48 – Hepatitis C vaccines

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The α Isoform of Protein Kinase CKI Is Responsible for Hepatitis C Virus NS5A Hyperphosphorylation

Hepatitis C virus (HCV) has been the subject of intensive studies for nearly two decades. Nevertheless, some aspects of the virus life cycle are still a mystery. The HCV nonstructural protein 5A (NS5A) has been shown to be a modulator of cellular processes possibly required for the establishment of viral persistence. NS5A is heavily phosphorylated, and a switch between a basally phosphorylated form of NS5A (p56) and a hyperphosphorylated form of NS5A (p58) seems to play a pivotal role in regulating HCV replication. Using kinase inhibitors that specifically inhibit the formation of NS5A-p58 in cells, we identified the CKI kinase family as a target. NS5A-p58 increased upon overexpression of CKI-α, CKI-δ, and CKI-ɛ, whereas the RNA interference of only CKI-α reduced NS5A hyperphosphorylation. Rescue of inhibition of NS5A-p58 was achieved by CKI-α overexpression, and we demonstrated that the CKI-α isoform is targeted by NS5A hyperphosphorylation inhibitors in living cells. Finally, we showed that down-regulation of CKI-α attenuates HCV RNA replication

Hepatitis C virus-specific directly acting antiviral drugs

The major targets for direct-acting antivirals (DAAs) are the NS3/4A protease, the NS5A protein, and the NS5B polymerase. The latter enzyme offers several target sites: the catalytic domain for nucleoside/nucleotide analogs and different allosteric sites for non-nucleoside inhibitors. Two protease inhibitors have already been approved and more than 40 new NS3/4A, NS5A, or NS5B inhibitors are in development pipeline. Not only these agents can achieve very high cure rates when combined with PEG-IFN and RBV, but have also started to provide promising results when combined in IFN-free, all-oral combinations. In addition to the more canonical drug targets, new alternative viral targets for small molecule drug development are emerging, such as p7 or NS4B. Current research is focusing on defining the most efficacious DAA combination regimens, i.e., those which provide the highest rates of viral eradication, broadest spectrum of action, minimal or no clinical resistance, shortest treatment duration, and good tolerability.status: publishe

A novel, inducible, eukaryotic gene expression system based on the quorum-sensing transcription factor TraR

Bacteria adapt their pattern of gene expression in response to a variety of external cues, including fluctuations in population density. This type of bacterial cell-to-cell communication is referred to as quorum-sensing. Quorum-sensing systems are present in many bacterial species and constitute a large collection of ligands and cognate receptors. The availability of such diversity offers interesting opportunities for biotechnological exploitation. We describe here the transformation of the quorum-sensing system of Agrobacterium tumefaciens into a transcription regulatory system that works in mammalian cells. The A. tumefaciens TraR protein was fused to the eukaryotic activation domain of NF-κB p65, generating a novel chimaeric transcriptional activator that stimulates gene transcription in different human cell lines from a minimal promoter containing the TraR DNA recognition sequence in the presence of the Agrobacterium quorum-sensing signal molecule N-(3-oxo-octanoyl)homoserine lactone (3-oxo-C(8)-HSL). The basal level of transcription was low in the absence of 3-oxo-C(8)-HSL, and gene expression was stimulated up to 1,000-fold at a saturating concentration of 3-oxo-C(8)-HSL

The crystal structure of the quorum sensing protein TraR bound to its autoinducer and target DNA

The quorum sensing system allows bacteria to sense their cell density and initiate an altered pattern of gene expression after a sufficient quorum of cells has accumulated. In Agrobacterium tumefaciens, quorum sensing controls conjugal transfer of the tumour- inducing plasmid, responsible for plant crown gall disease. The core components of this system are the transcriptional regulator TraR and its inducing ligand N-(3-oxo-octanoyl)-l-homoserine lactone. This complex binds DNA and activates gene expression. We have determined the crystal structure of TraR in complex with its autoinducer and target DNA (PDB code 1h0m). The protein is dimeric, with each monomer composed of an N-terminal domain, which binds the ligand in an enclosed cavity far from the dimerization region, and a C-terminal domain, which binds DNA via a helix–turn–helix motif. The structure reveals an asymmetric homodimer, with one monomer longer than the other. The N-terminal domain resembles GAF/PAS domains, normally fused to catalytic signalling domains. In TraR, the gene fusion is between a GAF/PAS domain and a DNA-binding domain, resulting in a specific transcriptional regulator involved in quorum sensing