No abstract

Hepatitis C virus (HCV) is a major global health burden accounting for around 170 million chronic infections worldwide. Although highly potent direct-acting antiviral drugs to treat chronic hepatitis C have been approved recently, owing to their high costs and limited availability and a large number of undiagnosed infections, the burden of disease is expected to rise in the next few years. In addition, HCV is an excellent paradigm for understanding the tight link between a pathogen and host cell pathways, most notably lipid metabolism. HCV extensively remodels intracellular membranes to establish its cytoplasmic replication factory and also usurps components of the intercellular lipid transport system for production of infectious virus particles. Here, we review the molecular mechanisms of viral replicase function, cellular pathways employed during HCV replication factory biogenesis, and viral, as well as cellular, determinants of progeny virus production.

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials.

No abstract

Numerous anti-hepatitis C virus (HCV) drugs targeting either the viral nonstructural 3 (NS3) protease or NS5B polymerase are currently in clinical testing. However, rapid resistance development is a major problem and optimal therapy will clearly require a combination of multiple mechanisms of action. Cyclosporine A (CsA) and its nonimmunosuppressant derivatives are among the more promising drugs under development. Based on work with subgenomic HCV replicons it has been thought that they act as NS5B-inhibitors. In this study we show that CsA inhibits replication of full-length HCV Japanese Fulminant Hepatitis (JFH1) genomes about 10-fold more efficiently than subgenomic replicons. This effect is dependent on the presence of NS2 in the viral polyprotein and mediated through cellular cyclophilin A. NS2 is either an additional target for CsA-dependent inhibition or modulates the antiviral activity against NS3 to NS5B proteins. CsA is thus the first anti-HCV drug shown to act through NS2.

No abstract

Picornavirus 2B, a non-structural protein required for effective viral replication, has been implicated in cell membrane permeabilization during the late phases of infection. Here, we have approached the molecular mechanism of this process by assessing the pore-forming activity of an overlapping peptide library that spanned the complete 2B sequence. At non-cytopathic concentrations, only the P3 peptide, spanning 2B residues 35-55, effectively assembled hydrophilic pores that allowed diffusion of low molecular mass solutes across the cell plasma membrane (IC(50) approximately 4x10(-7) M) and boundary liposome bilayers (starting at peptide to lipid molar ratios>1:10(4)). Circular dichroism data were consistent with its capacity to fold as a helix in a membrane-like environment. Furthermore, addition of this peptide to a sealed plasma-membrane model, consisting of retinal rod outer segments patch-clamped in a whole-cell configuration, induced ion channel activity within seconds at concentrations as low as 10(-8) M. Thus, we have established a "one-helix" 2B version that possesses the intrinsic pore-forming activity required to directly and effectively permeabilize the cell plasma membrane. We conclude that 2B viroporin can be classified as a genuine pore-forming toxin of viral origin, which is produced intracellularly at certain times post infection.

Hepatitis C virus (HCV) nonstructural protein (NS)5A is a RNA-binding protein composed of a N-terminal membrane anchor, a structured domain I (DI) and two intrinsically disordered domains (DII and DIII) interacting with viral and cellular proteins. While DI and DII are essential for RNA replication, DIII is required for assembly. How these processes are orchestrated by NS5A is poorly understood. In this study, we identified a highly conserved basic cluster (BC) at the N-terminus of DIII that is critical for particle assembly. We generated BC mutants and compared them with mutants that are blocked at different stages of the assembly process: a NS5A serine cluster (SC) mutant blocked in NS5A-core interaction and a mutant lacking the envelope glycoproteins (ΔE1E2). We found that BC mutations did not affect core-NS5A interaction, but strongly impaired core–RNA association as well as virus particle envelopment. Moreover, BC mutations impaired RNA-NS5A interaction arguing that the BC might be required for loading of core protein with viral RNA. Interestingly, RNA-core interaction was also reduced with the ΔE1E2 mutant, suggesting that nucleocapsid formation and envelopment are coupled. These findings argue for two NS5A DIII determinants regulating assembly at distinct, but closely linked steps: (i) SC-dependent recruitment of replication complexes to core protein and (ii) BC-dependent RNA genome delivery to core protein, triggering encapsidation that is tightly coupled to particle envelopment. These results provide a striking example how a single viral protein exerts multiple functions to coordinate the steps from RNA replication to the assembly of infectious virus particles.