Virology 471-473 (2014) 29–37

Contents lists available at ScienceDirect

Virology journal homepage: www.elsevier.com/locate/yviro

Infection of human amniotic and endothelial cells by Japanese encephalitis virus: Increased expression of HLA-F Gaurav Kumar a, Onkar Sanjay Date a,1, Kwang Sik Kim b, Ramanathapuram Manjunath a,n a b

Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India Department of pediatric infectious diseases, John Hopkins university school of medicine, Baltimore, MD 21287, USA

art ic l e i nf o

a b s t r a c t

Article history: Received 22 July 2014 Returned to author for revisions 5 September 2014 Accepted 26 September 2014 Available online 20 October 2014

Productive infection of human amniotic and endothelial cell lines with Japanese encephalitis virus (JEV) was established leading to the induction of NFκB and HLA-F, a non-classical MHC molecule. Induction of the HLA-F gene and protein in JEV-infected cells was shown to be NFκB dependent since it was blocked by inhibitors of NFκB activation. ShRNA targeting lentivirus-mediated stable knockdown of the p65 subunit of NFκB inhibited JEV-mediated induction of HLA-F both in the amniotic cell line, AV-3 as well as the human brain microendothelial cell line, HBMEC. The induction of HLA-F by treatment of AV-3 with TNF-α was also inhibited by ShRNA mediated knockdown of NFκB. TNF-α treatment of HEK293T cells that were transfected with reporter plasmids under the control of HLA-F enhancer A elements resulted in significant transactivation of the luciferase reporter gene. NFκB-mediated induction of HLA-F following JEV infection and TNF-α exposure is being suggested for the first time. & 2014 Elsevier Inc. All rights reserved.

Keywords: JEV TNF-α Amniotic Endothelial cells NFκB mediated HLA-F induction

Introduction Japanese encephalitis virus (JEV) is a neurotropic virus that is endemic in several parts of India and South East Asia (Halstead and Jacobson, 2003; Misra and Kalita, 2010). It is a positive single stranded RNA virus that belongs to the Flavivirus genus of the family Flaviviridae (Lindenbach and Rice, 2003) and causes encephalitis in newborn children and young adults. Its structural, pathological, immunological and epidemiological aspects have been well studied (Halstead and Jacobson, 2003; Heinz and Allison, 2000; Kurane, 2002). The ability of flaviviruses to upregulate the cell surface expression of MHC-I (Kurane, 2002; Shen et al., 1997) has been shown to be dependent on IFN-β as well as NFκB. We have also reported that JEV induces nonclassical MHC-I molecules such as H-2Qa-1b (Abraham et al., 2008) and soluble HLA-E (Shwetank et al., 2013) in addition to classical MHC-I molecules. Among the known human non-classical antigens – HLAE, -F and -G, only the HLA-F gene has been reported to be regulated by NFκB using in vitro gene reporter assays (Gobin and van den Elsen, 2000) and JEV is known to activate NFκB (Cheng et al., 2004). One study reports an increase in HLA-F RNA expression in rabies virus infected neuronal cells (Megret et al., 2007) but there are no other

reports of virus mediated alterations in HLA-F gene expression. Hence it was of interest to study if JEV infection would activate NFκB in human cells leading to functional HLA-F gene induction. We examined the effect of JEV infection on the expression of the HLA-F gene in AV-3 and WISH cells since they have been used as model cell lines to study intrauterine infection and amniotic inflammatory responses (Pavan et al., 2003; Potter et al., 1999). Amniotic epithelial cells and mid-trimester placental tissues are known to express the nonclassical MHC molecules, HLA-F and HLA-G (Adinolfi et al., 1982; Shobu et al., 2006). In addition, the bladder carcinomaderived epithelial cell line, ECV 304 (ECV) and immortalized human brain microvascular endothelial cells (HBMECs) were also included in the study since they have been used as models of the blood brain barrier (Untucht et al., 2011; Wilhelm et al., 2011). We show here that JEV establishes productive infection in all these cell lines and leads to induction of HLA-F at the gene and protein levels. This HLA-F induction was partially dependent on the activation of NFκB in JEVinfected cells as shown by inhibitor and p65 knockdown experiments. More importantly, HLA-F was shown to be inducible by TNFα, a cytokine that is known to be induced during JEV infections (Ravi et al., 1997).

Results n

Corresponding author. Tel.: þ 91 80 22932309; fax: þ 91 80 23600814. E-mail addresses: [email protected] (G. Kumar), [email protected] (O.S. Date), [email protected] (K.S. Kim), [email protected] (R. Manjunath). 1 Present address: Syngene International, Biocon Park, Plot 2 & 3, Electronic City, Bangalore, Karnataka 560099, India. http://dx.doi.org/10.1016/j.virol.2014.09.022 0042-6822/& 2014 Elsevier Inc. All rights reserved.

JEV infection and induction of HLA-F Productive JEV infection of several different mouse cell types including mouse embryonic fibroblasts as well as its ability to induce

30

G. Kumar et al. / Virology 471-473 (2014) 29–37

classical and nonclassical MHC molecules has been reported earlier (Abraham et al., 2010; Lobigs et al., 2003). Hence we tested the effects of JEV infection on the expression of the human nonclassical molecule, HLA-F in different human cell types. The human amnionderived cell types, AV-3 and WISH were infectable as seen by the presence of the JEV nonstructural protein, NS3 (Fig. 1A and B) as well as the intracellular presence of HB112, a flavivirus group specific antigen at 12 h and 24 h post-infection (Fig. S1) and PFU release ( 1–2  105/ml at 30 h p.i.). Similarly, both ECV and HBMECs were infected with JEV (Fig. 1C and D) confirming our previous study (Shwetank et al., 2013). As shown in Fig. 1, transcription of the HLA-F gene was progressively induced by JEV in all cell types in a timedependent manner and was followed by a concomitant increase in intracellular HLA-F antigen as measured by 3D11 monoclonal reactivity (Fig. 1, right panel). The position of the 3D11 immunoreactive band was authenticated as HLA-F by the detection of a 42 kDa band in cell lysates of a HLA-F negative cell line BeWo that expressed the antigen only after transfection with HLA-F pcDNA 3.1 (Fig. S2C). The band marked by the arrow corresponded to the predicted HLA-F molecular weight of 42 kDa approximately (Wainwright et al., 2000). Increased HLA-F gene transcription was not observed when AV-3 and WISH cells were infected for 36 h with UV inactivated virus suggesting that virus replication was needed (Fig. 1A and B). Similar results were observed with ECV and HBMEC cells using UV inactivated virus (data not shown). These data were further confirmed by real time PCR analysis as shown in Table 1. Induction of the HLA-F gene increased from 12 h p.i., and reached maximum levels between 24 h and 36 h of infection (Table 1) in the above cell lines. NFκB activation in JEV infected human amniotic and endothelial cell lines

independent pathways (Cheng et al., 2004). Binding of NFκB to the κB1 site present in the enhancer A module of the HLA-F promoter

has been demonstrated (Gobin et al., 1998). Hence we first tested if JEV was able to activate NFκB in human amniotic and endothelial cells. As shown, JEV infection of AV-3, WISH, ECV and HBMEC cells led to a time-dependent (Fig. 2A and B) and virus dose-dependent (Fig. S4) binding of NFκB to the κB1 oligomer. The position of the NFκB-oligo complex in JEV-infected cell extracts was verified by treatment of AV-3 cells with okadaic acid that is known to activate NFκB (Fig. 2, Pos). In addition, the supershifted band observed upon addition of anti-p65 NFκB antibody confirmed that the complex was NFκB1 specific (Fig. 2B) in all cell lines tested. The complex was not formed when the κB2 oligo was used in EMSA (Fig. 2B) confirming the earlier report that the HLA-F κB2 site does not bind NFκB (Gobin et al., 1998). Cells were then infected with JEV in the Table 1 Real Time PCR analysis of HLA-F gene transcription. Time of infection

Control 12 h 24 h 36 h

Cell linea AV-3

WISHb

ECV

HBMEC

1.0 7 0.10 3.2 7 0.05# 10.6 7 0.23n 13.0 7 0.20n

1.0 7 0.18 1.5 7 0.13 12.17 0.03n 12.2 7 0.03n

1.0 7 0.14 2.17 0.23 17.3 7 0.53n 14.4 7 0.11n

1.0 7 0.07 2.7 7 0.01# 15.7 7 0.24n n.dc

a

Data represent fold change over control. Wish cells infected for 36 h with UV inactivated virus gave a value of 1.570.2. c Not determined. n Po0.001. # Po0.01. b

A major role for NFκB in MHC induction was not evident in our previous studies using JEV-infected mouse embryonic fibroblasts (Abraham et al., 2010). However, flaviviruses such as West Nile Virus induce MHC antigen expression through NFκB dependent and

Fig. 1. HLA-F induction in JEV infected cells. As shown at the top, whole cell lysates (35 μg protein) from AV-3, WISH (Panel A and B), ECV and HBMEC (Panel C and D) cells were subjected to Western blotting using rabbit anti JEV-NS3 (JEV-NS3 Protein) or 3D11 anti HLA-F moAb (HLA-F Protein). Total RNA extracted from the cells was subjected to semiquantitative RT-PCR analysis (HLA-F gene) for HLA-F gene expression. In each panel, the top row represents the results obtained for Western blotting and RT-PCR analysis while the bottom row represents controls for β-tubulin protein and β-actin gene expression. JEV infection was done for 12, 24 and 36 h while ‘UV’ in panels A and B represents infection of AV-3 and WISH for 48 h with UV inactivated JEV. Numbers indicated at the bottom of each panel represents the fold change over control (Con) after normalization for β-tubulin protein or βactin gene expression.

Fig. 2. NFκB activation in JEV infected human cell lines. EMSA was performed with NF-κB1 oligomer using nuclear extracts from AV3, WISH, ECV and HBMEC cells as indicated either alone (Panel A) or in the presence of either anti-p65 antibody (Panel B) or NFκB inhibitor, Bay 11-7085 (Panel C). Panel A: Lanes U and Pos represent uninfected cells and cells treated with the positive control, okadaic acid while lanes 1–3 represent cells that were JEV infected for 12, 18 and 24 h respectively. Panel B: Lane U represents uninfected cells while lane ‘Neg’ represents EMSA results obtained with JEV infected cells and the NF-κB2 oligomer as a negative control. Uninfected cells (Lane U) or 24 h JEV-infected cells were incubated with the functional NF-κB1 oligomer, either alone (Lane 1) or in the presence of anti-p65 (Lane 2) and 15 fold excess anti-p65 antibody (Lane 3 for ECV only). Panel C: Lanes marked Neg1 and Neg2 indicate EMSA results obtained using the NF-κB2 oligomer for 24 h JEV-infected and normal uninfected cells respectively. Lanes 1–3 show 24 h JEV-infected cells that were either untreated (Lane 1) or treated with 10 mM (Lane 2) and 15 mM (lane 3) Bay. The protein–oligomer complex and the antibody-supershifted complex are indicated by arrows.

G. Kumar et al. / Virology 471-473 (2014) 29–37

31

presence of the NFκB inhibitor, Bay 11-7085 in order to check if the JEV-induced NFκB activation was inhibited in the human cell lines tested. Treatment of both WISH and AV-3 cells with Bay 11-7085 during virus infection clearly inhibited JEV-induced NFκB activation as seen in Fig. 2C. Similar results were obtained with ECV and HBMECs (Fig. 2C). Stable knockdown of p65 NFκB abrogates HLA-F induction NFκB is a heterodimeric DNA-binding protein complex consisting of p50/p65 subunits. NFκB activation is regulated by diverse stimuli in different cell types and mediates different functions (Perkins and Gilmore, 2006) that are believed to bridge inflammation and cancer. Hence virus-infected cells were treated with the NFκB inhibitor, Bay 11-7085 to check its effect on HLA-F expression. Intracellular expression of HLA-F antigen was found to decrease in all cell lines upon treatment with Bay as shown in Fig. 3A and B. HLA-F gene induction was also measured by real-time RT-PCR quantification in the infected cells treated with Bay 11-7085. Results obtained showed that HLA-F gene induction was also inhibited significantly by treating the cells with Bay 11-7085 (data not shown). Viral synthesis of JEV-NS3 non-structural protein remained unaffected by Bay (Fig. S3) treatment suggesting that NFκB might play a role in JEV mediated induction of HLA-F. RNA interference using lentiviral vectors coding for shRNA that downregulate p65 expression has been used to block NFκB signaling pathway in order to analyze its role in the function of VEGF and many other growth factors in tumor metastasis (Huang et al., 2000). In order to further confirm the role of NFκB, stable mutant HBMEC and AV-3 cell lines were generated by transduction and selection using lentiviral vectors expressing shRNA constructs that targeted three different p65 microRNA sequences called Shrna1, Shrna2 and Shrna3 as given in Material and methods. Nontargeting lentivirus particles served as controls. Western analyses revealed that the decrease in p65 expression was maximal in mutant HBMEC and AV3 cells that were transduced with Shrna1 lentiviral particles (Fig. 4A). Hence further analyses with JEV infection were done with Shrna1 mutant cells (Fig. 4B). Virus infection was confirmed by detection of the JEV-NS3 nonstructural protein in all cell lines by Western blotting. The JEV induction of HLA-F was significantly decreased upon p65 knockdown with Shrna1 as seen in Fig. 4B. Additionally, control Shrna and Shrna1 cells were treated with

Fig. 4. Effect of NFκB knockdown on HLA-F antigen expression in JEV infected cells. Panel A: HBMEC and AV-3 cells were transduced either with control non-targeting lentivirus (cShrna) or with three different lentivirus particle clones (Shrna1, Shrna2 and Shrna3) each targeting different microRNA sequences of RELA as given in Materials and methods. Sorted cells stably expressing the Shrna were lysed and the lysate protein (35 mg) was subjected to Western blotting using anti NF-κBp65 (Top) and anti β-tubulin (Bottom) antibodies. Panel B: As labeled at the bottom, HBMEC and AV-3 cells that were transduced with control non-targeting lentivirus (cShrna) or Shrna1 targeting lentivirus particle clone were left untreated (Con), infected with JEV for 24 h (Jev) or treated with TNF-α for 24 h (Tnfα). Cell lysates (35 mg protein) were subjected to Western blotting using anti HLA-F (Top), anti β-tubulin (Middle) anti JEV-NS3 (Bottom) antibodies. Numbers (Fold) indicate the fold change over control (Con) obtained after normalization for β-tubulin protein.

TNF-α as a positive control (Bouwmeester et al., 2004). Also, JEV infection activates production of pro-inflammatory cytokines such as TNF-α, IL-8, RANTES, IL-1 and IFNs (Chen et al., 2011; Ghosh and Basu, 2009) in different cell types. As shown in Fig. 4B, treatment with TNF-α increased the expression of 3D11 reactive HLA-F antigen and this increase was also inhibited in Shrna1 expressing cells both in HBMEC and AV-3. A separate experiment with AV-3 confirmed the decreased expression of p65 NFκB in shRNA mediated p65 knockout cells as seen in Fig. S5 showing clearly that NFκB played a role in HLA-F expression. TNF-α induces HLA-F gene expression

Fig. 3. Effect of NFκB inhibition on HLA-F expression in JEV infected cells. As labeled, whole cell lysates (35 mg) of uninfected and 24 h JEV infected HBMEC, AV-3 (Panel A), ECV and WISH (Panel B) cells were either left untreated (Con) or treated with Bay 11-7085 (Bay) for 24 h and subjected to Western blotting using anti HLA-F (Top row) and anti β-tubulin (Bottom row) antibodies.

Since TNF-α treatment resulted in increased expression of HLA-F antigen, it was of interest to check the effect of TNF-α treatment on HLA-F gene expression. It was observed that HLA-F gene expression was significantly induced when AV-3 and HBMEC cells were treated with 300 IU TNF-α for 24 h (Table 2). In addition, the TNF-α induced HLA-F gene expression could be inhibited by two inhibitors of NFκB activation pointing to its possible involvement in HLA-F gene induction. Similar inhibitory effects were also observed when the NFκB inhibitory peptide was tested on JEV infected cells but the results obtained with the control and inhibitory peptides were extremely variable (data not shown). This could possibly be due to the extensive induction of membranes by JEV that are essential for viral replication (Salonen et al., 2005; Uchil and Satchidanandam, 2003) but could interfere with the intracellular membrane transducing efficiency of the PTD in control and inhibitory peptides. Hence the results described thus far encouraged us to test the effect of JEV infection and TNF-α treatment on HLA-F gene expression in AV-3 cells that were stably knocked down for NFκB as described above. HLA-F gene expression increased in a time dependent manner in both instances of JEV infection as well as TNF-α treatment and the increase obtained at 24 h of treatment significantly decreased in

32

G. Kumar et al. / Virology 471-473 (2014) 29–37

Table 2 Effect of TNF-α on HLA-F induction. Treatment

Cell linea AV-3

HBMEC

TNF-α Plus Bay 15 mM

6.06 70.05 2.30 70.17n

7.167 0.16 2.83 7 0.20n

TNF-α Plus inhibitory peptideb

8.11 71.12 5.44 70.41n

6.63 7 2.29 1.577 0.02n

a

Data represent fold change over control untreated cells. Fold change observed upon incubation of TNF-α treated AV-3 cells with control peptide was 7.58 7 0.42. n Po 0.001. b

Fig. 6. TNF-α induces HLA-A and HLA-F. HEK293T cells were transiently transfected with luciferase reporter plasmid constructs containing the control construct pGL3basic alone or constructs containing enhancer A region of HLA-F (enhF) or enhancer A region of HLA-A (enhA) linked to pGL3-A140. The cells were treated with TNF-α (500 U/ml) for 2, 3 and 9 h in the upper panel (Expt 1) while they were treated with TNF-α (500 U/ml), IL-β (1 ng/ml) or IFNβ (500 U/ml) for 2 h in the lower panel (Expt 2). The luciferase activity values were normalized with β-galactosidase activity and are expressed as mean 7SD of n ¼3.

Fig. 5. Effect of NFκB inhibition on HLA-F gene expression. AV-3 cells that were transduced with control non-targeting lentivirus (cShrna) or Shrna1 targeting lentivirus particle clone were infected with JEV (JEV) at MOI 1 or treated with 500 U/ml TNF-α (TNF) for different periods of time as indicated. Cells were harvested and used for extraction of RNA and analysis of HLA-F gene expression by real time quantification. cShrna and Shrna1 transduced cells that were mock treated for 24 h were taken as controls and data were calculated as fold change over control Shrna RNA 7 SD of triplicates. Mock treated Shrna1 controls did not differ significantly from cShrna controls. Data represent mean fold change over control RNA 7 SD (**Po 0.001).

Shrna1 knockdown cells pointing to an involvement of NFκB in its induction (Fig. 5). Locus-specific variation in the binding of NFκB and other transcriptional factors to the κB1 and κB2 sites present in the enhancer A region of the HLA-F gene controls its expression (Gobin et al., 1998). Differential binding of the enhancer A elements by transcriptional factors and transactivation regulates MHC Class I gene expression in different cell types following cytokine exposure (Gobin and van den Elsen, 2000). Hence we checked if these two sites of the HLA-F enhancer A would mediate transactivation following TNF-α mediated NFκB activation. HEK293T cells were transfected with luciferase reporter plasmids driven by the enhancer A elements of HLA-F or HLA-A as given in Materials and methods. Luciferase activity was measured after treatment with TNF-α, IL-1β and IFNβ. As shown in Fig. 6 (Expt 1, top panel), TNF-α treatment of these transiently transfected HEK293T cells significantly increased the luciferase activity in a time dependent manner relative to the control plasmid. Similarly, IL-1β and IFNβ also resulted in increased luciferase activity after treatment for 3 h (Fig. 6, Expt 2, bottom panel). Enhancer A of HLA-A (enh A) served as the positive control since it has been shown that it would transactivate luciferase expression in Tera cells when co-transfected with p50 and p65 plasmid constructs (Gobin et al., 1998). Cell surface expression of HLA-F on JEV infected cells Cell surface expression of HLA-F has not been observed although present intracellularly in a number of cell lines (Lepin et al.,

2000; Wainwright et al., 2000). It has so far been reported to be expressed only on the cell surface of placental trophoblast cells (Ishitani et al., 2006), EBV transformed lymphoblastoid cells and some monocytic cell lines (Lee and Geraghty, 2003) as well as activated T lymphocytes (Goodridge et al., 2010; Lee et al., 2010). Hence it was of interest to check if JEV infection could upregulate cell surface HLA-F expression. As shown in Fig. 7, cell surface expression of HLA-F antigen increased only on HBMEC from 2.8% in uninfected cells to 18.4% in infected cells at 36 h after infection. No change was observed on AV-3 cells. Cell surface expression of total HLA (-A, -B and -C) antigens was also analyzed using the pan HLA-specific W6/32 antibody. Both cell types constitutively expressed total HLA on the surface which did not increase upon JEV infection confirming our earlier report (Shwetank et al., 2013).

Discussion HLA-F and HLA-G is expressed on mid-trimester placental cells and extravillous trophoblasts (Ishitani et al., 2006). Little is known regarding the role of HLA-F in these tissues. We have examined the status of HLA-F gene expression in human amniotic and endothelial cell lines since these cells are components of barrier systems (Kobayashi et al., 2010; Stamatovic et al., 2008). JEV has been shown to cross these barriers as shown by the occurrence of human transplacental infection by JEV (Chaturvedi et al., 1980). Intrauterine infection by JEV (Mathur et al., 1982) and West Nile virus has also been reported (Julander et al., 2006). Also, the effects of JEV infection on embryonic cell types per se have not been studied in detail. After our initial studies with AV-3, WISH, ECV and HBMECs, p65NFκB knockdown was performed with AV-3 and HBMECs due to the relatively better stability obtained in their TurboGFP expression following lentiviral infection. JEV was able to establish productive infection in AV-3 and WISH as well as ECV and HBMECs as judged by the expression of the NS3 non-structural protein and the flaviviral group specific antigen (Fig. 1 and S1) as well as release of fresh viral PFU. EMSA studies also showed that JEV infection activated NFκB in all the cell lines tested. Interestingly, only the κB1 oligo complex was observed with the nuclear extracts of infected cells. The κB2 oligo failed to show

G. Kumar et al. / Virology 471-473 (2014) 29–37

33

Fig. 7. JEV infection and cell surface expression of HLA antigens. As labeled, AV-3 and HBMEC were stained for the cell surface expression of HLA-F at 12 (Panels A, E), 24 (Panels B, F), and 36 h (Panels C, G), and total HLA class I (Panels D, H) at 36 h after JEV infection. Filled histograms represent cells alone, solid lines represent staining with control secondary (Panels A, B, C, E, F, G) or isotype (Panels D, H) antibody while dotted lines marked by arrows represent antigen-specific staining. Insets represent staining of uninfected cells. Numbers represent the percent antigen-specific positive cells (dotted lines) obtained in the marked M1 marker region after substracting the percent positive cells for control/isotype antibody (solid lines).

activation by EMSA studies in infected cells (Fig. 2). However, both the HLA-F gene and the protein were induced following JEV infection reflecting the possibility that the flanking sequences of the enhancer A region were helping in transactivation of the HLA-F gene as reported earlier (Gobin and van den Elsen, 2000). This conclusion is supported by our finding that Shrna1 knockdown failed to completely inhibit the expression of the HLA-F gene (Fig. 5) and intracellular protein in AV-3 cells (Fig. 4). Similarly, the effect of the NFκB inhibitor, Bay was incomplete in these cells suggesting that other pathways and cytokine factors could be controlling HLAF expression in infected cells. A separate binding site for IRF-1, for example has been shown to be involved in HLA-gene induction by IFN-γ (Gobin et al., 1999). Our study is the first to report that JEV induced NFκB activation can actually lead to HLA-F gene induction, not only in amniotic cells but also in endothelial cells. This effect is possibly mediated via TNF-α secretion since this cytokine has been shown to increase in JEV infections (Chen et al., 2011; Ravi et al., 1997). Although lower than JEV-infected cells, TNF-α induced HLA-F gene expression was also inhibited albeit partially by two NFκB inhibitors (Table 2) as well as by Shrna1 knockdown experiments (Fig. 5). Taken together, these experiments clearly support the involvement of NFκB in HLA-F induction. Our plasmid reporter assays showed that TNF-α treatment of transfected HEK293T cells was also able to transactivate the luciferase gene that was placed under the control of the enhancer A elements of the HLA-F gene (Fig. 6). The κB1 site in enh F of the pGL3-basic construct does not successfully transactivate the HLA-F gene although bound by NFκB (Gobin and van den Elsen, 2000). These studies were done in Tera cells that were cotransfected with p50 and p65 plasmid constructs along with enh F containing luciferase reporter plasmids. However, we observed that enh F was functional in our reporter assays using HEK293T cells that were treated with different cytokines and all these assays were validated using the enh A pGL3-basic construct. The reason for this observation is yet unclear but it is possible that additional

cytokine-induced transcriptional factors may bind κB1 and/or flanking sequences of enh A in HEK293T cells that would help in the transactivation. This possibility is supported by our observation that in addition to TNF-α, two other cytokines - IL-β and IFN-β also were also functional in transactivating the luciferase reporter gene. Both these cytokine genes are induced in infected cells (Shwetank et al., 2013 and data not shown). The increase in cell surface expression of HLA-F was modest and was observed only on a fraction of HBMEC cells (Fig. 7) although JEV induced HLA-F gene expression in AV-3 and HBMEC cells (Table 1). Similarly, TNF-α treatment did not result in upregulation of cell surface HLA-F expression (data not shown) although the gene was induced in AV-3 and HBMEC cells (Table 2). Significant changes in HLA-F cell surface expression were also not observed on ECV cells (data not shown). The reason for this anomaly is unclear at this time but it must be mentioned that cell surface expression of HLA-F has not been observed although present intracellularly in a number of cell lines (Lepin et al., 2000; Wainwright et al., 2000). It has so far been reported to be expressed only on the cell surface of placental trophoblast cells (Ishitani et al., 2006), EBV transformed lymphoblastoid cells and some monocytic cell lines (Lee and Geraghty, 2003) as well as activated T lymphocytes (Goodridge et al., 2010; Lee et al., 2010). HLA-F tetramers have been reported to bind to inhibitory NK receptors in vitro suggesting a possible role for cell surface HLA-F induction in immune evasion (Goodridge et al., 2013). However, the significance of intracellular increase of HLA-F that was observed upon JEV infection in amniotic cells is still unclear. It is possible that these molecules could play a similar immune-evasive role upon release from infected cells either as a consequence of apoptosis or shedding. JEV has also been shown to cause metalloproteinase dependent shedding of sHLA-E that has the ability to inhibit IL-2 induced ERK phosphorylation and proliferation in NK cell lines (Shwetank et al., 2014). Based on a detailed study on the contribution of its cytoplasmic tail in ER to golgi transport, a unique alternative

34

G. Kumar et al. / Virology 471-473 (2014) 29–37

function for intracellular HLA-F in monitoring the golgi for infections has also been proposed (Boyle et al., 2006). Further studies will help in substantiating these hypotheses in JEV infected cells. The expression of HLA-F has also been associated with poor survival in cancer patients (Zhang et al., 2013). Hence we believe that the increase in cell surface expression of HLA-F on JEV infected HBMEC as well as the intracellular HLA-F induction in amniotic cells could be significant. Overall, our observations about the induction of HLA-F by JEV and its regulation by TNF-α and NFκB activation suggest a possible role in viral disease.

Conclusions Both human amniotic and endothelial cells were infected by JEV leading to induction of the gene expression of the nonclassical MHC molecule HLA-F. Cell surface expression was induced only on human brain microvascular endothelial cells and not all cell types tested. Induction of HLA-F gene expression by JEV was dependent, at least partially on NFκB and could also be brought about by TNF-α treatment.

Materials and methods Media, antibodies, and cell lines Trizol, Bradford reagent, Coomassie Brilliant Blue stain, leupeptin, PMSF, Dulbecco's modified Eagle's medium (DMEM), sodium pyruvate and MEM vitamin solution were purchased from SigmaAldrich, India. FCS was obtained from Gibco, USA and Nu-Serum from BD Biosciences, USA. Revert Aid Moloney murine leukemia virus (M-MuLV) reverse transcriptase and Taq polymerase were obtained from MBI Fermentas, Lithuania while iQ SYBR Green Supermix 2  was purchased from Bio-Rad, USA. Anti HLA-F mouse monoclonal antibody, 3D11 for Western blotting and cell surface staining was a kind gift from Dr. Geraghty, Fred Hutchinson Cancer Research Center, Seattle, USA. Goat anti-mouse IgG (Fcγ chainspecific) secondary antibody conjugated to FITC was from Jackson Immunoresearch Labs, USA and goat anti rabbit IgG (HþL) conjugated to Alexa Fluor 488 was obtained from Invitrogen, USA. The Flavivirus mouse monoclonal antibody, HB112 was used either as culture supernatants or purified antibody. Anti-p65 NFκB rabbit monoclonal antibody was obtained from Cell Signaling Technology, USA. Rabbit anti β-tubulin IgG for Western blotting was obtained from Imgenex, India. Goat anti rabbit IgG-HRP conjugate was from Bangalore Genei, India. Purified human TNF-α was obtained from Peprotech, Israel The human amniotic epithelial cell line, AV-3 was obtained from NCCS, Pune while WISH was a gift from Dr. Ajit Kumar, Indian Institute of Science and BeWo, a HLA-F negative cell line was obtained from Dr A.J. Rao, Indian Institute of Science. They were grown in DMEM containing 10% FCS. Human Brain Microvascular Endothelial cells (HBMEC) transfected with a plasmid containing the SV40 Large T antigen were cultured in DMEM containing 10% FCS, 10% Nu-Serum, glutamine, non-essential aminoacids, sodium pyruvate and MEM vitamin solution (Stins et al., 2001; Takahashi et al., 1990). ECV304 (ECV) cell line was a kind gift from Dr. M. Jaggi, Dabar Research Center, Ghaziabad, India. ECV is a human bladder carcinoma-derived epithelial cell line that is used as an endothelial cell model (Brown et al., 2000; Wilhelm et al., 2011). ECV cells were cultured in DMEM containing 10% FCS. PS (porcine kidney) cells were grown in minimum essential medium (MEM) supplemented with 10% FCS and used for virus titrations, while C6/36 mosquito cells were grown in MEM supplemented with 10% FCS and 10%

tryptose phosphate broth and used for virus collection. HEK293T cells were grown in 10% DMEM. Virus and infection of cells JEV strain P20778, was propagated on a confluent layer of C6/36 cells that were grown to confluency at 28 1C. Culture supernatants from C6/36 cells infected with JEV were collected after every 8 h, from 24 to 80 h and stored at  70 1C until they were used for infection of cell lines. Virus titers were evaluated by plaque forming unit (PFU) assays on porcine kidney (PS) cells (Abraham et al., 2010). Cells were infected with virus at MOI of 10 in DMEM containing 1.5% FCS. After 2 h of infection, cells were washed once with incomplete medium and replenished with DMEM containing 2.5% FCS. Thereafter, cells were harvested at different times postinfection (p.i.) and used for RNA extraction, Western blotting, nuclear extract preparation and flow cytometric (FACS) analysis. Control cells were similarly mock infected with uninfected C6/36 cell culture supernatant. For NFκB inhibitor experiments, JEV-infected cells (7.5  105) were cultured in the presence of 10 μm and 15 μm of Bay 11-7085 (Sigma, India) for the times mentioned. All cells were pretreated with the inhibitor for 1 h before JEV infection. DMSO solvent controls were also included which did not differ significantly from untreated controls. In the case of TNF-α treatment, AV-3 and HBMEC cells were treated with a peptide inhibitor of NF-κBp65 (Ser276) (DRQIKIWFQNRRMKWKKQLRRPSDRELSE, Imgenex, USA) for 24 h along with 300 IU of TNF-α (Takada et al., 2004). Controls included the addition of control peptide with only the protein transducing domain (DRQIKIWFQNRRMKWKKQ) along with TNF-α. Plasmids SMARTvector 2.0 hCMV-turboGFP Lentiviral Human RELA shRNA particles were obtained from Thermo Fisher Scientific, USA. The three individual microRNA sequences targeted were ATCAGTCAGCGCATCCAGA, GGATTGAGGAGAAACGTAA and GCTATAACTCGCCTAGTGA. SMARTvector 2.0 Non-targeting hCMV-turboGFP Lentiviral particles were used as controls. Enhancer A-containing HLA-F (enh F) and HLA-A (enh A) plasmid reporter constructs that were cloned upstream of the firefly luciferase gene in pGL3-Basic (a kind gift from Dr. Peter J van den Elsen) were used in transient transfection assays. These contained both κB1 and κB2 motifs of the enhancer A sequence of either HLA-F or HLA-A genes and were cloned upstream of a 143bp HLA-An0201 promoter fragment in pGL3-A140 (Gobin et al., 1999). For the purpose of transfecting HLA-F negative BeWo cells, the HLA-F gene was amplified from HLA-F-pCMV6 sport vector (Open Biosystem, USA) with the Forward primer 50 -CGCGGATCCGCCACCATGGCGCCCCGAAGCCTC-30 and reverse primer 50 -GCTCCGGAATTCTCACACTGCAGCCTGAGAGTAGCTC-30 carrying BamHI and EcoRI Restriction sites using Q5 High-fidelity DNA polymerase (NEB, USA). The PCR product was digested with BamHI and EcoRI and cloned in pcDNA 3.1 to generate HLA-F pcDNA 3.1 which was used for transfection. Semiquantitative reverse transcriptase (RT PCR) analysis Total RNA was isolated from control and infected cells at different times p.i., using Trizol (Sigma-Aldrich, India) reagent according to the manufacturer0 s instructions. First strand cDNA was made from 2 μg of RNA using oligo dT primers and Revert Aid™ M-MuLV Reverse Transcriptase from MBI Fermentas, Canada. PCR was performed with different dilutions of cDNA using Taq-DNA polymerase

G. Kumar et al. / Virology 471-473 (2014) 29–37

and gene-specific primers in a total reaction volume of 50 μl using a Peltier thermal cycler from Bio-Rad, USA. PCR was performed for 35 cycles in the case of HLA-F and 23 cycles for β-actin. PCR cycles comprised of 95 1C for 4 min, 94 1C for 1 min, 60 1C for 1 min, 72 1C for 1 min and final extension at 72 1C for 10 min. Amplified PCR products were separated by agarose (2%) gel electrophoresis. The gene specific primers used for semi-quantitative PCR were HLA-F, 50 -TATTGGGAGTGGACCACAGGGTAC-30 and 50 -GGCACAAGTGCAATTCTGCTAC-30 ; β-Actin, 50 -ATCTGGCACCACACCTTCTACAATGAGCTGCG-30 and 5-CGTCATACTCCTGCTTGCTGATCCACATCTGC-30 . Several standardization experiments were performed to ensure the semiquantitative nature of the results obtained. Quantititative real time PCR analysis The real-time PCR for the relative quantification of genes was performed using a Bio-Rad iQ5 system (Bio-Rad, USA) and cDNA prepared from 2 mg of Trizol extracted RNA using random primers or oligo dT primers. Real-time PCR mixtures contained 10 ml of 2  iQ SYBR Green Supermix (Bio-Rad, USA), 0.75 ml of each primer (10 mM), 5 ml of 1:50 diluted cDNA and water in a final volume of 20 ml. The thermal cycling conditions were: 50 1C for 2 min and 95 1C for 2 min, followed by 45 cycles of 95 1C for 15 s and 60 1C for 1 min. The final stage consisted of 95 1C, 60 1C and 95 1C for 15 s each to check the melting curve for the formed amplicons and the specificity of the primers. ΔCt values for all genes were obtained using the Bio-Rad iQ5 Software from which relative fold ΔΔ change was determined using the threshold cycle (2  Ct) method where β-actin was used as a reference gene for normalization of the ΔCt values of target genes. The gene-specific primers used were: HLA-F, 50 -ATCGTTGCTGGCCTTGTTGTCCTT-30 and 50 GGCACAAGTGGAATTCTGCTAC-30 ; β-actin, 50 -CAAGATCATTGCTCCTCCTG-30 and 50 -AGGGTGTAACGCAACTAAGTCAT-30 . Data was analyzed by one-way ANOVA with Dunnett's post-test using GraphPad Version 5.00 and represented as mean fold change 7SD of three independent PCR analyses. EMSA 3  106 cells were harvested, washed twice with PBS and the cell pellet was resuspended in ice cold Buffer A (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) by gentle pipetting. The cells were allowed to swell on ice for 30 min, after which 10% solution of NP-40 was added and the tube was vigorously vortexed. The solubilised suspension was centrifuged at 4 1C for 2 min at 12,000 rpm in a microfuge and the nuclear pellet was resuspended in ice-cold buffer B (20 mM HEPES pH 7.9, 0.4 M NaCl, 10 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) containing 10 μg/ml leupeptin and aprotinin. The tubes were vigorously rocked at 4 1C for 30 min on a rocker. The nuclear extracts were centrifuged and the supernatant containing nuclear proteins were stored at 70 1C until analysis by Bradford assay and EMSA. Double-stranded oligonucleotides containing the HLA-F promoter NF-κB1 consensus (50 -GTTGAGAATTCCCCCTGCA-30 ) and NF-kB2 (50 GGTTGGAAGGCTCACTGCA-30 ) primer sequence (Gobin et al., 1998) were used. The oligonucleotides were end-labeled with [γ-32P] ATP using T4 polynucleotide kinase (MBI fermentas, Lithuania). EMSA was performed in a total volume of 20 μl at 4 1C. Nuclear extracts (35 μg) were equilibrated for 20 min in binding buffer (10 mM Tris– HCl, pH 8.0, 75 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.25 mM DTT) and 1 μg of poly-dI-dC (Sigma, India). The mixture was incubated with 32P-labeled oligonucleotide probe for 30 min on ice. Protein–DNA complexes were separated by electrophoresis on 5% native polyacrylamide gels followed by autoradiography for visualization in a BioImage Analyzer (FLA5100, Fuji Film, and Japan). For supershift assays, anti p65 NFκB antibody was added to nuclear

35

extracts and incubated for 30 min on ice before EMSA. Cells (1  106) were treated with 10 μM okadaic acid for 24 h as a positive control for NFκB activation. Inhibition of NFκB activation was done by treatment of cells (1  106) for 24 h with 10 μM Bay 11-7085, a reagent that inhibits IκB phosphorylation. Positive controls were performed separately to confirm the action of the inhibitors used (data not shown). Stable knockdown of p65 NFκB Stable knockdown of p65 NF-kB was performed using a set of three individual Human SMARTvector 2.0 hCMV-turboGFP Lentiviral Human RELA shRNA particles, each having an individual shRNA design targeting RELA. These particles expressed turbo-GFP and shRNA as part of a bicistronic transcript. The presence of a puromycin drug resistance marker allowed the selection of stable mammalian cell lines after transduction. Lentivirus transduction was done using incomplete DMEM containing polybrene at 10 mg/ ml. Briefly, AV-3 and HBMC cells were separately transduced with three different lentivirus particle clones each targeting different microRNA targeting sequences of RELA at MOI of 1 in a 48 well format according to the manufacturer's instructions. After 72 h of transduction cells were observed by fluoresence microscopy for turbo-GFP expression and antibiotic selection was performed using puromycin at 3 mg/ml. Cells stably expressing Turbo-GFP were sorted using MoFlow™XDP Beckman Coulter machine before use in knockdown experiments. Nontargeting lentivirus particles supplied by the company were used as controls. Transient transfection Transfection was done with 80% confluent HEK293T cells in a 12 well format using Lipofectamine 2000 (Invitrogen, USA) according to manufacturer's instructions. Briefly, DNA and lipofectamine complex were made in Opti-MEM and added drop wise on cells. DNA-lipid complexes were removed after 6 h and complete medium was added. A total of 1.5 mg DNA mix was used for transfection and contained 600 ng each of HLA-F / HLA-A enhancer A luciferase reporter plasmid, 600 ng of plasmids encoding various JEV nonstructural proteins and 300 ng pCMV-beta-gal plasmid. After 24 h of transfection, cells were lysed in reporter lysis buffer according to the manufacturer's instructions (Promega, USA). 10 mg of lysate was used to measure luciferase activity using Tecan 2000 infinite pro system, Austria, Germany. Transfection efficiency was normalized using βgalactosidase activity. All the experiments were done in triplicates and repeated at least three times. Western blot Western blotting was done by infecting the cells at MOI 10 for 12, 24 and 36 h. Mock-infected and JEV-infected cells (1  106) were harvested and washed twice with PBS, and lysed in 90 μl lysis buffer (50 mM Tris–HCl, 1% NP-40, 0.25% Sodium Deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Aprotinin, 1 mM Leupeptin, 1 mM NaF) and incubated at 4 1C for 30 min. The suspension was spun at 16,000g for 15 min. The supernatants were aliquoted and stored at  70 1C. Protein was estimated using Bradford reagent (Sigma-Aldrich, India) and 35 μg of protein was electrophoresed on 12% SDS-PAGE gels and blotted onto Millipore Immobilon-P PVDF membranes. Blocking was done for 2 h with 2.5% skimmed milk in PBS containing 0.2% tween-20. Membranes were washed with PBS containing 0.2% tween-20 after incubating overnight with optimal titers of primary rabbit anti HLA-F antibody. After incubating with secondary HRP conjugated secondary antibody for 2 h, the blot was developed using Immobilon western

36

G. Kumar et al. / Virology 471-473 (2014) 29–37

chemiluminescent HRP substrate (Millipore, India) and visualized using a Luminescent Image Analyzer (LAS 3000, Fuji Film, Japan). Flow cytometry Viable cells (0.5  106) were harvested using cell scrapers, stained and analyzed (Abraham et al., 2010) using a BD Canto II flow analyzer or FACSCalibur cytometer (Becton Dickinson, USA). Care was taken to avoid trypsinization to avoid possible cleavage of cell surface HLA-F during the harvesting procedure. Data was analyzed using WinMDI software (Version 2.8). Cell surface expression of total HLA class I was analyzed using the FITC conjugated mouse anti pan HLA class I-specific primary antibody, W6/32 and the appropriate FITC conjugated isotype antibody. Mouse anti HLA-F monoclonal antibody, 3D11 and Alexa fluor 488 conjugated goat anti rabbit IgG antibody were used as primary and secondary antibodies respectively to stain cell surface HLA-F. Control and virus infected cells were stained at different times p.i., and the data is representative of three independent experiments. For Intracellular staining of HB112, 0.5  106 cells were fixed with BD cytofix/cytoperm solution for 20 min at 4 1C. After washing twice with BD perm wash solution, tubes were incubated with primary antibody for 1 h at 4 1C. After washing twice with BD perm wash solution, they were incubated with secondary antibody for 1 h at 4 1C. Finally tubes were washed twice with BD perm wash and cells were fixed with 4% paraformaldehyde.

Acknowledgments This work was supported by a Grant from Council for Scientific and Industrial Research (CSIR), Grant number CSIR37(1436)/10/EMRII dtd 15-12-2010. Partial support was received from DBT, Government of India (Grant no: DBT/BF/PR/INS/2011-12/IISc dtd 28-092012) as a part of the “Pathogen Biology” and “DBT-IISc Partnership” Programs at the Indian Institute of Science, Bangalore. All Flow cytometry studies were performed using the BD Canto II flow analyzer, BD FACSCalibur and MoFlow™XDP Beckman Coulter machines located at the Central Instrument Facility, which also received funding from DBT. Cytometry operations were performed by Dr. Omana Joy, Dr. William Surin, Ms. Puja Pai and Mr. Santosh Kacham. Gaurav Kumar was a recipient of a fellowship from CSIR, Govt of India.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2014.09.022. References Abraham, S., Nagaraj, A.S., Basak, S., Manjunath, R., 2010. Japanese encephalitis virus utilizes the canonical pathway to activate NF-kappaB but it utilizes the type I interferon pathway to induce major histocompatibility complex class I expression in mouse embryonic fibroblasts. J. Virol. 84 (11), 5485–5493. Abraham, S., Yaddanapudi, K., Thomas, S., Damodaran, A., Ramireddy, B., Manjunath, R., 2008. Nonclassical MHC-I and Japanese encephalitis virus infection: induction of H-2Q4, H-2T23 and H-2T10. Virus Res. 133 (2), 239–249. Adinolfi, M., Akle, C.A., McColl, I., Fensom, A.H., Tansley, L., Connolly, P., Hsi, B.L., Faulk, W.P., Travers, P., Bodmer, W.F., 1982. Expression of HLA antigens, beta 2microglobulin and enzymes by human amniotic epithelial cells. Nature 295 (5847), 325–327. Bouwmeester, T., Bauch, A., Ruffner, H., Angrand, P.O., Bergamini, G., Croughton, K., Cruciat, C., Eberhard, D., Gagneur, J., Ghidelli, S., Hopf, C., Huhse, B., Mangano, R., Michon, A.M., Schirle, M., Schlegl, J., Schwab, M., Stein, M.A., Bauer, A., Casari, G., Drewes, G., Gavin, A.C., Jackson, D.B., Joberty, G., Neubauer, G., Rick, J., Kuster, B., Superti-Furga, G., 2004. A physical and functional map of the human TNF-alpha/ NF-kappa B signal transduction pathway. Nat. Cell Biol. 6 (2), 97–105.

Boyle, L.H., Gillingham, A.K., Munro, S., Trowsdale, J., 2006. Selective export of HLAF by its cytoplasmic tail. J. Immunol. 176 (11), 6464–6472. Brown, J., Reading, S.J., Jones, S., Fitchett, C.J., Howl, J., Martin, A., Longland, C.L., Michelangeli, F., Dubrova, Y.E., Brown, C.A., 2000. Critical evaluation of ECV304 as a human endothelial cell model defined by genetic analysis and functional responses: a comparison with the human bladder cancer derived epithelial cell line T24/83. Lab. Investig. 80 (1), 37–45. Chaturvedi, U.C., Mathur, A., Chandra, A., Das, S.K., Tandon, H.O., Singh, U.K., 1980. Transplacental infection with Japanese encephalitis virus. J. Infect. Dis. 141 (6), 712–715. Chen, C.J., Ou, Y.C., Chang, C.Y., Pan, H.C., Liao, S.L., Raung, S.L., Chen, S.Y., 2011. TNFalpha and IL-1beta mediate Japanese encephalitis virus-induced RANTES gene expression in astrocytes. Neurochem. Int. 58 (2), 234–242. Cheng, Y., King, N.J., Kesson, A.M., 2004. Major histocompatibility complex class I (MHC-I) induction by West Nile virus: involvement of 2 signaling pathways in MHC-I up-regulation. J. Infect. Dis. 189 (4), 658–668. Ghosh, D., Basu, A., 2009. Japanese encephalitis-a pathological and clinical perspective. PLoS Negl. Trop. Dis. 3 (9), e437. Gobin, S.J., Keijsers, V., van Zutphen, M., van den Elsen, P.J., 1998. The role of enhancer A in the locus-specific transactivation of classical and nonclassical HLA class I genes by nuclear factor kappa B. J. Immunol. 161 (5), 2276–2283. Gobin, S.J., van den Elsen, P.J., 2000. Transcriptional regulation of the MHC class Ib genes HLA-E, HLA-F, and HLA-G. Hum. Immunol. 61 (11), 1102–1107. Gobin, S.J., van Zutphen, M., Woltman, A.M., van den Elsen, P.J., 1999. Transactivation of classical and nonclassical HLA class I genes through the IFN-stimulated response element. J. Immunol. 163 (3), 1428–1434. Goodridge, J.P., Burian, A., Lee, N., Geraghty, D.E., 2010. HLA-F complex without peptide binds to MHC class I protein in the open conformer form. J. Immunol. 184 (11), 6199–6208. Goodridge, J.P., Burian, A., Lee, N., Geraghty, D.E., 2013. HLA-F and MHC class I open conformers are ligands for NK cell Ig-like receptors. J. Immunol. 191 (7), 3553–3562. Halstead, S.B., Jacobson, J., 2003. Japanese encephalitis. Adv. Virus Res. 61, 103–138. Heinz, F.X., Allison, S.L., 2000. Structures and mechanisms in flavivirus fusion. Adv. Virus Res. 55, 231–269. Huang, S., Robinson, J.B., Deguzman, A., Bucana, C.D., Fidler, I.J., 2000. Blockade of nuclear factor-kappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res. 60 (19), 5334–5339. Ishitani, A., Sageshima, N., Hatake, K., 2006. The involvement of HLA-E and -F in pregnancy. J. Reprod. Immunol. 69 (2), 101–113. Julander, J.G., Winger, Q.A., Rickords, L.F., Shi, P.Y., Tilgner, M., Binduga-Gajewska, I., Sidwell, R.W., Morrey, J.D., 2006. West Nile virus infection of the placenta. Virology 347 (1), 175–182. Kobayashi, K., Kadohira, I., Tanaka, M., Yoshimura, Y., Ikeda, K., Yasui, M., 2010. Expression and distribution of tight junction proteins in human amnion during late pregnancy. Placenta 31 (2), 158–162. Kurane, I., 2002. Immune responses to Japanese encephalitis virus. Curr. Top. Microbiol. Immunol. 267, 91–103. Lee, N., Geraghty, D.E., 2003. HLA-F surface expression on B cell and monocyte cell lines is partially independent from tapasin and completely independent from TAP. J. Immunol. 171 (10), 5264–5271. Lee, N., Ishitani, A., Geraghty, D.E., 2010. HLA-F is a surface marker on activated lymphocytes. Eur. J. Immunol. 40 (8), 2308–2318. Lepin, E.J., Bastin, J.M., Allan, D.S., Roncador, G., Braud, V.M., Mason, D.Y., van der Merwe, P.A., McMichael, A.J., Bell, J.I., Powis, S.H., O’Callaghan, C.A., 2000. Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. Eur. J. Immunol. 30 (12), 3552–3561. Lindenbach, B.D., Rice, C.M., 2003. Molecular biology of flaviviruses. Adv. Virus Res. 59, 23–61. Lobigs, M., Mullbacher, A., Regner, M., 2003. MHC class I up-regulation by flaviviruses: immune interaction with unknown advantage to host or pathogen. Immunol. Cell Biol. 81 (3), 217–223. Mathur, A., Arora, K.L., Chaturvedi, U.C., 1982. Transplacental Japanese encephalitis virus (JEV) infection in mice during consecutive pregnancies. J. Gen. Virol. 59 (Pt 1), 213–217. Megret, F., Prehaud, C., Lafage, M., Moreau, P., Rouas-Freiss, N., Carosella, E.D., Lafon, M., 2007. Modulation of HLA-G and HLA-E expression in human neuronal cells after rabies virus or herpes virus simplex type 1 infections. Hum. Immunol. 68 (4), 294–302. Misra, U.K., Kalita, J., 2010. Overview: Japanese encephalitis. Prog. Neurobiol. 91 (2), 108–120. Pavan, B., Fiorini, S., Ferretti, M.E., Vesce, F., Biondi, C., 2003. WISH cells as a model for the “in vitro“ study of amnion pathophysiology. Curr. Drug Targets Immune Endocr. Metab. Disord. 3 (1), 83–92. Perkins, N.D., Gilmore, T.D., 2006. Good cop, bad cop: the different faces of NFkappaB. Cell Death Differ. 13 (5), 759–772. Potter, S., Hansen, W.R., Keelan, J.A., Mitchell, M.D., 1999. Characterization of the amnion-derived AV3 cell line for use as a model for investigation of prostaglandin-cytokine interactions in human amnion. Prostaglandins Leukot. Essent. Fatty Acids 61 (6), 373–379. Ravi, V., Parida, S., Desai, A., Chandramuki, A., Gourie-Devi, M., Grau, G.E., 1997. Correlation of tumor necrosis factor levels in the serum and cerebrospinal fluid with clinical outcome in Japanese encephalitis patients. J. Med. Virol. 51 (2), 132–136.

G. Kumar et al. / Virology 471-473 (2014) 29–37

Salonen, A., Ahola, T., Kaariainen, L., 2005. Viral RNA replication in association with cellular membranes. Curr. Top. Microbiol. Immunol. 285, 139–173. Shen, J., T-To, S.S., Schrieber, L., King, N.J., 1997. Early E-selectin, VCAM-1, ICAM-1, and late major histocompatibility complex antigen induction on human endothelial cells by flavivirus and comodulation of adhesion molecule expression by immune cytokines. J. Virol. 71 (12), 9323–9332. Shobu, T., Sageshima, N., Tokui, H., Omura, M., Saito, K., Nagatsuka, Y., Nakanishi, M., Hayashi, Y., Hatake, K., Ishitani, A., 2006. The surface expression of HLA-F on decidual trophoblasts increases from mid to term gestation. J. Reprod. Immunol. 72 (1–2), 18–32. Shwetank, Date, O.S., Carbone, E., Manjunath, R., 2014. Inhibition of ERK and proliferation in NK cell lines by soluble HLA-E released from Japanese encephalitis virus infected cells. Immunol. Lett. http://dx.doi.org/10.1016/j.imlet.2014.07.010. Shwetank, Date, O.S., Kim, K.S., Manjunath, R., 2013. Infection of human endothelial cells by Japanese encephalitis virus: increased expression and release of soluble HLA-E. PLoS One 8 (11), e79197. Stamatovic, S.M., Keep, R.F., Andjelkovic, A.V., 2008. Brain endothelial cell-cell junctions: how to “open” the blood brain barrier. Curr. Neuropharmacol. 6 (3), 179–192. Stins, M.F., Badger, J., Sik Kim, K., 2001. Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microb. Pathog. 30 (1), 19–28.

37

Takada, Y., Singh, S., Aggarwal, B.B., 2004. Identification of a p65 peptide that selectively inhibits NF-kappa B activation induced by various inflammatory stimuli and its role in down-regulation of NF-kappaB-mediated gene expression and up-regulation of apoptosis. J. Biol. Chem. 279 (15), 15096–15104. Takahashi, K., Sawasaki, Y., Hata, J., Mukai, K., Goto, T., 1990. Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell Dev. Biol. 26 (3 Pt 1), 265–274. Uchil, P.D., Satchidanandam, V., 2003. Characterization of RNA synthesis, replication mechanism, and in vitro RNA-dependent RNA polymerase activity of Japanese encephalitis virus. Virology 307 (2), 358–371. Untucht, C., Rasch, J., Fuchs, E., Rohde, M., Bergmann, S., Steinert, M., 2011. An optimized in vitro blood-brain barrier model reveals bidirectional transmigration of African trypanosome strains. Microbiology 157 (Pt 10), 2933–2941. Wainwright, S.D., Biro, P.A., Holmes, C.H., 2000. HLA-F is a predominantly empty, intracellular, TAP-associated MHC class Ib protein with a restricted expression pattern. J. Immunol. 164 (1), 319–328. Wilhelm, I., Fazakas, C., Krizbai, I.A., 2011. In vitro models of the blood–brain barrier. Acta Neurobiol. Exp. (Wars) 71 (1), 113–128. Zhang, X., Lin, A., Zhang, J.G., Bao, W.G., Xu, D.P., Ruan, Y.Y., Yan, W.H., 2013. Alteration of HLA-F and HLA I antigen expression in the tumor is associated with survival in patients with esophageal squamous cell carcinoma. Int. J. Cancer 132 (1), 82–89.