Glycobiology, 2017, vol. 27, no. 11, 999–1005 doi: 10.1093/glycob/cwx079 Advance Access Publication Date: 26 September 2017 Original Article Cell Biology NFκB-mediated activation of the cellular FUT3, 5 and 6 gene cluster by herpes simplex virus type 1 Rickard Nordén1, Ebba Samuelsson, and Kristina Nyström Department of Infectious Diseases/Clinical Virology, Institute of Biomedicine, University of Gothenburg, Sahlgrenska Academy, Guldhedsgatan 10B, SE-413 46 Gothenburg, Sweden To whom correspondence should be addressed: Tel: +46-31-3424914; Fax: +46-31-7412435; e-mail: [email protected] 1 Received 5 March 2017; Revised 28 August 2017; Editorial decision 28 August 2017; Accepted 30 August 2017 Abstract Herpes simplex virus type 1 has the ability to induce expression of a human gene cluster located on chromosome 19 upon infection. This gene cluster contains three fucosyltransferases (encoded by FUT3, FUT5 and FUT6) with the ability to add a fucose to an N-acetylglucosamine residue. Little is known regarding the transcriptional activation of these three genes in human cells. Intriguingly, herpes simplex virus type 1 activates all three genes simultaneously during infection, a situation not observed in uninfected tissue, pointing towards a virus speciﬁc mechanism for transcriptional activation. The aim of this study was to deﬁne the underlying mechanism for the herpes simplex virus type 1 activation of FUT3, FUT5 and FUT6 transcription. The transcriptional activation of the FUT-gene cluster on chromosome 19 in ﬁbroblasts was speciﬁc, not involving adjacent genes. Moreover, inhibition of NFκB signaling through panepoxydone treatment signiﬁcantly decreased the induction of FUT3, FUT5 and FUT6 transcriptional activation, as did siRNA targeting of p65, in herpes simplex virus type 1 infected ﬁbroblasts. NFκB and p65 signaling appears to play an important role in the regulation of FUT3, FUT5 and FUT6 transcriptional activation by herpes simplex virus type 1 although additional, unidentiﬁed, viral factors might account for part of the mechanism as direct interferon mediated stimulation of NFκB was not sufﬁcient to induce the fucosyltransferase encoding gene cluster in uninfected cells. Key words: FUT3, FUT5, FUT6, Herpes simplex virus type 1, NFκB Introduction In general, viral infection of permissive cells results in a shut-off of the synthesis of most host genes. This is true also for large human viruses, such as human herpesviruses. Yet, the transcription of a select number of host cell genes persists during viral replication, sometimes at higher rates than before viral infection. Hence, the mechanisms utilized by herpesviruses for regulating expression of host cell genes are selective: host genes not necessary for virus multiplication tend to be switched off whereas transcription of host genes that promote virus replication or viral colonization are unchanged or up-regulated (Smiley 2004). One of the most prominent events in this context concerns a simultaneous activation of a family of human fucosyltransferase genes (FUT3, FUT5 and FUT6) whose transcription rate each may be increased by as much as three orders of magnitude following infection by a number of different herpesviruses (Nystrom et al. 2004, 2007; Norden et al. 2013). The human fucosyltransferase encoding genes located in tandem at chromosome 19p13.3, FUT3, FUT5 and FUT6 are closely related, sharing an 85% sequence similarity, and they are the result of gene duplication events (Fig. 1) (Dupuy et al. 2002). The genes have evolved into fucosyltransferases that are able to add a fucose to an N-acetylglucosamine, in either an α-1,3 or α-1,4 position to a type 1 or type 2 precursor (Dupuy et al. 1999). These genes are likely to be associated with viral pathogenesis, as adding an α-1,3 fucose to a © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 999 1000 Fig. 1. Organization of the gene cluster including NRTN, FUT6, FUT3, FUT5 and NDUFA11 on chromosome 19, location 19p13.3. The arrows show the position of and the direction of transcription of each gene. The data were obtained from assembly GRCh38.p7 deposited in GenBank provided by National Center for Biotechnology Information (NCBI). sialylated type 2 structure creates sialyl Lewis X (sLeX), which is known to bind selectins. Selectins are proteins at the inner endothelial wall of relevance for allowing various circulating cells to cross the endothelium and access adjacent tissue (Scott and Patel 2013). Consequently, a herpesvirus with the capacity to promote expression of sLeX would be equipped with tools that would assist the infected cell to leave the circulation, thereby enabling spread of the virus to the underlying tissue. This mechanism has been shown for another human virus, the retrovirus human T-cell leukemia virus-1 (HTLV-1), where virus-induced surface-associated sLeX on virus-containing cells promoted viral colonization of the skin (Kannagi 2001). At present, little is known about the molecular mechanisms that permit human viruses to dramatically activate the transcription of a few human genes concomitantly with an almost complete virusorchestrated shutdown of expression of the vast majority of the host genes. This problem is even more intriguing in the perspective of FUT3, FUT5 and FUT6 on chromosome 19p13.3. This is due to the synchronous transcriptional activation of FUT3, −5 and −6 in herpesvirus-infected cells that stands in contrast to the otherwise individual regulation of these genes in normal tissue. Thus, FUT3 and FUT6 but not FUT5 are normally expressed in the small intestines, FUT6 is expressed in the salivary glands and only FUT3 is expressed in the lungs and the stomach (Cameron et al. 1995). FUT5 expression is extremely limited in normal cells (Cameron et al. 1995). Even when these FUT genes are pathologically induced, i.e., in various tumor cells, only one or two, but never all of the three are up-regulated (Cameron et al. 1995; Hanski et al. 1996; Dabrowska et al. 2005; Escrevente et al. 2006; Barthel et al. 2009; Carvalho et al. 2010). The only known exception is hepatocytes (HepG2 cell line), where a study demonstrated that two hepatocyte nuclear factors (HNF1A and HNF4A) were able to indirectly activate all three FUT genes, although FUT5 expression in liver tissue may be weak (Cameron et al. 1995; Lauc et al. 2010). The aim of this study was to establish if the simultaneous induction of the FUT-gene cluster on chromosome 19 by herpes simplex virus type 1 (HSV-1) is a speciﬁc process regulated by the infectious cycle and to deﬁne the underlying mechanism for the observed transcriptional activation of fucosyltransferases in HSV-1-infected ﬁbroblasts. Results HSV-1 speciﬁcally activate FUT3, −5 and −6 gene expression Diploid ﬁbroblasts were infected with HSV-1 or mock infected for 9 h and the expression levels of NRTN, FUT6, FUT3, FUT5 and NDUFA11 were assessed by reverse transcription real-time PCR (RTqPCR). FUT3, −5 and −6 were all up-regulated in the HSV-1-infected R Nordén et al. cells compared to the mock-infected cells, whereas the adjacent genes NRTN and NDUFA11 were unaffected or down-regulated, respectively (Fig. 2A). Additionally, ﬁbroblasts were treated with the histone deacetylase (class I, II and IV) inhibitor Trichostatin (TSA), a compound that cause relaxation of heterochromatin structures that enables increased transcriptional activation, for 9 h in the absence of viral infection and the expression levels were assessed. The TSAtreatment-activated transcription of NRTN, FUT6, FUT3 and FUT5 whilst moderately lowered the transcription of NDUFA11 compared to untreated cells (Fig. 2B). TSA treatment was veriﬁed by measuring the expression levels of CD40 (Gregorie et al. 2009), which increased in all replicates after TSA treatment, although the number of replicates (n = 3) were too few to confer a statistically signiﬁcant result (P = 0.13) (Supplementary Fig. 1). Hepatocyte nuclear factors 1a and 4a are not expressed in ﬁbroblasts The only known transcription factors for induction of FUT5, −3 and −6 are hepatocyte nuclear factors 1α and 4α (HNF1α and HNF4α) in hepatocytes. Therefore, the expression of HNF1α and HNF4α were assessed in HSV-1-infected and mock-infected ﬁbroblasts by RT-qPCR. Fibroblasts were infected with 10 plaque forming units/cell (10 PFU/cell) of HSV-1 or mock infected for 9 h. The Huh7.5 hepatocellular cell line was used as a control as HNF1α and HNF4α are known to regulate expression of the FUT6, −3 and −5 gene cluster in this cell type. There was no detectable mRNA expression of HNF1α or HNF4α in ﬁbroblasts whereas high levels were found in the Huh7.5 control cells (Fig. 3). NFκB-inhibitors subverts HSV-1 induction of FUT5, −3 and −6 in ﬁbroblasts Human diploid ﬁbroblasts were infected with HSV-1 at 10 PFU/cell for 8 h in the presence of indicated concentrations of Panepoxydone, a drug that inhibits NFκB via inhibition of IkB phosphorylation in addition to FOXM1 inhibition (Arora et al. 2014). Panepoxydone blocked, in a dose dependent manner, the transcriptional activation of FUT3, −5 and −6 in HSV-1 infected cells (Fig. 4A). To further verify this ﬁnding, HSV-1-infected ﬁbroblasts were treated with increasing concentrations of NFκB-activation inhibitor IV for 8 h. A modest reduction in FUT3, −5 and −6 expression could be observed in both the infected cells as well as in the mock-infected cells (Fig. 4B). Thereafter, HSV-1-infected ﬁbroblasts were treated with the FOXM1-inhibitor Thiostreptone for 8 h. Inhibition of FOXM1 did not reduce expression of FUT3, −5 and −6 in the HSV-1-infected cells. Only a modest reduction of FUT3 expression could be observed in the mock-infected ﬁbroblasts (Fig. 4C). There was no visible effect on cell viability of any of the applied drug concentrations as assessed by a MTS-assay (Supplementary Fig. 2). Transcription of HSV-1 glycoprotein B (gB-1) was measured as a surrogate marker for viral replication. The drug treatments had no signiﬁcant effect on gB-1 transcription (Supplementary Fig. 3A, B and C). The p65 subunit of NFκB is crucial for FUT-induction in HSV-1-infected ﬁbroblasts Analysis of the putative promoter regions of FUT6, −3 and −5 using the online tool UniProbe (http://thebrain.bwh.harvard.edu/nfkb/), revealed that the p65 subunit has multiple potential binding sites in all three promoter regions (Supplementary Table I). To further verify the notion that NFκB is a crucial component for induction of FUT6, 1001 HSV-1-mediated transcription of FUT3, FUT5 and FUT6 via NFκB Fig. 2. HSV-1 speciﬁcally activates the FUT-gene cluster but not the adjacent genes in HEL cells whereas TSA-induced chromatin remodeling affects both FUT genes and the ﬂanking gene NRTN. (A) The mRNA expression of FUT6, FUT3 and FUT5 and the ﬂanking genes NRTN and NDUFA11 was determined using RT-qPCR in HEL cells either infected with HSV-1 (black bars) at a multiplicity of infection 10 (MOI 10) or mock infected (white bars) for 9 h. (B) Fold change in RNA expression of NRTN, FUT6, FUT3, FUT5 and NDUFA11 in HEL cells treated with 0.5 μM TSA compared to mock treated for 9 h, determined by RT-qPCR. Fig. 3. No expression of HNF1α or HNF4α could be detected in HEL cells. The mRNA expression of HNF1α and HNF4α was determined with RT-qPCR in HEL cells that were mock or HSV-1 infected (MOI 10) for 9 h. Uninfected Huh7.5 hepatocellular cell line was used as a positive control. −3 and −5 transcription in HSV-1-infected cells, the expression of the p65 subunit was blocked by short interfering RNA (siRNA) transfection. Human ﬁbroblasts were transfected with either p65 siRNA or a scrambled siRNA control and were then either infected with HSV-1 at 10 PFU/cell or mock infected. Down regulation of p65 mRNA after transfection with siRNA in HSV-1-infected cells was veriﬁed by RT-qPCR (Fig. 5A). There was approximately 70% reduction in p65 mRNA levels after siRNA transfection. The protein levels of p65 and actin in transfected ﬁbroblasts were determined by western blot (Fig. 5B). The band intensity was used to calculate the fold reduction in p65 levels using actin as a loading control (Fig. 5C). Approximately half of the p65 protein levels could be blocked by siRNA transfection. Next the IL-6 mRNA levels were measured in HSV-1-infected ﬁbroblasts that were either transfected with p65 siRNA or transfected with a scrambled siRNA control. A signiﬁcant reduction in IL-6 mRNA expression was observed upon p65 siRNA transfection in HSV-1-infected cells (Fig. 5D). Finally, Fig. 4. NFκB is required for HSV-1-mediated induction of FUT genes. The transcription levels of FUT3, FUT5 and FUT6 in HEL cells were determined with RT-qPCR. The cells were treated with (A) Panepoxydone, n = 3, (B) NFκB-activation inhibitor IV, n = 2 or (C) Thiostrepton, n = 2, at indicated concentrations for 1 h and thereafter mock or HSV-1 infected (10 MOI) for additional 7 h in the presence of the drugs. Statistics were done using two-way ANOVA and multiple comparison t-test, * < 0.05, ** < 0.01, *** < 0.001 and **** < 0.0001. the expression of FUT3, −5 and −6 was assessed after p65 siRNA transfection, both in HSV-1 and mock-infected ﬁbroblasts. There was a signiﬁcant reduction of expression from all three fucosyltransferase encoding genes when p65 was targeted by siRNA in the HSV1-infected cells (Fig. 5E). Transcription of HSV-1 glycoprotein B was higher in the cells that were transfected with p65 siRNA compared to the cells transfected with scrambled siRNA control prior to viral infection (Supplementary Fig. 3D). FUT3, −5 and −6 are not activated by interferon stimulation alone Uninfected human ﬁbroblasts were treated with NFκB stimulating interferon beta (IFNβ), interferon gamma (IFNγ) or IL1 beta (IL1β) at 1002 Fig. 5. The NFκB subunit p65 is important for mediating transcriptional induction of FUT3, FUT5 and FUT6 in HSV-1-infected HEL cells. HEL cells were transfected with siRNA targeting the NFκB subunit p65 or with a scrambled control siRNA. (A) Expression of p65 mRNA in HEL cells transfected with siRNA targeting p65 and subsequently infected with HSV-1 at a multiplicity of infection 10 (10 MOI) for 7 h. The mRNA levels were determined using RTqPCR and the transcription level compared to cells treated with a scrambled siRNA control. (B) The amount of expressed p65 protein in uninfected HEL cells transfected with siRNA targeting p65 and in cells transfected with a scrambled siRNA control was detected by western blot. Beta actin was used as a loading control. (C) The ratio of p65 protein expression, from three separate western blot experiments (n = 3), calculated using the Image Lab™ Software. (D) Transcription of IL-6 was determined using RT-qPCR in HEL cells transfected with siRNA targeting p65 or with a scrambled siRNA control and subsequently mock or HSV-1 infected (MOI 10), n = 4. (E) Transcription levels of FUT6, FUT3 and FUT5 mRNA in HEL cells transfected with siRNA targeting p65 or with a scrambled siRNA control and subsequently mock or HSV-1 infected for 7 h (MOI 10), n = 4, determined using RT-qPCR. Statistics were done using two-way ANOVA and multiple comparison t-test and unpaired t-test, * < 0.05, ** < 0.01, *** < 0.001 and **** < 0.0001. the indicated concentration and the gene expression of CXCL10, FUT3, FUT5 and FUT6 was determined. The CXCL10 gene is known to be a target for NFκB transcriptional regulation and was induced by three orders of magnitude upon stimulation with IFNβ and IFNγ, while IL1β stimulation triggered its activation by more than two orders of magnitude after 6 h of stimulation. Conversely, FUT3, −5 and −6 were expressed at lower levels after 6 h of stimulation with IFNβ, IFNγ and IL1β (Fig. 6). After 24 h of stimulation, the expression levels of FUT3, −5 and −6 were essentially the same in the interferon stimulated cells as in the un-stimulated control cells. Discussion Expression of the majority of human genes is down modulated upon infection with HSV-1 in order to facilitate expression of viral genes (Smiley 2004). However, as many as 500 genes have been described to be up-regulated more than 3-fold in HSV-1 infected ﬁbroblasts (Taddeo et al. 2004). FUT3, along with FUT5 and FUT6, located in tandem on chromosome 19 are uniquely induced during HSV-1 infection in as much as they are normally separately regulated in uninfected cells, and they are under control of individual promoters R Nordén et al. Fig. 6. Stimulation of NFκB using interferon treatment fail to induce the FUTgene cluster in the absence of HSV-1-infection. To control whether the FUT genes were induced by activation of the NFκB pathway, the expression of CXCL10, FUT6, FUT3 and FUT5 was determined by RT-qPCR in uninfected cells treated with 200 units/mL IFNβ, 10 ng/mL IFNγ or 50 ng/mL IL1β for 6 or 24 h. (Cameron et al. 1995; Dabrowska et al. 2005). Moreover, several HSV-1 proteins are involved in the reduction of heterochromatin formation on cellular chromosomes (Conn and Schang 2013). Despite this, we demonstrate that the up-regulation of FUT3, FUT5 and FUT6 in HSV-1-infected ﬁbroblasts is speciﬁc and does not involve the adjacent NRTN and NDUFA11 genes. Conversely, treating the cells with a histone deacetylase inhibitor will open the chromatin structure and facilitate access to the promoter regions for transcription factors, potentially leading to a general increase in transcription (Grunstein 1997). When uninfected ﬁbroblasts were treated with TSA, which inhibits histone deacetylases of class I, II and IV, transcription was induced not only from FUT3, FUT5 and FUT6 but also from NRTN, supporting the notion that in HSV-1-infected ﬁbroblasts the FUT-gene cluster is specifically targeted for transcriptional up-regulation. FUT3 and FUT6 are both described to be involved in cancer cell metastasis and apoptosis (Mas et al. 1998; Togayachi et al. 1999; Le Pendu et al. 2001; Barthel et al. 2009), and the promoter regions have been investigated in various cancer cell types (Dabrowska et al. 2005; Serpa et al. 2006; Higai et al. 2008). FUT5 expression is, however, very limited in normal tissue, and not much is known regarding its transcriptional activation. Studies on the FUT3 promoter suggest that the AP-1 transcription factor (Dabrowska et al. 2005) and promoter methylation (Serpa et al. 2006) are able to 1003 HSV-1-mediated transcription of FUT3, FUT5 and FUT6 via NFκB regulate FUT3 expression. Promoter methylation has previously been shown not to be involved in FUT up-regulation of HSV-1 infected ﬁbroblasts (Norden et al. 2010). AP-1 may however be relevant in HSV-1 infected cells as ICP0, an immediate early HSV-1 gene essential for a productive HSV-1 infection, stimulates AP-1 dependent transcription (Zachos et al. 1999; Diao et al. 2005). Thus, AP-1 may be involved in the transcriptional activation of FUT3, but not FUT5 or FUT6, which have dissimilar promoter regions (Dabrowska et al. 2005). In contrast, the FUT5 and FUT6 promoter could be regulated by HNF1α, HNF4α and Oct-1 (Higai et al. 2008; Kel et al. 2008; Lauc et al. 2010) but we show here that HNF1α and HNF4α are not expressed in ﬁbroblasts, either HSV-1infected or uninfected, thus making them unlikely as master regulators of the FUT-gene cluster in this cell type. It has previously been shown that the dsRNA-sensor protein kinase R (PKR) is necessary for HSV-1 induced activation of FUT3, −5 and −6 in ﬁbroblasts (Norden et al. 2009). In response to viral infection, PKR undergoes auto-phosphorylation, which drives signaling through multiple pathways, and one of the downstream targets is NFκB (Gil et al. 2000; Su et al. 2006). In this study, it is demonstrated that treatment of ﬁbroblasts with NFκB inhibiting drugs reduce HSV1-induced transcriptional activation of the FUT-gene cluster. The notion that NFκB is a key element is further strengthened as blocking the p65 subunit of NFκB by siRNA transfection also prevented HSV1-mediated FUT3, −5 and −6 transcription. Interestingly, blocking the p65 subunit with siRNA appeared to facilitate increased viral replication as the expression of the late gene gB-1 was signiﬁcantly higher in these cells, possibly reﬂecting a damped antiviral state in the absence of fully functional NFκB signaling. Thus, even though the replication of HSV-1 may be enhanced in cells with reduced expression of p65 the transcription of FUT3, FUT5 and FUT6 is down modulated, further highlighting the importance of NFκB for transcriptional regulation of these fucosyltransferases in HSV-1-infected cells. Furthermore, ubiquitination has been described to regulate NFκB (Chen and Chen 2013), and inhibition of ubiquitindegradation of proteins by proteasomal inhibitor MG-132 also prevents HSV-1 induced transcription of FUT3, −5 and −6 (Nystrom et al. 2009). A detailed study of the DNA binding capacity of the ﬁve different NFκB subunits (c-Rel, p65, RelB, p50/p105 and p52/ p100) has resulted in the development of an online tool to search promoter sequences for NFκB binding sites (Siggers et al. 2011). In silico analysis of a 3 kb region 5′ of the ATG start site of FUT3, −5, and −6 demonstrated p65 binding sites in the potential promoter region of all three genes. Together, these data indicate that transcription of FUT3, −5 and −6 is regulated by p65 in HSV-1-infected ﬁbroblasts. However, the residual transcription of FUT3, -5 and -6 mRNA in the uninfected cells was not signiﬁcantly affected by blocking p65 NFκB. This may be due to an additional effect by p52, which is regulated by the non-canonical NFκB pathway (Oeckinghaus and Ghosh 2009). Moreover, potential binding sites for p52 are also found in the putative promoter regions of FUT3, -5 and -6 as determined by the in silico analysis (Supplementary Table I). In addition, treatment of uninfected ﬁbroblasts with IFNβ, IFNγ or IL1β was not sufﬁcient to induce transcription of the FUTgene cluster. The interferon treatment was adequate for generating a strong CXCL10 response indicating that the NFκB pathway was activated through p50/p65, which is the main downstream effector complex in the NFκB pathway of these three drugs. IFNβ, IFNγ and IL1β mediated transcription through NFκB may differ from the observed p65-dependent pathway by which HSV-1 induced the FUT genes, as many varying regulatory events ranging from the seven different members of the IkB family to the posttranslational phosphorylation or acetylation allowing for a ﬁne-tuned NFκB response (Oeckinghaus and Ghosh 2009). Alternatively, additional viral factors may also be necessary for driving transcription of the FUT-gene cluster during HSV-1 infection in ﬁbroblasts. Thus, the phenomenon we describe here, where HSV-1 simultaneously induce transcription of FUT3, FUT5 and FUT6 via NFκB, may be the sum of several separate events, which only take place during herpesvirus infection. Materials and methods Virus and cells The Syn17+ strain of HSV-1 was used throughout the study. Plaque titration on green monkey kidney cells (GMK) was used to determine the viral concentrations, which allowed for calculation of plaque forming units per cell (PFU/cell). Human embryonic lung ﬁbroblasts were cultivated in Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal calf serum, 1% pencillinstreptomycin and 1% L-glutamine. Drug treatment and viral infection The cells were grown in 6-well plates (9 cm2 per well) until they were conﬂuent. Fresh growth media supplemented with drugs at indicated concentrations was added to the cells, and the plates were incubated for 1 h at 37°C and 5% CO2 in a humid chamber. Subsequently HSV-1 (Syn17+) was added at a multiplicity of infection 10 (MOI 10), and the virus was allowed to attach to the cells for 1 h at 37°C and 5% CO2 in a humid chamber. For mock infection, the cells were instead incubated with virus free growth medium. Unbound viral particles were removed by washing the cells with buffered NaCl solution. Growth medium supplemented with the indicated drug concentrations was again added to the cells and incubation was continued for 6 h. At the end of the incubation, the growth medium was removed and the cells were harvested by adding 600 μL lysis buffer, consisting of Nucleic acid puriﬁcation lysis solution (Applied Biosystems, Waltham, MA) and phosphate-buffered saline at a ratio of 1:1, to each well. The samples were subsequently stored at –20°C until RNA extraction. The following drugs were used in the study: panepoxydone (Sigma Aldrich, Saint Louis, MO), NFκB-activation inhibitor IV (Calbiochem, Billerica, MA), Thiostrepton (Thermo Fisher Scientiﬁc, Waltham, MA), IFNß1A (Thermo Fisher Scientiﬁc, Waltham, MA), IFNγ (Thermo Fisher Scientiﬁc, Waltham, MA) and IL1β (Sigma Aldrich, Saint Louis, MO). siRNA transfection The siRNA complexes Silencer®select (Thermo Fisher Scientiﬁc, Waltham, MA), targeting p65 and a scrambled siRNA control, were prepared according to the manufacturer instructions. Subsequently, 5 μL of the siRNA complexes were diluted in Opti-MEM® I (1×) reduced serum medium (Gibco, Thermo Fisher Scientiﬁc, Waltham, MA) to a ﬁnal volume of 90 μL. Additionally, 6 μL Lipofectamine®RNAiMAX reagent (Invitrogen, Carlsbad, CA) was added to 94 μL Opti-MEM® I (1×) reduced serum medium and incubated at room temperature for 10 min before combined with the prepared siRNA complexes. The ﬁnal mix was incubated for an additional 20 min at room temperature. The cells to be transfected with siRNA were grown in growth medium without antibiotics in a 12-well plate until they were 50% conﬂuent. The growth medium was removed and the cells washed once with EMEM (1% L-glutamine) before addition of 300 μL of 1004 EMEM (1% L-Glu) and 190 μL oligonucleotide-mix to each well. The cells were incubated for 4 h in 37°C and 5% CO2. Subsequently 250 μL EMEM (1% L-Glu, 2% fetal calf serum) was added to each well without removal of the transfection mixture. The incubation was allowed to continue for 72 h. The transfected cells were then either harvested for protein puriﬁcation by removing the growth medium and adding 240 μL 1× sample buffer (4 mL dH2O, 1 mL Tris–HCl (0.5 M, pH 6.8), 0.8 mL Glycerol, 1.6 mL SDS 10% (w/v), 0.4 mL 2-mercaptoethanol, 0.2 mL saturated bromphenol blue-solution) and removal of the cells using a cell scraper, or mock and HSV-1 infected as described above. Finally, the infected cells were harvested for nucleic acid puriﬁcation as described in the section “Drug treatment and viral infection”. Nucleic acid extraction The samples, stored in lysis solution, were thawed and the nucleic acids puriﬁed using the 6100 Nucleic acid PrepStation (Applied Biosystems, Waltham, MA) and a Total RNA isolation kit (Applied Biosystems, Waltham, MA) or RNeasy Mini Prep Kit (Qiagen, Hilden, Germany) both according to the manufacturer instructions. To further purify RNA, the total nucleic acids were subjected to DNase treatment using the TURBO DNA-free™ Kit (Thermo Fisher Scientiﬁc, Waltham, MA) according to the manufacturer instructions. The RNA concentration was determined using a NanoDrop®ND1000 spectrophotometer or a Qubit® 2.0 Fluorometer, and adjusted to 10-20 ng/μL using dH2O. Reverse transcription real-time PCR The gene expression levels were assessed by reverse transcription realtime PCR (RT-qPCR). All reactions were set up as follows: Superscript® III Platinum 2X mastermix (Invitrogen, Carlsbad, CA) was mixed with 0.5 mM forward and reverse primer, 0.3 mM probe, 20–40 ng RNA and H2O in a total volume of 20 μL. The reaction conditions were as follows: one step at 50°C for 30 min, one step at 95°C for 10 min, 40 PCR cycles at 95°C for 15 s and 60°C for 60 s. The primer and probe sequences for FUT3, FUT5, FUT6, 18S and gB-1 have been described previously (Nystrom et al. 2004; Norden et al. 2009), primers and probes for HNF1A, HNF4A, CXCL10, IL-6, CD40 and p65 were purchased from Thermo Fisher Scientiﬁc (Waltham, MA). The primer and probe sequences for NRTN and NDUFA11 were as follow: NDUFA11 forward primer, 5′-AAG GAG TGG CTA AGG TGG AC-3′; NDUFA11 reverse primer, 5′CAC CGA GGA AGT AGT TCA GG-3′; NDUFA11 probe, FAM5′-CCT CAC CAC CTG CAT CAG CGC CCA-3′-TAMRA; NRTN forward primer, 5′-CTG GAT GTG TCG AGA GGG C-3′; NRTN reverse primer, 5′-AGC TCC ATC GCA TCC GG-3′; NRTN probe, FAM-5′-CTT CTC AGC CAC CGC CTC GGA CCT GC- 3′TAMRA. The results from the RT-qPCR assays were calculated with the ΔCT method based on the formula CR = 2−ΔCT+X and 18S was used as a reference gene (Nystrom et al. 2004). Western blot The samples were heated to 95°C for 10 min and subjected to three sonication cycles (30 s ON/30 s OFF) using the Bioruptor® Pico Sonication system (Diagenode, Seraing, Belgium) before separation of the proteins in a gel (NuPage 4–12% Bis–Tris gel, Novex, Thermo Fisher Scientiﬁc, Waltham, MA). The proteins were subsequently blotted to a polyvinylidene ﬂuoride membrane (Millipore, Billerica, MA). The membrane was blocked in 5% Bovine Serum R Nordén et al. Albumine (BSA) (Sigma Aldrich, Saint Louis, MI) dissolved in TTBS (Tris buffered saline supplemented with 0.1% Tween) for 30 min with gentle agitation. The membrane was washed in TTBS and the primary antibody, Anti-NF-kB p65 antibody—ChIP Grade (Abcam®, Cambridge, UK) diluted 1:2000, was added. The AntiActin antibody—Loading Control (Abcam®, Cambridge, UK), diluted 1:10,000, was used as a loading control. After incubation for 1 h during gentle agitation the membrane was again washed and the secondary antibody Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (Abcam®, Cambridge, UK), diluted 1:6000, was added. The incubation and washing procedures were repeated. The Chemi Doc MP imager (Bio-Rad Laboratories Inc., Hercules, CA) together with Supersignal® West Dura Extended Duration Substrate (Thermo Fisher Scientiﬁc, Waltham, MA) was used according to the manufacturer instructions to visualize the proteins. To calculate the ratio between p65 and beta actin, the Image Lab™ Software (BioRad Laboratories Inc., Hercules, CA) was used. Toxicity test (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assays were performed to determine the cell viability after drug treatment. Human ﬁbroblasts were grown in 96-well plates and drugs were applied at the indicated concentrations. The cells were incubated at 37°C and 5% CO2 for 14 h in a humid chamber. Subsequently 20 μL Cell Titer 96® Aqueous One Solution Cell Proliferation Assay (Promega, Fitchburg, MA) was added to each well and the plate was incubated for 2 h as above before determining the absorbance at 492 nm. Statistical analysis The statistical analysis was performed using either unpaired t-test or two-way analysis of variance (two-way ANOVA) followed by multiple comparison using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA). A P-value <0.05 was considered signiﬁcant. Supplementary data Supplementary data are available at Glycobiology online Funding This work was supported by Stiftelsen Professor Lars-Erik Gelins minnesfond. Conﬂict of interest statement None declared. 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