, the two genes have distinct requirements for TATA-binding protein (TBP). On the basis of chromatographic and functional properties, the TBP, or TBP complex, required for U1 transcription appears to differ from previously described complexes required for RNA polymerase I, II or III transcription. The different TBP requirements of the U1 and U6 promoters are reflected by specific association with either TFIIB or TFIIIB respectively, thus providing a basis for differential RNA polymerase selection.We wish to thank Henk Stunnenberg, Dirk Bohmann, Francis Stewart and Peter Becker for comments on the manuscript, Jacky Schmidt and H.Stunnenberg for helpful suggestions and plasmids, the EMBL photolab, Susie Weston and Sam O'Loughlin for efficient oligonucleotide synthesis, and Maryka Kimmins and Fiona Berrie for typing the manuscript. We are particularly grateful to all other members of the group for constructive criticism and suggestions. J.B. was supported by an EEC fellowship, J.D.L. by a Human Frontiers Science Program fellowship and S.I.G. by an Alexander von Humboldt fellowship.Peer reviewe

Polymerase selectivity and the promoters of U snRNA genes

Capítulo en: Fritz Eckstein; David M. J. Lilley (eds.). Nucleic Acids and Molecular Biology. Berlin: Springer, 1992, p.174-186. (Nucleic Acids and Molecular Biology ; 6)The U small nuclear RNAs (snRNAs) form a functionally conserved family of RNAs found in eukaryotic cells. A subset of these RNAs, the spliceosomal snRNAs, function in the removal of introns from messenger RNA precursors. In most eukaryotes this group of snRNAs are U1, U2, U4, U5 and U6 but in trypanosomes, which produce mature mRNAs by transrather than cis-splicing, no homologues of U1 or U5 have been found (see Guthrie and Patterson 1988; Lamond et al. 1990 for reviews). Another ubiquitous member of the U snRNA family which will be discussed here is U3. U3 is found in the nucleolus rather than the nucleoplasm and is involved in the processing of ribosomal RNA precursors (Kass et al. 1990). The topic of this review will not, however, be the function of these RNAs, what will interest us is their transcriptionPeer Reviewe

Virogenomics: a novel approach to antiviral drug discovery

Target discovery in virology has been limited to the few open-reading frames encoded by viral genomes. However, several recent examples show that inhibiting host-cell proteins can prevent viral infection. The human genome sequence should, therefore, contain many more genes that are essential for viral propagation than viral genomes. A systematic approach to find these potential cellular antiviral targets is global host gene expression analysis using DNA microarrays. Several recent studies reveal both unique and common strategies by which viruses change the gene expression profile of the host cell. Moreover, work in progress shows that some of the host pathways discovered by expression profiling are important for viral replication. Thus, human genomics tools have the potential to deliver novel antiviral drugs

Tracking the Evolution of Multiple In Vitro Hepatitis C Virus Replicon Variants under Protease Inhibitor Selection Pressure by 454 Deep Sequencing ▿

Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV

Rapid viral response of once-daily TMC435 plus pegylated interferon/ribavirin in hepatitis C genotype-1 patients: a randomized trial.

Antiviral activity of TMC435, an oral, once-daily, HCV NS3/4A protease inhibitor, was evaluated with pegylated interferon-α2a/ribavirin (P/R) in HCV genotype-1 patients.Clinical Trial, Phase IIJournal ArticleRandomized Controlled TrialResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Antiviral Activity of TMC353121, a Respiratory Syncytial Virus (RSV) Fusion Inhibitor, in a Non-Human Primate Model.

BackgroundThe study assessed the antiviral activity of TMC353121, a respiratory syncytial virus (RSV) fusion inhibitor, in a preclinical non-human primate challenge model with a viral shedding pattern similar to that seen in humans, following continuous infusion (CI).MethodsAfrican green monkeys were administered TMC353121 through CI, in 2 studies. Study 1 evaluated the prophylactic and therapeutic efficacy of TMC353121 at a target plasma level of 50 ng/mL (n=15; Group 1: prophylactic arm [Px50], 0.033 mg/mL TMC353121, flow rate 2.5 mL/kg/h from 24 hours pre-infection to 10 days; Group 2: therapeutic arm [Tx50], 0.033 mg/mL TMC353121 from 24 hours postinfection to 8 days; Group 3: control [Vh1] vehicle, 24 hours post-infection to 8 days). Study 2 evaluated the prophylactic efficacy of TMC353121 at target plasma levels of 5 and 500 ng/mL (n=12; Group 1: prophylactic 5 arm [Px5], 0.0033 mg/mL TMC353121, flow rate 2.5 mL/kg/h from 72 hours pre-infection to 14 days; Group 2: prophylactic 500 arm [Px500], 0.33 mg/mL TMC353121; Group 3: control [Vh2] vehicle, 14 days). Bronchoalveolar lavage fluid and plasma were collected every 2 days from day 1 postinfection for pharmacokinetics and safety analysis.FindingsTMC353121 showed a dose-dependent antiviral activity, varying from 1 log10 reduction of peak viral load to complete inhibition of the RSV replication. Complete inhibition of RSV shedding was observed for a relatively low plasma exposure (0.39 μg/mL) and was associated with a dose-dependent reduction in INFγ, IL6 and MIP1α. TMC353121 administered as CI for 16 days was generally well-tolerated.ConclusionTMC353121 exerted dose-dependent antiviral effect ranging from full inhibition to absence of antiviral activity, in a preclinical model highly permissive for RSV replication. No new safety findings emerged from the study

Binding-Site Identification and Genotypic Profiling of Hepatitis C Virus Polymerase Inhibitors▿

The search for hepatitis C virus polymerase inhibitors has resulted in the identification of several nonnucleoside binding pockets. The shape and nature of these binding sites differ across and even within diverse hepatitis C virus genotypes. These differences confront antiviral drug discovery with the challenge of finding compounds that are capable of inhibition in variable binding pockets. To address this, we have established a hepatitis C virus mutant and genotypic recombinant polymerase panel as a means of guiding medicinal chemistry through the elucidation of the site of action of novel inhibitors and profiling against genotypes. Using a genotype 1b backbone, we demonstrate that the recombinant P495L, M423T, M414T, and S282T mutant enzymes can be used to identify the binding site of an acyl pyrrolidine analog. We assess the inhibitory activity of this analog and other nonnucleoside inhibitors with our panel of enzyme isolates generated from clinical sera representing genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a

In Vitro Activity and Preclinical Profile of TMC435350, a Potent Hepatitis C Virus Protease Inhibitor▿ †

The hepatitis C virus (HCV) NS3/4A serine protease has been explored as a target for the inhibition of viral replication in preclinical models and in HCV-infected patients. TMC435350 is a highly specific and potent inhibitor of NS3/4A protease selected from a series of novel macrocyclic inhibitors. In biochemical assays using NS3/4A proteases of genotypes 1a and 1b, inhibition constants of 0.5 and 0.4 nM, respectively, were determined. TMC435350 inhibited HCV replication in a cellular assay (subgenomic 1b replicon) with a half-maximal effective concentration (EC50) of 8 nM and a selectivity index of 5,875. The compound was synergistic with alpha interferon and an NS5B inhibitor in the replicon model and additive with ribavirin. In rats, TMC435350 was extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability was 44% after a single oral administration. Compound concentrations detected in both plasma and liver at 8 h postdosing were above the EC99 value measured in the replicon. In conclusion, given the selective and potent in vitro anti-HCV activity, the potential for combination with other anti-HCV agents, and the favorable pharmacokinetic profile, TMC435350 has been selected for clinical development