Reduced lipid droplet content in cells reduces and delays the host response to dsDNA.

<p><b>A.</b> HeLa and <b>B.</b> Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to transfection with 0.5 μg poly dA:dT per well for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFN-β and (ii) IFN- λ or <b>C.</b> Viperin mRNA (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).</p

Lipid droplet content alters the host cell response to sendai virus.

<p> Hela cells were pre-incubated in either low serum or control media for 48 hrs prior to infection with 50 HAU/ ml SeV. Following 8 and 24 hrs infections of SeV, RTq-PCR was performed to quantitate mRNA expression of <b>A.</b> IFN-β and <b>B.</b> IFN-λ. (*, <i>p<</i> 0.05; **, <i>p<</i> 0.01; ***, <i>p<</i> 0.001).</p

Primer table.

<p>Primer table.</p

Lipid droplet content does not impact nucleic entry into Huh-7 cells.

<p><b>A. A.</b> Huh-7 cells were either incubated in control media or 2% FCS media, 48 hrs prior to transfection with rhodamine conjugated poly I:C. <b>B.</b> Cells were imaged, using a Nikon T<i>i-</i>E inverted microscope and quantification of fluorescence intensity performed using NIS Elements AR v.3.22.</p

Lipid droplet mass is reduced by using lowered serum media, with little effect on their biogenesis.

<p><b>A.</b> Reduced serum (10% to 2% FCS) was applied to Huh-7 and HeLa cells for 48 hrs. Bodipy 505/515 (i) and Oil Red O (ii) were used to stain neutral lipid in both Huh-7 and HeLa cells. Images of fixed cells were captured using a Nikon Eclipse Ti-E microscope at 20X and 40X magnification respectively. DAPI counterstaining was also used to visualise cell nuclei. <b>B.</b> Following visualisation of bodipy stained cells, quantification of fluorescence intensity was performed using NIS Elements AR v.3.22. ***p < 0.001 <b>C.</b> Cells were grown in low serum conditions (2% FCS) for 72 hrs. Rapamycin (RAPA) and Chloroquine (CQ) were used as a positive control for the induction of autophagy. Membranes were probed with anti-LC3 specific antibody and anti-rabbit HRP. Membranes scanned using Amersham 600 chemiluminescence imager (i); Densitometry was performed using Image J analysis (ii) <b>D.</b> LD number was reduced in Huh-7 and HeLa cells by reducing FCS in media to 2% for 48 hrs prior to experiment, representative by time 0. Cells were then returned to 10% FCS media at commencement for experiment, and fixed at the indicated time points. Cells were stained with Bodipy 505/515 and DAPI prior to imaging using a Nikon Eclipse Ti-E microscope at 20X magnification (i), and subsequent image analysis using NIS elements (ii).</p

LD content alters the regulation of ISGs in a cell-type dependent manner.

<p><b>A.</b> Huh-7 and HeLa cells were transiently transfected 24 hrs prior to stimulation with 500 ng/well of ISRE-Luc and 5 ng/well of pRL-TK in 12-well tissue culture plates. Luciferase measurements were taken 5 hrs post stimulation with IFN-β (ns = not statistically significant). <b>B.</b> HeLa and <b>C.</b> Huh-7 cells were pre-incubated in either low serum or control media 48 hrs prior to stimulation with 1000 units/ml of. IFN-β for the indicated times. RTq-PCR was used to quantify mRNA expression of (i) IFIT-1 (ii) OAS-1 and (iii) Viperin (ns = not statistically significant, *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001).</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 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 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

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