No abstract

The DXO family of proteins participates in eukaryotic mRNA 5′-end quality control, removal of non-canonical NAD + cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5′-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations observed upon DXO1 knockout in plants, which include growth and pigmentation deficiency, global transcriptomic changes and accumulation of RNA quality control siRNAs. Conversely, these phenotypes depend on the plant-specific N-terminal extension of DXO1. Pale-green coloration of DXO1-deficient plants and our RNA-seq data reveal that DXO1 affects chloroplast-localized processes. We propose that DXO1 mediates the connection between RNA turnover and retrograde chloroplast-to-nucleus signaling independently of its deNADding properties.

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNAstimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3 0 labeled with a Cy3 dye and 5 0 labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.

A universal and effective method for long-term storage of bacteriophages has not yet been described. We show that randomly selected tailed phages could be stored inside the infected cells at -80°C without a major loss of phage and host viability. Our results suggest the suitability of this method as a standard for phage preservation.

Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ( 1 H and 15 N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family (11). HCV infection, affecting 3% of the world population, leads to chronic hepatitis in up to 85% of the cases, in 10 to 20% of the cases it develops into cirrhosis that requires constant treatment and provokes permanent infirmity, while 1 to 5% of chronically infected patients are diagnosed with hepatocellular carcinoma (9). No efficient treatment exists; even the new dual therapy with pegylated interferon alpha 2a or 2b and ribavirin is effective only in up to 60% of the cases, depending on the genotype of the virus and the duration of the treatment (21). To date, no vaccine against HCV has been developed in spite of numerous attempts and advanced trials, principally because of the high variability of the RNA genome and association of HCV particles with host lipoproteins and immunoglobulins (1, 15). Thus, nonstructural proteins involved in viral replication are being examined as targets of antiviral therapy. One of them is NS3 (serine protease/RNA helicase), whose helicase activity is indispensable for replication of the viral RNA (25). The helicase part of NS3 folds into three domains of comparable size (domains 1, 2, and 3) that form a triangular molecule. Five structures of the NS3 helicase have been resolved by X-ray crystallography. The latest resolved structure shows two helicases bound to a single DNA molecule and reveals an apparent interface between two protein molecules (33). The existence of oligomeric structures o...