Virology 484 (2015) 241–250

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Differential utilisation of ceramide during replication of the flaviviruses West Nile and dengue virus Turgut E. Aktepe a, Helen Pham b,1, Jason M. Mackenzie a,n a b

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia Department of Microbiology, La Trobe University, Melbourne, VIC, Australia

art ic l e i nf o

a b s t r a c t

Article history: Received 18 March 2015 Returned to author for revisions 5 April 2015 Accepted 12 June 2015

It is well established that þ ssRNA viruses manipulate cellular lipid homoeostasis and distribution to facilitate efficient replication. Here, we show that the cellular lipid ceramide is redistributed to the West Nile virus strain Kunjin virus (WNVKUN) replication complex (RC) but not to the dengue virus serotype 2 strain New Guinea C (DENVNGC) RC. We show that prolonged chemical inhibition of serine palmitoyltransferase with myriocin had a significant deleterious effect on WNVKUN replication but enhanced DENVNGC replication. Additionally, inhibition of ceramide synthase with Fumonisin B1 had a detrimental effect on WNVKUN replication and release of infectious virus particles but contrastingly an enhancing effect on DENVNGC replication and virus production. These observations suggest that ceramide production via the de novo and salvage pathway is a requirement for WNVKUN replication but inhibitory for DENVNGC replication. Thus, although these two viruses are from the same genus, they have a differential ceramide requirement for replication. & 2015 Elsevier Inc. All rights reserved.

Keywords: West Nile virus Dengue virus Virus replication Ceramide

Introduction The role of ceramide in the regulation of cell survival and apoptosis has been well recognised in cell biology (Arboleda et al., 2009), however how it aids in efficient flavivirus replication has received little attention until recently (Martin-Acebes et al., 2014; Perera et al., 2012). Like many viruses, members of the Flaviviridae are observed to alter cellular lipid levels during infection (Heaton et al., 2010; Heaton and Randall, 2010; Khan et al., 2014; Mackenzie et al., 2007; Martin-Acebes et al., 2011, 2014; Perera et al., 2012; Soto-Acosta et al., 2013; Tani et al., 2010; Yamane et al., 2014). The flavivirus genus contains more than 70 highly pathogenic viruses such as West Nile virus (WNV), dengue virus (DENV), Japanese encephalitis virus (JEV) and yellow fever virus (YFV). Transmitted to humans through a mosquito vector, hundreds of thousands of deaths are associated with flavivirus infections each year, mainly due to the lack of specific therapeutic and vaccine strategies. To successfully replicate its positive-sense RNA genome, flaviviruses utilise many host factors contributing to transcription and translation, but also have an intimate association with cellular membranes. At the end of the latent period, flavivirus replication

n

Corresponding author. Tel.: +61 3 9035 8376 Fax: þ61 3 8347 1540. E-mail address: [email protected] (J.M. Mackenzie). 1 Present address: Dorevitch Pathology, Melbourne, VIC, Australia.

http://dx.doi.org/10.1016/j.virol.2015.06.015 0042-6822/& 2015 Elsevier Inc. All rights reserved.

is observed in highly curved, complex membrane structures called convoluted membrane (CM), paracrystalline arrays (PC) and vesicle packets (VP) (Mackenzie et al., 1996; Westaway et al., 1997), which are derived from the endoplasmic reticulum (ER) and also contain markers of the Golgi apparatus (Gillespie et al., 2010; Junjhon et al., 2014; Mackenzie et al., 1999; Miorin et al., 2013; Welsch et al., 2009). These virally induced structures act as platforms for efficient viral replication; however the exact mechanism for the formation of these structures is not entirely understood (Gillespie et al., 2010; Mackenzie et al., 1999; Westaway et al., 1997). Viral replication is a complex process that requires many host factors including the lipid metabolism and lipid redistribution (Stapleford and Miller, 2010). The lipid composition of cellular membranes determines their degree of membrane fluidity, plasticity and topology in facilitating membrane curvature and signalling, or to recruit viral and cellular factors to the replication complexes (RC) (Martín-Acebes et al., 2011). Recent studies have shown changes (either increase or decrease) in specific lipids, including sphingolipids, during WNV, DENV and Hepatitis C virus (HCV) infection, (Heaton and Randall, 2010; Khan et al., 2014; Martin-Acebes et al., 2014; Perera et al., 2012). Sphingolipids are composed of a long chain sphingoid base, amide linked fatty acids and a polar (carbohydrate) head group at the 1-position, except for sphingomyelin (SM), which has a phosphorylcholine head group (Hannun and Bell, 1989). Removal of phosphorylcholine from SM by the hydrolytic activity of acid

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sphingomyelinase (ASMase) results in the production of ceramide (Utermöhlen et al., 2008). Ceramide is also generated via the de novo pathway by serine-palmitoyl-coenzyme A transferase (Cutler et al., 2004). The presence of the cone-shaped ceramide on one leaflet of the lipid bilayer enhances membrane bending (negative curvature) and the tendency to form hexagonal phases (Goñi and Alonso, 2002; Krönke, 1999; Utermöhlen et al., 2008), that are reminiscent of the WNVKUN-induced membrane architecture (Westaway et al., 1997). Although many studies have reported an overall increase of ceramide during WNVKUN and DENV infection (Martin-Acebes et al., 2014; Perera et al., 2012), the effects of altering endogenous ceramide levels on viral replication have not yet been analysed. In this study we have interrogated the importance of ceramide during WNV strain Kunjin virus (WNVKUN) and DENV serotype

2 New Guinea C (DENVNGC) replication. We have revealed that although WNVKUN and DENVNGC belong to the same genus, they both require different lipid mechanisms to efficiently replicate their genomes. We have shown that depleting endogenous levels of ceramide severely attenuates WNVKUN replication, whereas the same treatment has an enhancing effect on DENVNGC replication.

Results Ceramide redistributes to WNVKUN replication sites but not to DENVNGC replication sites Ceramide is a cone-shaped lipid with the ability to shape membranes by inducing a negative curvature (Utermohlen et al.,

Fig. 1. Intracellular ceramide is redistributed to the sites of WNVKUN replication but not substantially to DENVNGC replication sites. Mock-infected Vero cells were fixed for IFA and immunolabelled with antibodies raised the sphingolipid ceramide (panels a and e), and to the ER (anti-calnexin, panel b and d) or Golgi Apparatus (anti-Giantin, panels f and h). Additionally Vero cells were infected with WNVKUN (panels m–p) or DENVNGC (panels u–x) for 24 and 48 h, respectively, before fixation for IFA. The cells were subsequently labelled with antibodies to Ceramide (green, panels m, p, u and x), WNVKUN NS4A (red, panels n and p), DENVNGC NS2B-3 (red, panels v and x) and viewed on a Zeiss confocal microscope. Panels were merged for visualisation in Adobes Photoshops CS6 and co-localisation was determined by the JaCOP plugin software in ImageJ.

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2008). The transport of ceramide from the ER to the Golgi apparatus is an important step for the conversion of ceramide to SM and other complex glycosphingolipids (Riboni et al., 2010). Recently, it was observed that sphingolipids play a crucial role in the biogenesis and composition of infectious WNV particles (Martin-Acebes et al., 2014) and thus we aimed to identify the cellular localisation of ceramide in WNVKUN- and DENVNGCinfected cells and the contribution of ceramide to intracellular virus replication. To initially determine the subcellular distribution of ceramide, mock-infected Vero cells were fixed and immunelabelled with antibodies raised against the cellular proteins Calnexin (ER resident protein) and Giantin (Golgi apparatus protein) as cellular markers, and with anti-ceramide antibodies (Fig. 1a–h). IF analysis revealed low levels of ceramide colocalisation with the ER stained with anti-Calnexin antibodies (Fig. 1a–d, Rr¼0.400), however we observed abundant levels of ceramide within the Golgi apparatus, co-localising with Giantin (Fig. 1e–h, Rr¼ 0.712). This result confirmed the observations that the majority of intracellular ceramide resides within the Golgi apparatus where it acts as the central metabolic hub of sphingolipid catabolism (Hannun and Obeid, 2008). To investigate the distribution of ceramide during flavivirus replication, Vero cells were infected with WNVKUN or DENVNGC and the intracellular distribution of ceramide was investigated by IF analysis. As shown in Fig. 1m–p, infection with WNVKUN led to the redistribution of ceramide to sites of viral replication as co-localisation was observed with ceramide and the viral protein, NS4A (Fig. 1m–p, Rr¼ 0.788). In contrast, little obvious change in ceramide distribution was observed in DENVNGC-infected cells (Fig. 1u–x). Intracellular ceramide did not co-localise with the DENVNGC viral protein NS2B-3 (Fig. 1u–x, Rr¼0.426), but instead was observed to have a similar distribution to mock cells (Fig. 1e–h). Thus it appeared that there was a differential recruitment, and potential requirement, of ceramide during the replication cycle of

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WNVKUN and DENVNGC implying that these two flaviviruses might utilise ceramide differently for efficient intracellular replication.

Acute inhibition of serine pamityltransferase activity did not affect flavivirus replication but enhanced DENVNGC virus production. To determine if the observed redistribution of ceramide was essential for WNVKUN and/or DENVNGC replication, we utilised the chemical myriocin to potently inhibit the enzyme serine pamitylransferase (SPT) activity, an enzyme that catalysis the first committed step of sphingolipid biosynthesis, thus reducing intracellular sphingolipid intermediates (Miyake et al., 1995). To determine the effects of acutely depleted ceramide levels during flavivirus replication, Vero cells were pre-treated with 6.2 mM, 12.5 mM or 25 mM myriocin and subsequently infected with either WNVKUN or DENVNGC for a further 24 and 48 h, respectively, in the presence of myriocin. Effects of myriocin were determined via western blotting for the WNVKUN viral protein NS5 and the DENVNGC viral protein NS1 and via the measurement of the amount of released infectious virus particles (Fig. 2). As it can be observed the acute depletion of cellular ceramide, via myriocin, did not have a significant effect on WNVKUN nor DENVNGC protein production independent of the concentrations used (Fig. 2A and B, respectively). Interestingly though we did observe a dose-dependent increase in DENVNGC protein production (Fig. 2C; n¼3). However, DENVNGC-infected cells treated with 6.2 mM myriocin did show a significant increase in viral particles secreted compared to MeOH- and WNVKUN-infected cells, however these amounts declined at the higher myriocin concentrations (Fig. 2D). Intriguingly, these results have shown that the production of infectious WNVKUN particles is unaltered when de novo ceramide was inhibited, however a slight (although not significant) increase in DENVNGC virus release was observed at higher myriocin concentrations (Fig. 2D).

Fig. 2. Acute myriocin treatment differentially affects flavivirus replication and virion production. Vero cells were infected with WNVKUN or DENVNGC and protein lysates and cell culture supernatants were collected at 24 and 48 h.p.i., respectively (A–C) protein lysates were analysed by western blot using antibodies raised against WNVKUN protein NS5 and DENVNGC protein NS1, and the internal control actin and PDI (panel A and B). Ratios shown were determined by densitometry analyses compared to actin (for WNVKUN) or PDI (for DENVNGC) after analysis from replicate experiments (n¼ 3; þ/–SEM). (D) Cell culture supernatants were analysed by plaque assay (WNVKUN) and foci forming unit assay (DENVNGC) for infectious virion secretion. Error bars ( þ/  SEM) indicate duplicate analysis of triplicate experiments, and asterisks (***) indicate a significant change compared to MeOH treatment as determined by paired t-test (Graph Prism 6).

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These results demonstrate that acute depletion of ceramide via myriocin do not attenuate flavivirus replication significantly but did appear to promote DENVNGC release at lower concentrations. Prolonged myriocin treatment attenuates WNVKUN replication, however increases DENVNGC replication Ceramide is the central metabolic hub for a number of sphingolipids, and as such can be recycled from sphingosine and ceramide-1-phosphate (Mao and Obeid, 2008; Shinghal et al., 1993). Due to the ability of ceramide to be recycled, a prolonged treatment of myriocin was performed to deplete endogenous ceramide levels (Meszaros et al., 2013). IF analysis was performed on Vero cells treated with 12.5 mM myriocin for 7 days and infected with either WNVKUN or DENVNGC for 24 or 48 h, respectively (Fig. 3). Prolonged myriocin treatment

clearly decreased endogenous ceramide levels as we observed only trace levels of ceramide in myriocin-treated cells compared to MeOH-treated cells (compare Fig. 3a–d with e–h). Interestingly we observed a slight increase in ceramide in cells treated 7d with myriocin and infected with WNVKUN (Fig. 3m–p). Consistent with our previous observation the detected ceramide co-localised with WNVKUN NS4A protein (Fig. 3m–p, Rr¼ 0.912). Interestingly, in DENVNGC-infected cells treated with myriocin for 7 days, we observed a slight increase in detectable ceramide levels (Fig. 3u–x) compared to myriocin-treated uninfected cells (Fig. 3e–h). In addition, the detected ceramide labelling had a similar distribution to untreated cells and in contrast to WNVKUN-infected cells, DENVNGC NS4A did not co-localise with ceramide under any of the treatment conditions (Fig. 3q–x, Rr¼0.217). It should be noted that increased ceramide biosynthesis has been observed in both WNV strain New York 1999 (WNVNY99) and DENV strain 16681

Fig. 3. Prolonged treatment (7d) with myriocin depletes intracellular ceramide levels. Mock- (panels a–h), WNVKUN- (panels i–p) and DENVNGC-infected (panels q–x) Vero cells were fixed for IFA and immunolabelled with anti-Ceramide (panels a, e, i, m, q, and u), and anti-WNVKUN NS4A (panels b, f, j, n, r, and v) antibodies. Cell nuclei were counterstained with DAPI and viewed on a Zeiss confocal microscope under similar conditions. Panels were merged for visualisation in Adobes Photoshops CS6 and colocalisation was determined by the JaCOP plugin software in ImageJ.

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(DENV16681)-infected cells (Martin-Acebes et al., 2014; Perera et al., 2012), perhaps accounting for these observations. In summary, our observations suggest that the increased ceramide is not due to the de novo pathway. To determine the effect of chronic and prolonged ceramide depletion on flavivirus replication, Vero cells were treated with 12.5 mM of myriocin for 7 days and subsequently infected with WNVKUN or DENVNGC as previously described. Viral RNA, lysates and intracellular and extracellular viral particles were harvested to evaluate WNVKUN and DENVNGC viral replication and viral titres (Fig. 4). Compared to MeOH treated Vero cells, ceramide depletion by prolonged myriocin treatment did not show a transcriptional difference in viral (þ) and ( ) sense RNA for both WNVKUN and DENVNGC replication. However we did observe a slight increase in viral ( ) sense RNA in WNVKUN-infected cells and a slight increase in viral (þ) sense RNA DENVNGC-infected cells (Fig. 4A). These increases resulted in a change in the ratio of (þ) vs (  ) sense RNA, showing a reduced ratio in WNVKUN-infected cells but an increased ratio in DENVNGC-infected cells Prolonged treatment of Vero cells with myriocin had a significant effect on WNVKUN replication where a 30 78% decrease in viral protein production was observed (Fig. 4C and E). Accordingly, we also observed a significant decrease in WNVKUN intracellular and extracellular infectious viral particles compared to the vehicle treatment (Fig. 4F). Although we did observe a decrease in total infectious viral particles, ceramide depletion did not attenuate viral secretion, where the ratio of intracellular vs extracellular virus remained constant compared to MeOH treatment (Fig. 4G). Interestingly, the 7 day treatment with myriocin led to a 157 1.5% increase in DENVNGC protein production, contrary to what we observed with WNVKUN (Fig. 4D and E) and a significant increase in extracellular infectious viral particles (Fig. 4F). However, similar to WNVKUN, the ratio of extracellular to intracellular virus remained same between myriocin and MeOH treated cells (Fig. 4G). Thus, although WNVKUN and DENVNGC are from the same genus of viruses, our results suggest that decreasing endogenous ceramide levels have differing effects on WNVKUN and DENVNGC replication.

Inhibition of the de novo and salvage ceramide pathways with Fumonisin B1 differentially effects WNVKUN and DENVNGC replication. To determine the contribution of both the de novo and salvage ceramide pathways during flavivirus replication we utilised the chemical inhibitor Fumonisin B1 (FB1). FB1 is a mycotoxin that inhibits ceramide synthesis via inhibition of the cellular enzyme ceramide synthase. FB1 is structurally similar to the long-chain (sphingoid) base backbones of sphingolipids, inhibiting ceramide synthase and sphingosine–sphinganin-transferase activity (Wang et al., 1991). To assess the contribution of de novo and salvage pathways on flavivirus replication, Vero cells were pre-treated with either 30 nM or 40 nM FB1 and infected with WNVKUN or DENVNGC as described previously (Fig. 5). Similar to our observations with prolonged myriocin treatments (Fig. 4), FB1 treatment resulted in a signification reduction in WNVKUN viral protein levels (34.7 78.6% at 30 nM and 11.779.2% at 40 nM) and release of infectious viral particles (66.3 718.8% at 30 nM and 64.9 718.0% at 40 nM; Fig. 5A, C and D). Whereas, depletion of endogenous ceramide levels with FB1 resulted in a significant increase in DENVNGC protein production at 30 nM (124.9 72.6%), and a significant increase of secreted infectious virus at 30 nM (143.6 714.3%) and 40 nM (137.6736.9%) FB1, respectively. These results with FB1 treatment once again reiterate our observations that ceramide appears vital for WNVKUN replication and secretion, however there is a reverse correlation observed for DENVNGC

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infection, where depleted levels of ceramide increase DENVNGC replication.

Discussion Ceramide is a lipid consisting of a fatty acyl group of varying length bound to the amino group of a sphingoid base, usually sphingosine. Ceramide itself also is the major hydrophobic constituent backbone of all complex sphingolipids. The biosynthesis of ceramide involves a multitude of pathways including both de novo and salvage pathways of production (Castro et al., 2014). Ceramide is involved with numerous functions within membranes, such as membrane rigidity, membrane permeability, lipid–protein interaction, cell signalling and both within cell death and survival pathways (Castro et al., 2014). Interestingly, a recent study showed that the WNVNY99 envelope is enriched in sphingolipids and inhibition of sphingomyelinase reduced the release of infectious virions (Martin-Acebes et al., 2014). Accordingly, a whole cell lipidomic analysis of DENV16681-infected mosquito cells showed a significant increase in ceramide levels (Perera et al., 2012). In addition to WNV and DENV, down regulation of sphingolipid and glycerosphingolipid biosynthesis was observed to suppress HCV RNA replication and production (Heaton et al., 2010; Hirata et al., 2012), although interestingly ceramide enrichment of the plasma membrane also strongly inhibited HCV entry (Voisset et al., 2008). The above studies highlight that although these viruses belong to the same family, each virus appears to utilise ceramide at different stages for replication. In this study we aimed to determine the contribution of ceramide for the replication of WNVKUN and DENVNGC. Interestingly we have shown that the down regulation of ceramide has contrary effects on the replication of these two flaviviruses. We observed that during WNVKUN replication, ceramide co-localises with the viral RC (most likely redistributed from the Golgi apparatus, as markers of the Golgi apparatus are also present in RCs (Mackenzie et al., 1999)), however this co-localisation was not observed during DENVNGC replication (Fig. 1). The co-localisation of ceramide within the WNVKUN RC could be essential for replication to aid in virally induced membrane formation as ceramide has the capacity to alter membrane curvature, or to mediate membrane fluidity for internal membrane transport or to control cellular apoptosis pathways, as ceramide and its by-products play a central role in cell survival. Although ceramide is redistributed to the sites of WNVKUN replication, acute chemical inhibition of SPT, a central enzyme in the ceramide de novo synthesis pathway, by myriocin did not affect WNVKUN or DENVNGC replication, observations that support recent studies with DENVNGC (Carocci et al., 2015; Fraser et al., 2014). Although increases in DENVNGC viral protein production and a significant increase in secreted DENVNGC particles at 6.25 μM myriocin were observed (Fig. 2). However, we did observe a significant effect of myriocin during a prolonged treatment (i.e. 7 days) affecting WNVKUN and DENVNGC differently (Fig. 4). Under these treatment conditions there was a significant decrease in WNVKUN protein and infectious virus production yet a significant increase in DENVNGC protein and virus production (Fig. 4). In addition, we observed a decrease in the ratio of (þ) vs (  ) viral RNA during prolonged myriocin treatment of WNVKUN-infected cells, implying less ( þ) RNA released for translation and packaging; a hypothesis consistent with the decreased protein and virus produced under these conditions. Contrastingly, we observed an increase in the ration between (þ) vs ( ) viral RNA in myriocintreated DENVNGC-infected cells; again consistent with increases in DENVNGC protein and virus. These observations support the role of sphingolipids during WNV replication and biogenesis of infectious

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Fig. 4. Prolonged treatment (7d) with myriocin accentuates the differential effect of ceramide depletion on WNVKUN and DENVNGC replication. Vero cells were infected with WNVKUN or DENVNGC and viral RNA, protein lysates and intracellular and extracellular virus were collected at 24 and 48 h.p.i., respectively. (A and B) RNA samples from infected MeOH- or myriocon-treated cells were analysed for WNVKUN and DENVNGC genome levels using qPCR. Ratio between ( þ ) and (  ) sense viral RNA was determined and plotted in (B). Error bars indicate þ / S.E.M. of replicate analysis of triplicate experiments. (C) Protein lysates were analysed by western blot using antibodies raised against WNVKUN protein NS5 and DENVNGC protein NS1, and the internal control PDI and actin. (D) Ratios shown were determined by densitometry analyses compared to PDI, after analysis from replicate experiments (n¼ 3 for DENVNGC and n¼ 6 for WNVKUN; þ /  S.E.M.). (D) Cell culture supernatants were analysed by plaque assay for intracellular (n¼ 3) and extracellular (n ¼6) virus (error bars ¼ þ/  S.E.M.) indicate duplicate analysis of experiments. (E) The ratio between intracellular vs extracellular virus was determined for WNVKUN- and DENVNGC-infected cells treated with either MeOH or myriocin. In all cases the significant change compared to MeOH treatment is indicated and was determined by paired t-test (Graph Prism 6).

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Fig. 5. Treatment of cells with Fumonisin B1 has a differential on WNVKUN and DENVNGC replication. Vero cells were infected with WNVKUN or DENVNGC and protein lysates and cell culture supernatants were collected at 24 and 48 h.p.i., respectively. (A and B) Protein lysates were analysed by western blot using antibodies raised against WNVKUN protein NS5 and DENVNGC protein NS1, and the internal controls GAPDH (for WNVKUN) or PDI (for DENVNGC). (C) Ratios shown were determined by densitometry analyses compared to PDI after analysis from replicate experiments (n ¼3; þ /–SEM). (D) Cell culture supernatants were analysed by plaque assay for infectious virion secretion and error bars ( þ /  SEM) indicate duplicate analysis of triplicate experiments. In all cases the significant change compared to MeOH treatment is indicated and was determined by paired t-test (Graph Prism 6).

virus particles (Martin-Acebes et al., 2014), but are functionally in contrast to the observations of Perera et al (2012) who showed an increase in ceramide levels during DENV16681 infection of mosquito cells. One explanation could be the use of different cells during our study (a possibility we are currently exploring) or alternatively the up-regulation of ceramide may not be a strict requirement for DENV replication; a concept our current data supports. To extend the observations with myriocin, we utilised the suppressive affect of FB1 on multiple ceramide synthesis pathways, including the de novo and salvage pathways. FB1 treatment also attenuated WNVKUN replication and secretion (Fig. 5), indicating that WNVKUN relies on a certain threshold level of ceramide for efficient replication and virus production. Conversely, the FB1induced decrease of ceramide also enhanced DENVNGC replication, promoting an increase in DENVNGC viral particles and replicative proteins (Fig. 5). Thus, these results confirmed and validated the inherent role for ceramide in the replication of WNVKUN but the somewhat inhibitory effect of ceramide during DENVNGC replication. Again a possible explanation for this observation could be that WNV requires ceramide as a molecule whereas DENV may not require ceramide but its downstream products. As ceramide is the central hub for sphingolipid synthesis, DENV may utilise ceramide to mediate downstream lipid biosynthesis. Our chemical-induced inhibition of ceramide may in fact promote an upregulation of additional sphingolipids aiding in DENV replication. We have proposed a model of ceramide biosynthesis modulation in Fig. 6 and our on-going studies aim to determine the products of ceramide metabolism critical to DENV replication.

Fig. 6. Proposed model for the utilisation and modulation of ceramide biosynthesis by the flaviviruses WNV and DENV. Our study and the work of others (indicated) suggest that DENV and WNV utilise ceramide differently. WNV incorporates ceramide into the virus envelope and is dependent on ceramide for efficient replication (indicated by green solid arrows). However the requirement of ceramide for DENV is less crucial with a possible inhibitory influence (indicated by red dashed arrows) that is potentially modulated via inhibition of sphingosine kinase 1 activity.

Although we have not clearly determined the exact role for ceramide during flavivirus replication, our study extends and supports previous studies identifying the important role of lipids during flavivirus replication (Heaton and Randall, 2010; Mackenzie et al., 2007; Martin-Acebes et al., 2014; Perera et al., 2012; Soto-Acosta et al., 2013; Tani et al., 2010). One of the most obvious roles would be to organise functional virus replication factories by

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influencing the membrane curvature required for biogenesis and RC formation. Our results have indicated that ceramide effects the ratio between (þ ) and (  ) viral RNA species suggesting that ceramide may influence the release of (þ ) RNA from the RC, although how this may be facilitated is currently unknown. Ceramide invokes a negative curvature on cellular membranes and could contribute to the induction of the Flaviviridae RC, additional to other lipids such as cholesterol (Konan and Sanchez-Felipe, 2014; Mackenzie et al., 2007; Paul et al., 2013). Additionally, ceramide can induce membrane rigidity and stability and has the propensity to form ceramide-rich domains, akin to cholesterol-rich domains (Castro et al., 2014). In fact, many studies have shown that cholesterol and ceramide are the major constituents that govern membrane dynamics, and membrane fluidity is dependent on the interactions between ceramide and cholesterol and conditions where one lipid is displaced by the other (Castro et al., 2014). The membrane composition is obviously important for WNV, as sphingolipids are enriched in the virus envelope (Martin-Acebes et al., 2014) implying that assembly at the ER membrane may occur at ceramide-rich domains described above, and we have previously shown a crucial requirement for cholesterol during WNVKUN replication (Mackenzie et al., 2007). The other important function for ceramide is its role in cell survival and stress responses (Garcia-Ruiz et al., 2015). High levels of accumulated ceramide can induce apoptosis and the production of sphingosine-1-phosphate via conversion of ceramide to sphingosine; which is in fact a rheostat for cell viability. In addition, ceramide can induce the ER stress response and we have shown that WNVKUN infection also triggers this response, although the virus can modulate the downstream effector pathways (Ambrose and Mackenzie, 2011, 2013). Previous studies have also revealed that DENV replication reduces the activity of sphingosine kinase-1 and elevates ceramide levels (Carr et al., 2013; Perera et al., 2012). It is interesting to consider why the flaviviruses would promote ceramide levels ultimately leading to apoptosis, but this end effect may simply be a by-product of the requirements during replication. More research is required to dissect these requirements during flavivirus replication. In conclusion, our data together with previous studies suggest that flaviviruses utilise sphingolipids in different aspects of replication. Although it may be possible to clinically target sphingolipids in order to treat certain flavivirus infections, this may have confounding effect on infections with other flavivirus group members. Targeting of ceramide may therefore be a specific treatment approach towards certain flaviviral infections rather than a broad spectrum treatment against all flaviviruses. In light of this further research is also required to determine why viruses of the same genus differentially utilise the same lipid for replication.

Materials and methods Cells and virus stock Vero C1008 (Vero) cells were maintained in Dulbecco's modified Eagle medium (DMEM: Life Technologies) supplemented with foetal calf serum (FCS; Thermofisher), penicillin/streptomycin (50 U/mL and 50 mg/mL, respectively; GIBCO), 200 mM GlutaMAX (Glx; GIBCO). Cells were grown at 37 1C with 5% CO2. WNVKUN stocks were propagated from a WNVKUN MRM61C secondary stock in Vero C1008 cells at 37 1CC in DMEM supplemented with 0.2% (w/vol) bovine serum albumin (BSA; Sigma Aldrich) for 48 h. DENV2NGC was kindly provided by David Jans (Monash University) and propagated in Aedes albopictus C6/36 cells at 28 1C in DMEM for 5 days. Virus containing supernatant

was collected and centrifuged at 3400 rpm for 10 min to remove cell debris; aliquots were stored at 80 1C.

Drug treatment and infection Myriocin treatment: Vero cells were seeded in 12- and 24-well plates with DMEM/2%FCS. Cells were either mock treated with the solvent vehicle methanol (MeOH) or treated with 6.2, 12.5 or 25 mM myriocin (Sigma Aldrich) in DMEM/2%FCS for 24 h for DENVNGC or 48 h for WNVKUN infections. Mock- and infectedVero cells were also mock treated with MeOH or treated with 12.5 mM myriocin for 6 or 7 days in DMEM/2%FCS for DENVNGC or WNVKUN infections, respectively. Fumonisin B1 treatment: Vero cells were mock treated with the vehicle solvent Dimethyl sulfoxide (DMSO), or 30 nM or 40 nM Fumonisin B1 (FB1; Sigma Aldrich) for 24 or 48 h for DENVNGC or WNVKUN infection, respectively. The treated cells were infected as previously described. All drugs were used at sub-cytotoxic concentrations as determined by the CytoTox 96s Non-Radioactive Cytotoxicity Assay (Promega).

Antibodies Mouse anti-flavivirus-NS1 (clone 4G4) and mouse anti-WNVNS5 (clone 5H1.1) monoclonal antibodies (Hall et al., 2009; Macdonald et al., 2005) were generously provided by Roy Hall (University of Queensland). Rabbit anti-WNV-NS4A polyclonal antisera (Mackenzie et al., 1998) was generously provided by Alexander Khromykh (University of Queensland) and is able to recognise both WNV and DENV NS4A protein. Goat anti-NS2B-3 antibodies were generously provided by Paul Young (University of Queensland). Mouse anti-ceramide was purchased from Enzo Life Sciences. Mouse anti-actin and rabbit anti-giantin were purchased from Abcam. Rabbit anti-PDI and rabbit anti-calnexin were purchased from Calbiochem, Rabbit anti-GAPDH was from Cell Signalling Technology. Alexa Fluor 488-, 594- and 647-conjugated antirabbit and anti-mouse specific IgG were purchased from Molecular Probes (Invitrogen, Leiden, The Netherlands).

Immunofluorescence analysis (IFA) Vero cell monolayers seeded on coverslips were infected with WNVKUN or DENVNGC and incubated at 37 1C for 20 h or 42 h, respectively. Cells were washed twice with PBS and fixed with 4% paraformaldehyde (PFA; Sigma Aldrich, St. Louis, Mo.) in PBS for 15 min. PFA was removed, washed 2 times with PBS and treated with 0.2 M glycine for 10 min, subsequently washed with PBS. Cells were permeabilized with 0.1% Saponin (Sigma Aldrich) with 1% in fatty-acid free BSA (FAF-BSA; Sigma Aldrich) in PBS for 1 h. Primary antibodies were incubated with blocking buffer (PBS containing 1% FAF-BSA and 0.1% Saponin) for 1 h at RT, washed 4 times with PBS containing 0.1% FAF-BSA and 0.1% Saponin and incubated with species specific secondary antibodies in blocking buffer. Cells were washed in PBS, stained for 3 min with 4 mg/mL 40 ,6-diamidino-2-phenylindole (DAPI) to counterstain the nucleus, washed with PBS and Milli-Q H2O and mounted with Ultramount (Fronine) onto coverslips. Cells were visualised on the Zeiss confocal microscope (LSM 700 and LSM 710) and figures were assembled using Adobe PhotoshopTM. All images were taken under identical acquisition settings. Co-localisation was determined by Pearson's coefficient using the ImageJ JACoP plugin software A coefficient value exceeding 0.500 is considered as co-localisation.

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RNA extraction and qPCR analyses Cells were lysed in Trizol Reagent (Life Technologies) and RNA extracted as indicated by the manufacturers. 1 mg of total RNA was treated with RQ1 DNase (Promega) at 37 1C for 30 min to remove any contaminating cellular DNA, and cDNA generated with a Sensifast cDNA synthesis kit (Bioline) using both oligo d (T) primers and random hexamers. Gene-specific cDNAs were amplified using primers to the WNVKUN and DENVNGC genome and the internal control RPL13A (primer sequences are available from the authors upon request) with ITaq Universal Sybr Green (Bio-Rad) on an MX3000 real-time PCR machine (Agilent). Fold induction of the WNVKUN and DENVNGC genomes was calculated by comparing threshold cycle values (CT) to the internal control RPL13A. Western blot analysis Cells were washed with PBS and then lysed with SDS lysis buffer (0.1% SDS, 50 mM Tris–HCl pH 8.0, 150 mM NaCl) containing protease inhibitors (Protease inhibitor cocktail III, Astra Scientific). Lysates were mixed with 4X laemmli loading buffer (200 mM Tris– HCl pH 6.8, 8% SDS 40% glycerol and 0.08% bromophenol blue), boiled at 95 1C for 5 min and loaded on a 10% SDS polyacrylamide gel. Proteins were separated at 150 V till the dye front reached the end of the gel and transferred to a Hi-Bond ECL nitrocellulose membrane (Amersha) at 100 V for 60 min. The nitrocellulose membrane was blocked in 5% skim milk/PBS-T (PBS containing 0.05% Tween) at 4 1C overnight. Following blocking, membranes were washed four times for 10 min each in PBS-T and then incubated with primary antibodies diluted in 5% BSA-PBS-T for 4 h at RT. Membranes were once again washed with PBS-T as above and incubated with species–specific secondary antibodies conjugated to either Alexa Fluor 488 or 647 at RT for 2 h. Membranes were washed twice in PBS-T and a further two more times in PBS for 10 min each. Bio-Rad Pharos FX system was used to scan membranes. Quantity one software (Bio-rad) was used to quantitate western blots. Plaque assay For intracellular virus, treated- and/or infected-cells were lysed via three times freeze-thaw on dry ice and cellular debris was removed by centrifugation a 10,000 rpm for 5 min. The resulting supernatant was collected for analysis. For extracellular virus, tissue culture fluid (t.c.f) samples were collected from treatedand/or infected-cells. To remove cell debris, t.c.f was centrifuged at 10000 rpm for 5 min. Supernatants were then diluted 10-fold in DMEM/0.2% BSA and used to infect previously seeded Vero cells, in duplicates for 1 h at 37 1C. Following the incubation period, semisolid overlay (0.3% w/v low-melting point agarose, 2.5% w/v FCS, P/S, Glx, HEPES and NaCO3) was added to cells and solidified at 4 1C for 30 min. Cells were incubated at 37 1C for 3 days, fixed with 10% formalin (Sigma Aldrich) for 1 h and stained with 0.1% Toluidine blue at RT for 1 h. Plaques were enumerated and plaque-forming units per mL (pfu/mL) calculated. Foci forming unit assay To enumerate DENVNGC particles, intracellular and extracellular virus was collected as described for WNVKUN. Supernatants were then diluted 10-fold in DMEM and used to infect previously seeded Vero cells, in duplicates for 2 h at 37 1C. Semi-solid lowmelting-point agarose overlay (0.3% w/v low-melting point agarose, 2.5% w/v FCS) was added to cells and solidified at 4 1C for 30 min. Cells were then incubated at 37 1C for 3 days. Overlay was

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removed and fixed with ice-cold 80% acetone in PBS at  20 1C for 30 min. Fixative was removed; cells were completely dried and blocked with 10% BSA/PBS-T for 1 h at 37 1C. Blocking solution was removed and cells were incubated with primary antibody diluted in 10% BSA/PBS-T at 37 1C for 1 h. Cells were washed 4 times for 5 min with PBS-T and incubated with secondary antibody diluted in PBS-T for 1 h at 37 1C in the dark. Cells were washed once with PBS then 3 times with ddH2O and visualised using the Zeiss LSM 700 confocal microscope. Foci were enumerated and foci forming units (FFU) were calculated.

Acknowledgments We thank Alexander Khromykh, Roy Hall and Paul Young for generously providing the WNV and DENV antibodies, and David Jans for providing DENVNGC. We also thank Daniel Watterson (University of Queensland) for help and advice with the DENV foci-forming assay. This research was supported by a Project grant (No. 1004619) to J.M. from the National Health and Medical Research Council of Australia.

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