The interferon-stimulated gene viperin has been shown to have antiviral activity against hepatitis C virus (HCV) in the context of the HCV replicon, although the molecular mechanisms responsible are not well understood. Here we demonstrate that viperin plays an integral part in the ability of interferon to limit replication of cell culture derived HCV (JFH-1) that accurately reflects the complete viral life cycle. Using confocal microscopy and Fluorescence Resonance Energy Transfer (FRET) analysis we demonstrate that viperin localizes and interacts with HCV NS5A at the lipid droplet interface. In addition viperin also associates with NS5A and the pro-viral cellular factor, VAP-A at the HCV replication complex. The ability of viperin to limit HCV replication was dependent on residues within the C-terminus as well as an N-terminal amphipathic helix. Removal of the amphipathic helix redirected viperin from the cytosolic face of the ER and the lipid droplet to a homogenous cytoplasmic distribution, coinciding with a loss of antiviral effect. C-terminal viperin mutants still localized to the lipid droplet interface and replication complexes but did not interact with NS5A proteins as determined by FRET analysis. In conclusion we propose that viperin interacts with NS5A and the host factor VAP-A to limit HCV replication at the replication complex. This highlights the complexity of host control of viral replication by interferon stimulated gene expression.

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.

Hepatitis C virus (HCV) NS5A is essential for viral genome replication within cytoplasmic replication complexes and virus assembly at the lipid droplet (LD) surface, although its definitive functions are poorly understood. We developed approaches to investigate NS5A dynamics during a productive infection. We report here that NS5A motility and efficient HCV RNA replication require the microtubule network and the cytoplasmic motor dynein and demonstrate that both motile and relatively static NS5A-positive foci are enriched with host factors VAP-A and Rab5A. Pulse-chase imaging revealed that newly synthesized NS5A foci are small and distinct from aged foci, while further studies using a unique dual fluorescently tagged infectious HCV chimera showed a relatively stable association of NS5A foci with core-capped LDs. These results reveal new details about the dynamics and maturation of NS5A and the nature of potential sites of convergence of HCV replication and assembly pathways.