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The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.

Hepatitis C virus (HCV) afflicts more than 170 million people worldwide causing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. The recent development of complete cell-culture systems for HCV has accelerated the pace of hepatitis research. Specifically, these techniques have provided new insights into the virus lifecycle that are reviewed here. This should pave the way for developing bespoke and effective antiviral therapies and vaccines. Exciting progress has recently been made in understanding the replication of hepatitis C virus, a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. The development of complete cell-culture systems should now enable the systematic dissection of the entire viral lifecycle, providing insights into the hitherto difficult-to-study early and late steps. These efforts have already translated into the identification of novel antiviral targets and the development of new therapeutic strategies, some of which are currently undergoing clinical evaluation.

Solid state NMR sample preparation and resonance assignments of the U-[ 13 C, 15 N] 2 × 10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15 N, 13 C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts. Abbreviations: ssNMR – solid state NMR; MAS – magic angle spinning; Crh – catabolite repression histidinecontaining phosphocarrier protein; HPr – histidine-containing phosphocarrier protein; CcpA – carbon control protein A; HprK/P – HPr kinase/phosphatase; PEG – polyethylene glycol; DSS – 2,2-dimethylsilapentane-5sulfonic acid; CP – cross polarization; PDSD – proton driven spin diffusion; TPPM – two pulse phase modulation; RFDR – radio frequency-driven recoupling; DQ – double quantum; SPC-5 – supercycled POST-C5; r.f. radio frequency; HORROR – homonuclear rotary resonance; SPECIFIC-CP – spectrally induced filtering in combination with cross-polarization.

Active F1-ATPase prepared from pig heart mitochondria can react with about 2 mol of DTNB (5,5'-dithiobis-2-nitrobenzoic acid) or CPDS (6,6'-dithiodinicotinic acid). The reactivity of these thiol reagents decreases if ATP is absent or if F1-ATPase has been submitted to thermal treatment that increases the specific activity without eliminating any contaminating protein. Affinity chromatography on a Sepharose-DTNB column has shown that the thermal treatment of F1-ATPase induces a conformational change of the enzyme that completely prevents it from being retained on the column while the normal active enzyme can be specifically bound to the Sepharose-DTNB column. A comparative study of the thiols of F1-ATPase reacting with CPDS measured by spectrophotometric estimation of the thione released from CPDS and by [14C]CPDS binding to F1-ATPase suggests involvement of a vicinal dithiol in active F1-ATPase. After CPDS reaction, this vicinal dithiol may become an internal disulfide bridge.