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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]
Received 5 March 2017; Revised 28 August 2017; Editorial decision 28 August 2017; Accepted 30 August 2017
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
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]
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
HSV-1 specifically activate FUT3, −5 and −6 gene
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 (,
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,
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
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.
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
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
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
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
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
This work was supported by Stiftelsen Professor Lars-Erik Gelins
Conflict of interest statement
None declared.
HSV-1, herpes simplex virus type 1; IFNβ, interferon beta; IFNγ, interferon
gamma; IL1β, interleukin beta; PKR, protein kinase R; RT-qPCR, reverse
transcription quantitative polymerase chain reaction; sLex, sialyl Lewis x;
siRNA, short interfering RNA; TSA, trichostatin A.
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