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

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 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

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