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

Profiling of immune related genes silenced in EBV-positive gastric carcinoma identified novel restriction factors of human gammaherpesviruses.

EBV-associated gastric cancer (EBVaGC) is characterized by high frequency of DNA methylation. In this study, we investigated how epigenetic alteration of host genome contributes to pathogenesis of EBVaGC through the analysis of transcriptomic and epigenomic datasets from NIH TCGA (The Cancer Genome Atlas) consortium. We identified that immune related genes (IRGs) is a group of host genes preferentially silenced in EBV-positive gastric cancers through DNA hypermethylation. Further functional characterizations of selected IRGs reveal their novel antiviral activity against not only EBV but also KSHV. In particular, we showed that metallothionein-1 (MT1) and homeobox A (HOXA) gene clusters are down-regulated via EBV-driven DNA hypermethylation. Several MT1 isoforms suppress EBV lytic replication and release of progeny virions as well as KSHV lytic reactivation, suggesting functional redundancy of these genes. In addition, single HOXA10 isoform exerts antiviral activity against both EBV and KSHV. We also confirmed the antiviral effect of other dysregulated IRGs, such as IRAK2 and MAL, in scenario of EBV and KSHV lytic reactivation. Collectively, our results demonstrated that epigenetic silencing of IRGs is a viral strategy to escape immune surveillance and promote viral propagation, which is overall beneficial to viral oncogenesis of human gamma-herpesviruses (EBV and KSHV), considering that these IRGs possess antiviral activities against these oncoviruses

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 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

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

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