Oyster viperin retains direct antiviral activity and its transcription occurs via a signalling pathway involving a heat-stable haemolymph protein

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1 Journl of Generl Virology (215), 96, DOI 1.199/jgv..3 Oyster viperin retins direct ntivirl ctivity nd its trnscription occurs vi signlling pthwy involving het-stble hemolymph protein Timothy J. Green, 1,2 Peter Speck, 2 Lu Geng, 3 Dvid Rftos, 1 Michel R. Berd 3 nd Krl J. Helbig 3 Correspondence Timothy J. Green tim.green@mq.edu.u 1 Deprtment of Biologicl Sciences nd Sydney Institute of Mrine Science, Mcqurie University, NSW 219, Austrli 2 School of Biologicl Sciences, Flinders University, GPO Box 21, Adelide, SA 51, Austrli 3 School of Biologicl Sciences, University of Adelide, SA 51, Austrli Received 28 July 215 Accepted 24 September 215 Little is known bout the response of non-model invertebrtes, such s oysters, to virus infection. The vertebrte innte immune system detects virus-derived nucleic cids to trigger the type I IFN pthwy, leding to the trnscription of hundreds of IFN-stimulted genes (ISGs) tht exert ntivirl functions. Invertebrtes were thought to lck the IFN pthwy bsed on the bsence of IFN or ISGs encoded in model invertebrte genomes. However, the oyster genome encodes mny ISGs, including the well-described ntivirl protein viperin. In this study, we chrcterized oyster viperin nd showed tht it loclizes to cveolin-1 nd inhibits dengue virus repliction in heterologous model. In second set of experiments, we hve provided evidence tht the hemolymph from poly(i : C)-injected oysters contins het-stble, protesesusceptible fctor tht induces hemocyte trnscription of viperin mrna in conjunction with upregultion of IFN regultory fctor. Collectively, these results support the concept tht oysters hve ntivirl systems tht re homologous to the vertebrte IFN pthwy. INTRODUCTION IFNs re clss of cytokines tht induce vertebrte cells into n ntivirl stte (Rndll & Goodbourn, 28). Typiclly, virus-infected cells secrete IFNs to lert other cells in the body to the presence of virus (Robertsen, 26). IFNs induce n ntivirl stte by binding to IFN receptors, which re present on ll nucleted cells (Biron & Sen, 21). Receptor enggement ctivtes signl trnsduction vi the Jnus kinse/signl trnsducer nd ctivtor of trnscription (JAK/STAT) pthwy, leding to the trnscription of hundreds of IFN-stimulted genes (ISGs) (Schoggins & Rice, 211). The products of these ISGs exert numerous ntivirl effector functions, mny of which re still not fully described (Schoggins & Rice, 211). Viperin (virus inhibitory protein, endoplsmic reticulum-ssocited, IFN inducible) is one of few ISGs tht hs been shown to hve direct ntivirl ctivity ginst rnge of RNA nd DNA viruses (Mttijssen & Pruijn, 212), nd is one of the erliest nd most significntly upregulted genes in response to virl infection in humns (reviewed by Helbig & Berd, 214). Viperin ws first isolted from fibroblst cells nd ws shown to be n The GenBnk/EMBL/DDBJ ccession numbers for the four oyster viperin sequences determined in this study re KT KT inducible cytoplsmic ntivirl protein tht is induced by IFNs nd humn cytomeglovirus (HCMV) (Chin & Cresswell, 21). Subsequently, viperin hs been chrcterized in vriety of vertebrte species nd shown to be highly conserved evolutionry host protein (Goossens et l., 215; Helbig et l., 211; Milic et l., 215; Wng et l., 27; Zhng et l., 214). Viperin loclizes to the endoplsmic reticulum (ER) nd lipid droplets (reviewed by Mttijssen & Pruijn, 212) nd inhibits the relese of influenz virus nd humn immunodeficiency virus by ltering the formtion of lipid rfts, which re the known sites of virus budding (Nsr et l., 212; Wng et l., 214b). Viperin lso inhibits the repliction of both heptitis C virus nd dengue virus by intercting with virl nonstructurl proteins (Helbig et l., 211, 213). The evolutionry origins nd divergence of mjor immune response pthwys hve generlly been inferred from comprisons between vertebrtes nd model invertebrte species, such s Drosophil melnogster, Anopheles gmbie, Cenorhbditis elegns nd Cion intestinlis (Roblino et l., 24). The bsence of IFN or its mjor effectors from the genomes of these model invertebrtes hs been used to imply tht the IFN pthwy is vertebrte innovtion (Loker et l., 24; Roblino et l., 25). However, non-model invertebrte species might hve 3 G 215 The Authors Printed in Gret Britin 3587

2 T. J. Green nd others ntivirl systems tht re homologous to the vertebrte type I IFN response (Green & Montgnni, 213; He et l., 215). In prticulr, trnscriptome sequencing of the Pcific oyster () infected with ostreid herpesvirus type 1 (OsHV-1) hs reveled n ncient IFN pthwy in the oyster genome with key components [Toll-like receptor (TLR), Rig-like Receptor (RLR), IFN regultory fctors (IRFs), JAK/STAT nd ISGs] conserved nd highly upregulted in response to OsHV-1 infection (He et l., 215; Renult et l., 211; Rosni et l., 214; Segrr et l., 214b). Mny of these ntivirl genes re upregulted in C. gigs tissues following injection with poly(i : C) to mimic virus infection (Green et l., 214, b), nd this inducible immune response cn inhibit OsHV-1 infection (Green & Montgnni, 213). Viperin is lso reported to be one of the erliest nd most upregulted of the C. gigs genes in response to OsHV-1 (Rosni et l., 214 nd poly(i : C) (Green et l., 214b), but it is unknown whether oyster viperin hs direct ntivirl ctivity. The concept tht non-model invertebrtes, such s oysters, hve type I IFN response is still contrry to estblished views in innte immunology. Mny comprtive immunologists re scepticl tht oysters hve n IFN response becuse bioinformtics nlysis of ll fully sequenced invertebrte genomes (including the oyster) hve filed to identify n IFN cytokine (He et l., 215; Loker et l., 24). In ddition, it is unknown whether invertebrte genes tht shre sequence homology to vertebrte ISGs hve lso retined their ntivirl functions over long evolutionry time period. Therefore, the first objective of this study ws to chrcterize oyster viperin nd determine whether it hd direct ntivirl ctivity. The second objective ws to confirm tht expression of oyster viperin is induced vi cytokine. RESULTS Sequence nlysis of oyster viperin Utilizing the oyster genome dtbse ( com), primers were designed to mplify the complete coding sequence of C. gigs viperin. The full-length coding sequence of oyster viperin ws mplified nd sequenced from four individul oysters nd confirmed to be 15 bp, encoding 35. Comprison of these four nucleotide sequences reveled single 3 bp insertion in the N terminus nd 13 single-nucleotide polymorphisms (SNPs) in the C terminus of C. gigs viperin. Only two of these SNPs were ssocited with mino cid substitutions t 25 nd 273. In the vertebrte phylum, viperin mino cid sequences re highly conserved. A comprison of C. gigs viperin with humn (; GenBnk ccession no. NP_542388), fish (; GenBnk ccession no. NP_12727), chicken (; GenBnk ccession no. ACA83729) nd lncelet ( floride; GenBnk ccession no. EEN65148) viperin reveled % mino cid identity. The C-terminl region (essentil for some of the ntivirl ctivity of this protein) of viperin ws highly conserved between vertebrtes nd C. gigs (Fig. 1). The mphipthic helix in the N-terminl region of humn viperin llows it to tether to the ER nd lipid droplets. However, n mphipthic helix could not be predicted in C. gigs viperin (Amphipseek, lthough it does retin the rdicl S-denosyl methionine (SAM) domin (Conserved Domins serch, dsrna induces oyster viperin expression Mmmlin viperin is rpidly induced (within 2 h) in response to viruses, IFN nd bcteril by-products, such s dsrna nd lipopolyscchride (LPS) (reviewed by Helbig & Berd, 214). We chose to investigte oyster viperin expression in response to poly(i : C), which is synthetic dsrna molecule. The use of synthetic dsrna in plce of replicting virus llowed full nlysis of cellulr response to dsrna in the bsence of ny interference tht my occur vi specific virl proteins. In contrst to vertebrtes, injection of poly(i : C) in the oyster s dductor muscle resulted in the delyed expression of viperin (Fig. 2). Hemocyte expression of oyster viperin remined stble t 3 nd 9 h p.i. (Pw.5, Fig. 2) nd then incresed rpidly to pek t 27 p.i. (Fig. 2, Pv.5). At 27 h p.i., viperin mrna ws lso elevted in dductor muscle, gill nd mntle tissues (Pv.5) but not in digestive glnd or gond tissues (Fig. 2b, Pw.5). Stimultion of primry hemocyte cell cultures with different concentrtions of poly(i : C) or LPS reveled dose threshold for C. gigs viperin expression (Fig. 3). Expression of hemocyte viperin ws induced by poly(i : C) t concentrtion of 24. mg ml 21 (Pv.5) but not t 2.4 nd.24 mg ml 21 (Pw.5). Stimultion of hemocytes with three different concentrtions of LPS filed to induce the expression of C. gigs viperin (Fig. 3, Pw.5). Hemolymph protein/peptide induces hemocyte viperin expression Our results demonstrted tht injection of poly(i : C) into the oyster dductor muscle resulted in elevted viperin expression in the mjority of tissue comprtments. Three possibilities exist for the systemic expression of oyster viperin: (i) cells within the dductor muscle secrete cytokine; (ii) poly(i : C) diffuses from the site of injection to other tissue comprtments; or (iii) stimulted hemocytes re migrting from the dductor muscle to other tissue comprtments. We therefore undertook series of experiments to show tht cells within the dductor muscle re secreting cytokine. First, dult C. gigs were induced into n ntivirl stte by intrmusculr injection with poly(i : C) or sewter (control). The circulting hemocytes from oysters injected with poly(i : C) hd elevted expression levels of mny genes in the IFN pthwy, including TLR, retinoic cid inducible gene I-like helicse 3588 Journl of Generl Virology 96

3 Oyster viperin hs direct ntivirl ctivity Fig. 1. Viperin hs remrkbly high evolutionry conservtion in nimls. C. gigs viperin (GenBnk ccession no. KT334231) hs 68 % mino cid identity to viperin sequences isolted from humn (; GenBnk ccession no. NP_542388), fish (; GenBnk ccession no. NP_12727), chicken (; GenBnk ccession no. ACA83729) nd lncelet ( floride; GenBnk ccession no. EEN65148). The viperin mino cid sequences were ligned by CLUSTAL W using the phylogenetic softwre pckge MEGA v.6.6. The rdicl SAM domin is underlined. Asterisks indicte conserved mino cids. (RLH), IRF, JAK, STAT5A, STAT6, suppressor of cytokine signlling (SOC), protein kinse R (PKR) nd viperin (Fig. 4, Pv.5). The cellulr frction of the hemolymph ws retined from C. gigs injected with poly(i : C) [stimulted cell-free hemolymph (CFH)] or sewter (non-stimulted CFH) s culture medium for primry hemocyte cultures. The second group of experiments using primry hemocyte cultures reveled tht component of

4 T. J. Green nd others () 2 ΔΔC T viperin Sewter Poly(l : C) b b c bc (b) 2 ΔΔC T viperin Hemocytes (H) Brnchi (B) Mntle (M) Adductor (Ad) Digestive glnd (DG) Gond (G) Time (h) H B M Ad DG G Tissue (27 h) Fig. 2. Normlized expression of C. gigs viperin in response to poly(i : C) injection. () Viperin ws significntly upregulted in hemocytes t 27 nd 54 h post-injection. Different lower-cse letters denote significnt chnges in viperin expression compred with the sewter control (P,.5). (b) At 27 h post-injection, viperin ws significntly upregulted in hemocytes (H), brnchi (B), mntle (M) nd dductor tissue (Ad) (P,.5) but ws not induced in digestive glnd (DG) nd gond (G) tissue (P..5). Asterisks denote significnt differences compred with controls (*P,.5). stimulted CFH ctivted expression of IRF, JAK, STAT6 nd viperin (Fig. 4b, Pv.5). The upregultion of RLH in Fig. 4(b) is n nomly: RLH ws only upregulted in one experiment, wheres IRF, JAK, STAT6 nd viperin were consistently upregulted in three independent experiments. Fig. 4(c) demonstrtes tht the hemolymph compound tht induces viperin expression must be potent becuse diluting stimulted CFH (2 %, v/v) did not reduce viperin expression in primry hemocytes (Pw.5). Furthermore, this hemolymph component ppered to be het-stble, protese-susceptible fctor becuse the bility of the stimulted CFH to induce viperin expression ws retined fter digestion with RNse A or het inctivtion (Fig. 4c, Pw.5), but ws eliminted by proteinse K digestion (Fig. 4c, Pw.5). Oyster viperin loclizes to cveolin-1 but not to lipid droplets Mny viperin molecules to dte hve been shown to loclize to lipid droplets nd/or the ER, including murine, humn, crocodile nd fish viperin (Helbig et l., 211; Hinson & Cresswell, 29b; Milic et l., 215; Wng et l., 214), nd in most cses this hs been ttributed to the mphipthic helix of the N terminus of viperin (reviewed by Helbig & Berd, 214). Oyster viperin hs considerbly shorter N terminus thn humn viperin, nd our nlysis of oyster viperin suggested tht it is unlikely to form n mphipthic helix in its N terminus (see bove). To determine the locliztion of oyster viperin, we performed number of expression studies using the Huh-7 cell line, which is known to hve prominent lipid droplet formtion, mking viperin s potentil locliztion to this orgnelle esier to observe. To ssess the bility of both oyster nd humn viperin to loclize to lipid droplets, we co-trnsfected FLAG-tgged oyster nd humn viperin into Huh-7 cells in conjunction with MCherry conjugted to dipocyte differentition-relted protein (MCherry ADRP), which is resident lipid droplet mrker. As cn be seen in Fig. 5(), humn viperin co-loclized extensively with the lipid droplet; however, oyster viperin expression ppered more cytoplsmiclly punctte in nture nd did not co-loclize with ADRP. Due to the punctte nture of oyster viperin, we nlysed its potentil co-locliztion with number of orgnelle mrkers displying similr locliztion pttern, including those for lysosome (LAMP1), erly 2 ΔΔC T viperin b Sewter Poly(I : C) 24. μg ml 1 Poly(I : C) 2.4 μg ml 1 b Poly(I : C).24 μg ml 1 LPS 24. μg ml 1 LPS 2.4 μg ml 1 LPS.24 μg ml 1 Fig. 3. Poly(I : C) but not LPS is responsible for inducing hemocyte expression of C. gigs viperin. Primry cell cultures of C. gigs hemocytes were estblished from individul oysters nd exposed to three different concentrtions of poly(i : C) nd LPS. Different lower-cse letters denote significnt chnges in viperin expression compred with sewter control (P,.5). 359 Journl of Generl Virology 96

5 Oyster viperin hs direct ntivirl ctivity () 8 Poly(l : C) (b) Stim. CFH (c) In vivo 5 In vitro Sewter NS CFH ΔΔC T TLR RLH STING IRF JAK STAT5A STAT6 SOC PKR Viperin 2 ΔΔC T TLR RLH STING IRF JAK STAT5A STAT6 SOC PKR Viperin 2 ΔΔC T viperin In vitro b b b Stim. CFH 2 % CFH NS CFH 85 C RNAse Prot. K Fig. 4. Evidence tht hemolymph from oysters injected with poly(i : C) induces the expression of viperin nd IRF in hemocytes from nïve oysters. () In vivo injection with poly(i : C) results in the upregultion of IFN-relted genes in C. gigs hemocytes. The cell-free hemolymph (CFH) ws retined nd used s culture medium for primry hemocyte cell cultures. (b) Viperin, JAK, STAT6 nd IRF re upregulted in nïve hemocytes cultured using poly(i : C)-stimulted hemolymph (Stim. CFH). NS, Not stimulted. (c) Viperin induction ws retined when hemolymph ws treted with RNse A or het treted t 85 8C, but ws eliminted when digested with proteinse K (Prot. K). Poly(I : C)-stimulted hemolymph retined ctivity when diluted (2 %, v/v). Asterisks in (, b) nd different lower-cse letters (c) denote significnt differences compred with controls (*P,.5). b nd lte endosoml comprtments (Rb5 nd -7), mitochondri (Cox IV) nd the peroxisome (pex19); however, no colocliztion ws observed (dt not shown). Interestingly, oyster viperin ws observed to co-loclize with cveolin-1 in Huh-7 cells (Fig. 5b). Cveolin-1 is mrker of cveole, specilized form of lipid rft (Prton & Simons, 27). To exmine the divergent locliztions of humn nd oyster viperin in vitro, we co-expressed humn viperin GFP in conjunction with FLAG-tgged oyster viperin in Huh-7 cells. Fig. 5(c) shows tht the two viperin molecules displyed extensive co-locliztion to puttive lipid droplets, s well s to punctte cytoplsmic loci. Humn viperin hs been shown to dimerize previously (Hinson & Cresswell, 29b), independent of its N terminus, nd, given the inbility of oyster viperin to loclize to lipid droplets in the bsence of humn viperin expression (Fig. 5), we cn presume tht oyster viperin mintins the bility to dimerize, nd is ble to do so with humn viperin, reloclizing to lipid droplets in vitro. Oyster viperin inhibits dengue virus repliction Cell lines for mrine bivlves do not exist (Yoshino et l., 213), nd the methodology to culture OsHV-1 in primry cells isolted from C. gigs hs not yet been developed. Therefore, we utilized heterologous model to investigte whether oyster viperin directs ntivirl ctivity. Humn viperin restricts the repliction of number of humn virl pthogens, including dengue virus (DENV-2; Helbig et l., 213). We compred the bility of oyster nd humn viperin to restrict DENV-2 repliction in Huh-7 cells. Cells were trnsiently trnsfected with either FLAG expression control vector, FLAG-tgged oyster viperin or FLAG-tgged humn viperin expression plsmid, nd then infected with DENV-2 t 24 h post-trnsfection. Both oyster nd humn viperin were ble to restrict DENV-2 repliction t 24 h post-infection (p.i.) by 6 nd 54 % respectively (Fig. 6, Pv.5). No significnt difference ws observed between the bility of oyster nd humn viperin to restrict DENV-2 repliction in vitro. The bility of oyster viperin to restrict DENV-2 repliction demonstrted tht the lck of n mphipthic helix in the N terminus of this protein did not inhibit its nti-denv ctivity. DISCUSSION A growing body of evidence supports the concept tht some invertebrtes my hve n ncient ntivirl pthwy tht is homologous to the vertebrte type I IFN response (reviewed by Wng et l., 215). Despite numerous studies describing C. gigs genes tht re homologous to vertebrte ISGs (Green & Montgnni, 213; He et l., 215; Renult et l., 211; Rosni et l., 214), there re outstnding questions regrding whether these invertebrte genes hve similr biologicl function nd whether C. gigs type I IFN cytokine exists. In the current study, we showed oyster viperin hs direct ntivirl ctivity (Fig. 6) nd provided evidence tht intrmusculr poly(i : C) injection induces hemolymph protein/peptide (cytokine) tht induces hemocyte expression of viperin in conjunction with upregultion of IRF nd JAK/STAT (Fig. 4b). As herpesviruses pose the biggest thret to the globl production of Pcific oysters (Renult et l., 214; Segrr et l., 21) nd other mrine molluscs (reviewed by Green et l., 215), these results re of considerble interest in progressing novel therpeutics for the quculture industry

6 T. J. Green nd others () Viperin ADRP Merge Oyster Humn (b) O. viperin Cveolin-1 Merge (c) O. viperin H. Viperin Merge Fig. 5. C. gigs viperin co-loclizes to cveolin-1 but not to lipid droplets. () Huh-7 cells were trnsfected with either FLAG-tgged oyster viperin (O. viperin) or FLAG-tgged humn viperin (H. viperin) in conjunction with MCherry ADRP. ADRP is resident lipid droplet mrker. No co-locliztion ws observed between oyster viperin nd ADRP. (b) FLAG-tgged oyster viperin hs considerble co-locliztion to cveolin-1-gfp. (c) Co-expression of FLAG-tgged oyster viperin nd GFP-tgged humn viperin in Huh-7 cells reveling tht humn viperin molecules dimerize with oyster viperin loclizing to lipid droplets. Viperin hs been chrcterized from mny different nimls within the subphylums of Vertebrt (Goossens et l., 215; Helbig et l., 213; Milic et l., 215; Wng et l., 214b; Zhong et l., 215) nd Cephlochordt (Lei et l., 215). To the best of our knowledge, this is the first study to chrcterize viperin isolted from n niml without notochord (non-chordtes). The mino cid sequence of C. gigs viperin hs high homology to humn nd teleost viperin (Fig. 1) nd hs similr domin rrngement with the conserved motif of CxxxCxxC tht exists in rdicl SAM enzymes nd conserved C-terminl domin. However, we were unble to predict n mphipthic helix in the N-terminl region. Vertebrte viperin requires the mphipthic helix for its ssocition with the ER nd its bility to loclize to lipid droplets (Hinson & Cresswell, 29, b). The mphipthetic helix of humn viperin is importnt for its direct ntivirl ctivity ginst heptitis C virus (Helbig et l., 3592 Journl of Generl Virology 96

7 Oyster viperin hs direct ntivirl ctivity Reltive fold chnge in DENV RNA Control O. viperin H. viperin Fig. 6. C. gigs viperin hs direct ntivirl ctivity ginst DENV- 2 in heterologous model. Huh-7 cells were trnsfected with either oyster viperin (O. viperin), humn viperin (H. viperin) or n empty control vector, 24 h prior to infection with DENV-2 (m.o.i.51). Cells were hrvested for RNA t 24 h p.i. nd reverse trnscriptse PCR ws performed to detect virl RNA levels in comprison with the controls. C. gigs viperin hd the sme level of ntivirl ctivity s humn viperin ginst DENV-2. Asterisks denote significnt difference compred with control (*P,.5). 25) nd chikunguny virus (Teng et l., 212) but not ginst DENV-2 (Helbig et l., 213). The bsence of predictble mphipthic helix in the N terminus of C. gigs viperin is the most likely explntion for its filure to loclize with lipid droplets in Huh-7 cells (Fig. 5). Insted, C. gigs viperin co-loclized to cveolin-1 (Fig. 5b), which is mrker for cveole. Cveole re flsk-shped indenttions of the plsm membrne enriched in cholesterol, cveolin nd signlling fctors (Prton & Simons, 27), nd re exploited by some niml viruses s direct portl for endocytic entry to host cells (Smith & Helenius, 24). The mechnism for virus entry into molluscn cells is unknown, but other mrine invertebrte viruses, such s white spot syndrome virus, rely on cveole-medited endocytosis to enter crustcen cells (Dun et l., 214; Hung et l., 213). Cveole re lso involved in ntivirl signlling by llowing signlling molecules to cluster within the cveole domin, thus fcilitting protein interctions mong signlling components nd enhncing signl trnsduction (Gbor et l., 213). Cveolin-1 serves s the scffolding protein tht recruits signlling molecules, such n IFN receptors, to cveole (Tkok et l., 2). The role of cveolin nd cveole in molluscn ntivirl signlling nd recruitment of ntivirl proteins is lso unknown, but cveolin nd viperin trnscripts re both highly expressed in C. gigs infected with OsHV-1 (He et l., 215; Rosni et l., 214). We confirmed tht C. gigs viperin hs the sme level of ntivirl ctivity s humn viperin ginst DENV-2 (Fig. 6), lthough C. gigs viperin did not loclize to the sme cellulr comprtment s vertebrte viperin. Helbig et l. (213) confirmed tht the nti-denv effect of humn viperin is medited within the C-terminl 17. The C-terminl region of humn viperin ws shown to restrict erly DENV-2 RNA production/ccumultion by intercting with DENV-2 cpsid, non-structurl protein (NS3) nd virl RNA (Helbig et l., 213). Phylogenetic nlysis reveled tht humn nd C. gigs viperin shre 88 % mino cid identity within the C-terminl region. Herpesviruses re renowned for their bility to modulte the host s immune response nd co-opt host ntivirl proteins to fcilitte the infectious process (Aresté & Blckbourn, 29; White et l., 212). Both HCMV (humn herpesvirus-5) nd humn herpesvirus-1 [herpes simplex virus type 1 (HSV-1)] hve been shown to counterct viperin s ntivirl ctivities (Seo et l., 211; Shen et l., 214). The HCMV vmia protein hs been demonstrted to interct with viperin, resulting in the relocliztion of viperin from the ER to the mitochondri, where it reduces cellulr ATP genertion, resulting in ctin cytoskeleton disruption nd enhncement of HCMV infection (Seo et l., 211). HSV-1 does not co-opt viperin; rther, the endoribonuclese ctivity of its UL41 protein hs been shown to restrict viperin mrna ccumultion nd to bolish its bility to limit HSV-1 infection (Shen et l., 214). It is presumed tht OsHV-1 cn lso modulte the immune response of C. gigs, s the virl genome encodes four inhibitors of poptosis tht re highly expressed during the erly stges of infection (Green et l., 215b; Segrr et l., 214, b). There re fewer dt vilble regrding the bility of OsHV-1 to modulte the other evolutionrily conserved ntivirl proteins, such s viperin. Interestingly, younger developmentl stges of C. gigs induce viperin to higher expression levels when infected with OsHV-1 (unpublished dt) nd these erlier developmentl stges lso hppen to be more susceptible to OsHV-1 infection (Pul-Pont et l., 214; Peeler et l., 212). Further reserch is therefore wrrnted to determine whether OsHV- 1 diverts viperin from its ntivirl role nd co-opts it to fcilitte the infection process. In vertebrtes, viperin is highly inducible gene nd its expression rpidly increses following virl infections nd tretment with poly(i : C) nd LPS (reviewed by Helbig & Berd, 214; Mttijssen & Pruijn, 212). We conducted severl in vitro experiments to determine which pthogenssocited lignds nd sensors re responsible for inducing C. gigs viperin. In contrst to vertebrtes, C. gigs viperin ws induced by poly(i : C) but not by LPS (Fig. 3). These results suggest tht virl repliction products, such s dsrna, re responsible for inducing viperin expression in C. gigs hemocytes vi TLR or retinoic cid inducible gene I-like helicse (RLH) sensor. In vivo experiments reveled viperin expression is delyed in C. gigs hemocytes following poly(i : C)-injection when compred with other nimls from the vertebrte phylum. Vertebrte cells usully express viperin within 2 h following stimulus, nd its expression typiclly peks between 4 nd6hfollowingstimultionwithpoly(i:c)(goossens et l., 215; Zhng et l., 214), wheres hemocyte expression of C. gigs viperin remined stble for the

8 T. J. Green nd others first 9 h fter poly(i : C)-injection nd did not pek until 27 h post-injection (Fig. 2). Poly(I : C)-injection in the dductor muscle lso resulted in the upregultion of C. gigs viperin in the mjority of tissue comprtments (Fig. 2b). Severl plusible explntions for the delyed but systemic expression of C. gigs viperin re tht: (i) stimulted hemocytes migrte from the site of poly(i : C) injection to other tissue comprtments; (ii) poly(i : C) diffuses from the site of injection to other tissue comprtments; or (iii) poly(i : C) induces the cells within the dductor muscle to secrete type I IFN cytokine. We therefore devised severl experiments to indirectly test whether C. gigs hs type I IFN response. Genes with cler homology to type I IFNs hve been identified in tetrpods (mphibins, reptiles, birds nd mmmls) nd fishes but not in non-vertebrte chordtes (tunictes or lncelets). This hs previously been tken to suggest tht IFNs first evolved in erly vertebrtes (Lngevin et l., 213). However, nother interprettion is tht functionlly ctive IFNs from other niml groups simply lck sufficient sequence conservtion with their vertebrte counterprts to be identified by homology serches. Comprisons of mmmlin nd teleost IFNs revel low overll mino cid similrity (v25 %) nd dissimilr gene domin rchitecture of type I IFNs even within the vertebrte phylum (Robertsen, 26). Our results showed tht C. gigs injected with poly(i : C) produced hemolymph compound tht ctivted viperin expression in primry hemocyte cell cultures in conjunction with upregultion of IRF nd JAK/STAT (Fig. 4b). Furthermore, this compound(s) is likely to be het-stble protein/peptide (cytokine) becuse its ctivity ws retined fter digestion with RNse A nd het inctivtion (Fig. 4c) but ws eliminted by proteinse K digestion. Previous reserch hs shown tht ll type I IFNs from vertebrtes re het stble (Oritni et l., 23). Conclusion In summry, our results provide further support to the concept of n ncient type I IFN response existing in the common metzon ncestor. We demonstrted tht C. gigs viperin hs direct ntivirl ctivity nd provided evidence tht viperin expression is induced by non-specific dsrna vi hemolymph protein/peptide (cytokine). Reserch is currently underwy to purify this hemolymph protein/peptide(s) tht ctivtes viperin expression. The existence of type I IFN response in the oyster cretes exciting new possibilities for future reserch into novel therpeutic tretments for virl diseses tht re thretening globl quculture production. METHODS Oyster chllenge experiments. Juvenile oysters (C. gigs) hd notch filed in their shell djcent to their dductor muscle to llow delivery of poly(i : C) (5 mg ml 21 in sewter; Sigm) ccording to previous published procedures (Green & Brnes, 29; Green et l., 214b). At h, oysters were injected with 5 ml poly(i : C) or sewter (control) nd plced in replicted quriums (slinity 35 p.p.t., 19 uc, erted). Six oysters per tretment were smpled t, 3, 9, 27 nd 54 h post-injection. Smpling consisted of excising oyster tissues, snp freezing in liquid nitrogen nd storge t 28 uc for RNA extrction. The oysters were not fed for the durtion of the experiment. Primry cell culture nd pthogen-ssocited moleculr ptterns (PAMP) stimultion. Experiments investigting the effects of PAMPs on nïve hemocytes were crried out using primry hemocyte cell cultures tht were estblished from individul oysters. Six primry cell cultures were estblished from individul C. gigs ccording to previously published procedures (Morg et l., 211; Renult et l., 211). Briefly, hemolymph ws withdrwn from the pericrdil cvity using sterile 21-guge needle nd syringe. Hemolymph from individul oysters ws kept seprte nd divided into seven replicte wells of 24-well tissue culture plte (.4 ml per well). Hemocytes were llowed to dhere to the tissue culture wells for 3 min t 22 uc before the cellulr frction of the hemolymph ws removed from ech well, filtered (.2 mm) nd retined on ice. Adhered hemocytes were wshed three times with sterile sewter before.4 ml cellulr hemolymph with 2 % (v/v) penicillin/streptomycin ws replced s the culture medium. Three concentrtions of the PAMPs poly(i : C) nd LPS (5.,.5 nd.5 mg.ml 21 ) were prepred in sewter nd 2 ml PAMP suspension (control5sewter) ws dded to ech well. Hemocytes from ech individul oyster were therefore exposed to three concentrtions of poly(i : C) nd LPS. Hemocytes were incubted for 6 h t 22 uc in humid incubtor nd then used for RNA extrction. This set of experiments ws repeted on two seprte occsions. Stimultion of nïve hemocytes with hemolymph collected from oysters injected with poly(i : C). Six dult oysters were injected with either 1 ml poly(i : C) or with sewter s bove. Hemolymph from poly(i : C)-injected nd control oysters ws collected t 27 h post-injection using 21-guge needle nd syringe, pooled nd filtered (.2 mm), nd the cellulr frction of the hemolymph ws retined on ice. Primry hemocyte cultures were estblished from individul oysters (n56) s described bove. Pooled hemolymph from poly(i : C)- nd sewter-injected dult oysters ws used s the culture medium to determine whether hemolymph component induced viperin expression (Fig. 7). Additionl tretments included digestion of the hemolymph from poly(i : C)-injected oysters with RNse A (3.3 mg ml 21,37uC, 1.5 h), proteinse K (.1 mg ml 21,37uC, 1.5 h) nd het inctivtion (85 uc for 15 min) before using s culture medium. Hemolymph from poly(i : C)-injected oysters ws lso diluted with hemolymph from control oysters (2 %, v/v) before being used s culture medium. Hemocytes were incubted for 6 h t 22 uc in humid incubtor nd then used for RNA extrction. This set of experiments ws repeted on two seprte occsions. RNA extrction nd quntittive reverse trnscription PCR (RTqPCR). Totl RNA ws purified from oyster smples using TriSure (Bioline) nd reverse trnscribed using Tetro cdna synthesis kit (Bioline). Quntittive rel-time PCR ws performed in ViiA7 thermocycler (Applied Biosystems), s described previously, using the primers in Tble 1, which included the internl reference gene eef1 (Green et l., 214b). Oyster viperin coding sequence nd synthesis. The complete coding sequence of oyster viperin ws mplified from cdna smples of oysters injected with poly(i : C) (n54, 27 h p.i.), using the primers 59-ACATGGCTATTACGCSGTAC-39 nd 59-CCAGGATTACAAATC- GAC-39. PCR mplicons were DNA sequenced t the Austrlin Genome Reserch Fcility. The N-terminl FLAG-tgged oyster viperin ws generted in two steps. The consensus nucleotide sequence of oyster viperin ws directly synthesized (GenScript USA) 3594 Journl of Generl Virology 96

9 Oyster viperin hs direct ntivirl ctivity (i) Oysters stimulted with dsrna [poly(i : C)] or sewter (control). (n = 6 oysters per tretment) for 24 h in erted quri. (ii) Hemolymph collected, pooled nd.2 µm filtered. Cell-free hemolymph used s culture medium. (iii) Hemocytes collected from unstimulted oysters (n = 6 oysters). (iv) Hemocytes from individul oysters split into individul wells nd exposed to both poly(i : C)-stimulted nd control hemolymph. (v) Hemocytes cultured t 22 C for 6 h. RNA purified using TriSure. Poly(I : C), positive control; sewter, negtive control. Fig. 7. Methodology used to evlute the effect of hemolymph from oysters treted with poly(i : C) or sewter (control) on viperin expression in hemocytes from nïve oysters. The cellulr frction of the hemolymph ws collected from C. gigs stimulted with poly(i : C) or sewter (control). The cellulr frction of the hemolymph ws then used s culture medium to determine whether hemolymph component induced hemocyte expression of viperin. into the puc57 vector. puc57-viperin ws PCR mplified using the primers 59-TTATGCTAGCATGGACTACAAGGATGACGACGATA- AGATGGCTATTACGCAGTACGTCAGC-39 nd 59-TTATCTCGAG- TTACCAATCGAGCTTCATATCGGC-39. The resulting PCR mplicon ws double digested with Nhe I nd Xho I nd subcloned into the pci-neo expression vector (Promeg). Vector sequences were verified by DNA sequencing. Immunostining nd co-locliztion studies. The humn heptocellulr crcinom cell linehuh-7 were cultured on geltin-coted glss coverslips nd trnsiently trnsfected with plsmids expressing humn or oyster viperin tht were FLAG tgged t their N terminus in the vector pci-neo. Co-trnsfection ws performed with vectors expressing either MCherry ADRP or cveolin-1 GFP. Cells were fixed in 4 % prformldehyde t 24 h post-trnsfection (Eyre et l., Tble 1. Primer pirs used in RT-qPCR expression nlysis The GenBnk ccession number is provided for ech gene. STING, Stimultor of IFN genes; viperin, virus inhibitory protein, ER-ssocited, IFN inducible. Gene nme Accession no. Sense primer (59R39) Antisense primer (59R39) EFU ABI2266 GAGCGTGAACGTGGTATCAC ACAGCACAGTCAGCCTGTGA TLR GCAGGACTCCACTTTCTCAC GTTGGCACCCAGGTAAAGG RLH EKC3834 CAACAACATGGGAAGTATGGTG TCGGTCTGTTAACTGCGGAC STING EKC29965 CTGCTATTGTCCGCCATC GAATGGGCGTGGCATACTC IRF EKC43155 CGAAACGCAGAAACTGTTC ATTTGCCTTCCATCTTTTGG SOC EKC24772 CAAGAGAGAATCTGTGGGAAC GCATCTTAGCACTAATTCTCTC JAK EKC41693 AGAACACCTACCTTCCTGTG TGAGCCACGTCACTTATCATC STAT5A EKC3789 AGCTCAGAGTCCTCTGTG ACACTGTTAGTCTGGATACTC STAT6 EKC39332 AGCAGCAGACAGGCAACAC ACTGGGCTCATTTGCTGGTC PKR EKC3487 GAGCATCAGCAAAGTGTTGAG GTAGCACCAGGAGATGGTTC Viperin EKC2825 GCTTTGACCCGGAAACCAAC TGACACCAATCCCGAACTCG

10 T. J. Green nd others 27, 214), wshed in PBS nd permebilized in 1 % NP-4, before blocking in 5 % (w/v) BSA in PBS. FLAG-tgged viperin ws visulized using n nti-flag mouse ntibody (Sigm) nd either got nti-mouse IgG Alex Fluor 488 or got nti-mouse IgG Alex Fluor 555 (Moleculr Probes) nd counterstined with DAPI. Florescence ws visulized using Nikon TiE inverted microscope where seril Z-sections were cquired nd deconvoluted using the 3D AutoQunt Blind Deconvolution plug-in of NIS Elements AR v Imges re single representtive Z-sections. DENV ssy. Huh-7 cells were seeded in 12-well dish nd infected 24 h fter seeding t n m.o.i. of 1 for 9 min t 37 uc with DENV-2 in volume of 3 ml per well s described previously (Milic et l., 215). Cells were then wshed with PBS three times before being reincubted in culture medium. At 24 h p.i., the cells were hrvested for RNA purifiction s described bove, nd rel-time PCR ws performed utilizing the DENV-2-specific primers 59-ATCCTCCTA- TGGTACGCACAAA-39 nd 59-CTCCAGTATTATTGAAGCTGCT- ATCC-39 in combintion with primers for the internl RPLPO (lrge ribosoml protein) reference gene: 59-AGATGCAGCAGATCC- GCAT-39 nd 59-GGATGGCCTTGCGCA-39. ACKNOWLEDGEMENTS The uthors cknowledge the funding provided by Mcqurie University postdoctorl reserch scheme (MQ grnt ), Austrlin Sefood Coopertive Reserch Centre (CRC project no. 211/ 758) nd the Austrlin Ntionl Helth nd Medicl Reserch Council (NHMRC progrmme grnt APP15326). The uthors lso cknowledge Gry Zippel, Kevin McAsh nd Ewn McAsh for providing oysters for reserch. REFERENCES Aresté, C. & Blckbourn, D. J. (29). Modultion of the immune system by Kposi s srcom-ssocited herpesvirus. Trends Microbiol 17, Biron, C. A. & Sen, G. C. (21). Interferons nd other cytokines. In Fields Virology, 4th edn, pp Edited by D. M. Knipe, P. M. 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