Background & Aims Direct-acting antivirals that target nonstructural protein 5A (NS5A), such as daclatasvir, have high potency against the hepatitis C virus (HCV). They are promising clinical candidates, yet little is known about their antiviral mechanisms. We investigated the mechanisms of daclatasvir derivatives. Methods We used a combination of biochemical assays, in silico docking models, and high-resolution imaging to investigate inhibitor-induced changes in properties of NS5A, including its interaction with phosphatidylinositol-4 kinase IIIα and induction of the membranous web, which is the site of HCV replication. Analyses were conducted with replicons, infectious virus, and human hepatoma cells that express a HCV polyprotein. Studies included a set of daclatasvir derivatives and HCV variants with the NS5A inhibitor class-defining resistance mutation Y93H. Results NS5A inhibitors did not affect NS5A stability or dimerization. A daclatasvir derivative interacted with NS5A and molecular docking studies revealed a plausible mode by which the inhibitor bound to NS5A dimers. This interaction was impaired in mutant forms of NS5A that are resistant to daclatavir, providing a possible explanation for the reduced sensitivity of the HCV variants to this drug. Potent NS5A inhibitors were found to block HCV replication by preventing formation of the membranous web, which was not linked to an inhibition of phosphatidylinositol-4 kinase IIIα. Correlative light-electron microscopy revealed unequivocally that NS5A inhibitors had no overall effect on the subcellular distribution of NS5A, but completely prevented biogenesis of the membranous web. Conclusions Highly potent inhibitors of NS5A, such as daclatasvir, block replication of HCV RNA at the stage of membranous web biogenesis—a new paradigm in antiviral therapy.

Background: Corticosteroids may cause immediate or delayed hypersensitivity. In 1989, based on structural and clinical characteristics, we put forward a classification of corticosteroids into four cross‐reacting groups, namely group A, B, C, and D, the latter later subdivided into two subgroups, i.e. D1 and D2. The constituents on the D‐ring of the corticosteroid‐molecule are considered to have a central role for binding to skin proteins and for cross‐reactions patterns; however, halogenation of the molecules is also interfering.Objective: To study the clinical data and analyse simultaneous positive reactions obtained in a large group of corticosteroid‐allergic patients.Methods: Patch tests were performed with the baseline series, to which hydrocortisone butyrate and prednisolone caproate were added, as well as with the corticosteroids to which the patients had been exposed. Three hundred and forty subjects with a presumed or proven corticosteroid allergy were further investigated with an extended series containing 72 molecules.Results: Out of 11 596 patients investigated, 315 subjects reacted positively to at least 1 corticosteroid‐molecule, with most of them presenting with multiple positive reactions.Conclusion: A prevalence of corticosteroid allergy of 2.7% was found. Despite validity of the ABCD (sub)classification in many cases, possible adjustments may have to be considered.

Capsular polysaccharides are well-established virulence factors of pathogenic bacteria. Their biosynthesis and export are regulated within the transmembrane polysaccharide assembly machinery by the autophosphorylation of atypical tyrosine-kinases, named BY-kinases. However, the accurate functioning of these tyrosine-kinases remains unknown. Here, we report the crystal structure of the non-phosphorylated cytoplasmic domain of the tyrosine-kinase Wzc from Escherichia coli in complex with ADP showing that it forms a ring-shaped octamer. Mutational analysis demonstrates that a conserved EX₂RX₂R motif involved in subunit interactions is essential for polysaccharide export. We also elucidate the role of a putative internal regulatory tyrosine and we show that BY-kinases from proteobacteria autophosphorylate on their C-terminal tyrosine cluster via a single-step intermolecular mechanism. This structure-function analysis also allows us to demonstrate that two different parts of a conserved basic region called the RK-cluster are essential for polysaccharide export and for kinase activity respectively. Based on these data, we revisit the dichotomy made between BY-kinases from proteobacteria and firmicutes and we propose a unique process of oligomerization and phosphorylation. We also reassess the function of BY-kinases in the capsular polysaccharide assembly machinery.