The antiviral protein viperin inhibits HCV replication via interaction with NS5A

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

Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin

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.Karla J. Helbig, Jillian M. Carr, Julie K. Calvert, Satiya Wati, Jennifer N. Clarke, Nicholas S. Eyre, Sumudu K. Narayana, Guillaume N. Fiches, Erin M. McCartney, Michael R. Bear

Generation of a chimeric hepatitis C replicon encoding a genotype-6a NS3 protease and assessment of boceprevir (SCH503034) sensitivity and drug-associated mutations

Background: Genotype (gt)6 HCV is common amongst HCV-positive populations of the Asia-Pacific region but cell culture models for this gt have only recently been developed. Boceprevir (SCH503034) is a clinically available inhibitor of the HCV NS3 protein. We investigated the efficacy of boceprevir for inhibiting replication of a chimeric gt1b replicon encoding a gt6a NS3 protease and defined the development of mutations in the protease when boceprevir treatment was applied. Methods: We constructed a chimeric gt1b subgenomic replicon encoding a gt6 NS3 protease (NS3p) sequence (gt6NS3p-gt1b). The boceprevir EC value against replication of this replicon was determined using quantitative reverse transcriptase PCR. Next-generation sequencing was used to identify nucleotide changes associated with boceprevir resistance. The replication capacities of chimeric replicons containing mutations associated with boceprevir resistance were determined by colony formation efficiency assays. Results: The boceprevir EC value for the gt6NS3p-gt1b replicon was 535 ±79 nM. Boceprevir-resistant gt6NS3p-gt1b replicon cell lines could be selected and they demonstrated drug-associated amino acid changes that have previously been reported in other HCV gts. Interestingly, no mutations were observed at A156, a position defined for boceprevir resistance in gt1 NS3p, while mutation at N122, which is rarely reported in boceprevir-resistant gt1 proteases, was frequently observed. Re-introduction of these mutations into the chimeric replicon altered their replication capacity, ranging from complete abolishment of replication (A156T) to increasing replication capacity (V36A, N122S). This report provides the first characterization of gt6 HCV resistance to boceprevir. Conclusions: A chimeric HCV replicon encoding gt6 NS3 protease is sensitive to boceprevir and develops drug-resistant mutations at amino acid sites previously reported for other gts. Mutation at N122 also appears to be associated with boceprevir resistance in the gt6 NS3 protease

Viperin mRNA is induced in DENV-2 infected cells.

<p>Cells were infected with DENV-2 (MOI = 1 or MOI = 3 for MDM) and at various time points pi intracellular RNA was extracted and viperin mRNA and DENV −ve strand RNA quantitated by real time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change. Values represent average ± SEM (n = 3). (<b>A</b>) A549; (<b>B</b>) Huh-7; (<b>C</b>) Huh-7.5; (<b>D</b>) MDM. * Significantly different in comparison to 0 h time point, p<0.05.</p

Viperin protein is induced in DENV-2 infected cells.

<p><b>A.</b> Primary MDM were left uninfected, treated with 500 U/ml IFN-α or DENV-2 infected. At 48 h pi cells were lysed and viperin protein analysed by western blot. Blots were re-probed for β-actin and images visualised by chemiluminesence. Images were quantitated using Carestream Molecular Imaging Software and viperin signal normalised against β-actin. <b>B.</b> Primary MDM were DENV-2 (i) or mock (ii) infected and at 24 h pi were fixed and immunostained for viperin and DENV, with detection of stained complexes with anti-rabbit 647 (red) and anti-mouse 488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy. <b>C.</b> Immunolabeling for viperin was quantitated in cells from mock-infected MDM and compared with antigen negative bystander and DENV-2 antigen positive cells of the DENV-2 infected MDM cultures. Values represent average ± SEM. (n = 111 mock; 27 DENV-antigen positive; 136 DENV-antigen negative bystander cells). * = significantly different, p<0.05, Students unpaired t-test. Results of a single experiment are shown which was replicated.</p

Viperin is anti-viral in primary MDM.

<p>Primary MDM were generated from peripheral blood and transduced with lentiviral particles expressing control td-Tomato or WT viperin. At 24 h post transduction, cells were infected with DENV-2 (MOI = 3). (<b>A</b>) Supernatant was sampled and infectious virus release quantitated by plaque assay. Values represent average ± SEM (n = 3). * p<0.001; (<b>B</b>) Viperin lenti-transduced MDM were DENV-2 or mock infected and at 48 h pi cells were fixed and immunolabelled for viperin and DENV with detection of complexes with Alexa-647 (red) and Alexa-488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy.</p

Induction of viperin is needed to restrict early viral replication.

<p>Viperin shRNA or control shRNA expressing Huh7 cells were infected with DENV-2 (MOI = 0.1). (<b>A</b>) Supernatant was sampled and infectious virus release quantitated by plaque assay. Values represent average ± SEM (n = 3); At the indicated time point pi cells were lysed, RNA extracted and analysed by real time RT-PCR for (<b>B</b>) viperin mRNA; (<b>C</b>) IFIT1 mRNA. Values represent average ± SEM (n = 4). Results were normalised against control RPLPO mRNA levels and expressed as fold change relative to mock infected cells. * = significant at p<0.05, Students unpaired t-test.</p

Viperin interacts with DENV-2 CA and NS3 protein.

<p>Huh-7 cells were co-transfected with a DENV-2 CA-GFP and viperin-mCherry expression vector (<b>A</b>), or DENV-2 NS3-GFP and expression plasmids for either viperin-mCherry (<b>B</b>), viperin 5′Δ33-mCherry (<b>C</b>) or viperin 3′Δ17-mCherry (<b>D</b>). Slides were analysed on a Zeiss Axioplan microscope and FRET determined by acceptor photobleaching. DIF was calculated from comparison of aligned pre and post-bleach images, from 5–10 regions per cell of at least 10 cells from 2 different experiments. Data are represented as average ± SEM with a significance of p<0.05 for (A), (B) and (C).</p

Viperin is anti-viral against DENV-2 and requires C-terminal regions of the protein.

<p>(<b>A</b>) HeLa cells were transfected with either a viperin-FLAG expression plasmid (<b>i</b>) or a control vector (<b>ii</b>) and at 24 h post transfection infected with DENV-2 (MOI = 1). At 24 h pi cells were fixed and immunolabelled with anti-FLAG (viperin) and anti-dsRNA antibodies with detection of stained complexes with anti-rabbit 647 (red) and anti-mouse 488 (green), respectively. Nuclei were stained with Hoechst (blue) and images collected by confocal microscopy. (<b>B</b>) Huh-7 cells were transfected to express WT viperin or viperin mutants and at 24 h post transfection infected with DENV-2 (MOI = 0.1). 24 h pi RNA was extracted and DENV-2 −ve strand PCR quantitated by real-time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change. Values represent average ± SEM (n = 3). * = significantly different to no viperin control, ** = significantly different to no viperin control and WT viperin, p<0.05, Students t-test. Similar experiments to (B) were performed in (<b>C</b>) Huh-7 or (<b>D</b>) A549. Cells were transfected using WT viperin or a 3′Δ17 viperin expression construct and infected as in (B). Supernatant was sampled and analysed for infectious virus release by plaque assay and RNA extracted from infected cells and DENV −ve strand RNA quantitated by real time RT-PCR. Results were normalised against control RPLPO mRNA levels and expressed as fold change relative to 3′Δ17 viperin control. Values represent average ± SEM (n = 3). * = significantly different to WT viperin, p<0.05, Students unpaired t-test.</p