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Herpes Simplex Virus Type 1 Induces

Nuclear Accumulation of
Hyperphosphorylated Tau in
Neuronal Cells
Gema A
´
lvarez,
1
Jesu´ s Aldudo,
1,2
*
Marı ´a Alonso,
3,4
Soraya Santana,
5
and Fernando Valdivieso
1,2
1
Departamento de Biologı ´a Molecular and Centro de Biologı ´a Molecular ‘‘Severo Ochoa’’
(C.S.I.C.-U.A.M.), Madrid, Spain
2
Centro de Investigacio´n Biome´dica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),
Madrid, Spain
3
Servicio de Microbiologı ´a y Enfermedades Infecciosas, Hospital General Universitario Gregorio Maran˜o´n,
Madrid, Spain
4
CIBER Enfermedades Respiratorias-CIBERES, Spain
5
Drug Discovery Unit, NEURON BioPharma, Granada, Spain
Herpes simplex virus type 1 (HSV-1) is a neurotropic vi-
rus that remains latent in host neurons. Viral DNA repli-
cation is a highly structured process in which the redis-
tribution of nuclear proteins plays an important role.
Although tau is most widely known as a microtubule-
associated protein found in a hyperphosphorylated state
in the brains of patients with Alzheimer’s disease (AD),
this protein has also been detected at other sites such
as the nucleolus. Here, we establish that HSV-1 infection
gives rise to an increase in tau phosphorylation and that
hyperphosphorylated tau accumulates in the nucleus,
forming defined structures in HSV-1-infected neuronal
cells reminiscent of the common sites of viral DNA repli-
cation. When tau expression in human neuroblastoma
cells was specifically inhibited using an adenoviral vec-
tor expressing a short hairpin RNA to tau, viral DNA rep-
lication was not affected, indicating that tau is not
required for HSV-1 growth in neuronal cells. Given that
HSV-1 is considered a risk factor for AD, our results sug-
gest a new way in which to understand the relationships
between HSV-1 infection and the pathogenic mecha-
nisms leading to AD. V VC
2012 Wiley Periodicals, Inc.
Key words: HSV-1; phosphorylation; neurodegeneration;
infection
Herpes simplex virus type 1 (HSV-1) infection
provokes complex biochemical and morphological
changes within infected cells that culminate in the pro-
duction of new virus particles. The earliest morphologi-
cal changes occur in the nucleus and include the margin-
ation of host chromatin, the disaggregation of the nucle-
olus, and the appearance of dense intranuclear bodies
(Knipe, 1989).
HSV-1 DNA replication is a highly structured pro-
cess, in which large globular replication compartments
(VRCs) containing the viral replication and transcription
machinery are generated (de Bruyn Kops and Knipe,
1988). The formation of VRCs requires viral DNA syn-
thesis and the accumulation of viral and cellular regula-
tory proteins. The inhibition of viral DNA replication
induces the generation of small structures showing a dot-
ted distribution (known as prereplicative sites) that con-
tain several viral and cellular proteins that form VRCs
(Quinlan et al., 1984; Rice et al., 1994). The functional
homogeneity of the VRCs resembles functional com-
partments of the nucleus, such as the nucleolus and
ND10 (Monier et al., 2000). It is likely that the mecha-
nism responsible for VRC formation is similar to the
principles involved in the assembly and function of the
compartmentalized nucleus.
HSV-1 has been associated with Alzheimer’s dis-
ease (AD). Several researchers have suggested that HSV-
1 infection of the brain is a significant risk factor for this
disease, at least in the case of late-onset sporadic AD
(Itzhaki et al., 1997; Pyles, 2001; Lin et al., 2002;
G. A
´
lvarez and J. Aldudo contributed equally to this work.
Contract grant sponsor: Ministerio de Educacio´n y Ciencia; Contract
grant sponsor: Obra Social Caja Madrid; Contract grant sponsor: Comu-
nidad Auto´noma de Madrid; Contract grant sponsor: Ministerio de Sani-
dad y Consumo (Instituto de Salud Carlos III); Contract grant sponsor:
Asociacio´n de Familiares de Enfermos de Alzheimer (AFAL).
*Correspondence to: Jesu´s Aldudo, Centro de Biologı ´a molecular
‘‘Severo Ochoa,’’ Universidad Auto´noma de Madrid, C/Nicola´s Cabrera
1, 28049 Madrid, Spain. E-mail: jaldudo@cbm.uam.es
Received 20 July 2011; Revised 4 November 2011; Accepted 12
November 2011
Published online 18 January 2012 in Wiley Online Library
(wileyonlinelibrary.com). DOI: 10.1002/jnr.23003
Journal of Neuroscience Research 90:1020–1029 (2012)
' 2012 Wiley Periodicals, Inc.
Wozniak et al., 2008). Recently, the virus has been
linked to the pathological features of AD brains (Woz-
niak et al., 2007, 2008; Zambrano et al., 2008; Piacen-
tini et al., 2010; Lerchundi et al., 2011; Santana et al.,
2011). Thus, HSV-1 infection could predispose individ-
uals to increased inflammation, thereby promoting the
formation of amyloid plaques and neurofibrillary tangles
(NFT). Hyperphosphorylation of tau seems to be an
early event preceding the formation of NFT in the
brains of AD patients (Bancher et al., 1989), and it has
been hypothesized that tau hyperphosphorylation might
lead to a loss of function that could eventually result in
neuronal death (Lovestone and Reynolds, 1997). Tau is
a predominantly neuronal microtubule-associated protein
(MAP) involved in microtubule dynamics (Harada et al.,
1994). Although tau was first described as an MAP, this
protein has also been observed at other sites such as
ribosomes (Papasozomenos and Binder, 1987) and the
nucleus (Brady et al., 1995; Thurston et al., 1997; Lefeb-
vre et al., 2003). The functional significance of the ribo-
somal or nuclear location of tau is as yet unclear.
There is a growing body of evidence that HSV-1
infection is associated with AD, and given the important
alterations that infection causes in ribosomes (including
unusual phosphorylation of ribosomal proteins and asso-
ciation of viral and cellular proteins to the ribosomal
fraction; Diaz et al., 2002) and the nucleolus (including
modification of nucleolar morphology, delocalization of
nucleolar proteins, and association of several viral pro-
teins to nucleoli; Hiscox, 2002), where tau is found, we
decided to investigate the effects of HSV-1 infection on
the phosphorylation state of tau in neuronal cells. The
experiments described here show that HSV-1 infection
induces an intranuclear accumulation of hyperphos-
phorylated tau at viral replication sites.
MATERIALS AND METHODS
Materials
The cyclin-dependent kinase inhibitors GW8510 and
roscovitine, the glycogen synthase kinase-3b (GSK-3b) inhib-
itor lithium chloride, the viral entry inhibitor heparin, the
nucleic acid stain 4
0
,6-diamidino-2-phenylindole (DAPI), and
the viral DNA polymerase inhibitor phosphonoacetic acid
(PAA) were from Sigma (St. Louis, MO). The protein kinase
A inhibitor H-89 was from Calbiochem (La Jolla, CA).
Antibodies
Tau monoclonal antibodies 7.51 (Novak et al., 1991),
PHF1 (Greenberg et al., 1992), and PG5 (Jicha et al., 1999)
were used as previously described. PG5 antibody recognizes
an epitope containing phosphorylated Ser
409
, and PHF1 anti-
body recognizes an epitope containing phosphorylated Ser
396
and Ser
404
. Tau monoclonal antibodies TAU-1 (clone
PC1C6; catalog No. MAB3420) and TAU-5 (catalog No.
MAB361) were purchased from Chemicon, Millipore (Teme-
cula, CA). TAU-1 recognizes an epitope containing dephos-
phorylated Ser
195
, Ser
198
, Ser
199
, and Ser
202
. Rabbit polyclonal
anti-tau (pS409) was obtained from Invitrogen (Carlsbad, CA;
catalog No. 44-760). Mouse monoclonal antitubulin antibody
was supplied by Sigma (catalog No. T5168). Antibodies that
recognize HSV-1 were supplied by Dako (Carpinteria, CA;
rabbit polyclonal anti-HSV-1; catalog No. B0114), BD Bio-
sciences, Clontech (San Jose, CA; rabbit polyclonal anti-
VP16; catalog No. 3844-1), and Abcam (Cambridge, MA;
mouse monoclonal anti-ICP4; catalog No. ab6514).
Cell Cultures
SK-N-MC human neuroblastoma cells were obtained
from the American Type Culture Collection and grown as
monolayers in minimal Eagle’s medium supplemented with
10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine
and 50 lg ml
21
gentamicin. Vero cells were passaged in Dul-
becco’s modified Eagle medium supplemented with 5% FCS,
2 mM glutamine, and 50 lg ml
21
gentamicin. All cells were
grown at 378C in a 5% CO
2
atmosphere.
HSV-1, Infection Conditions, and Plaque Assays
The wild-type HSV-1 strain Kos 1.1 (kindly provided
by Dr. L. Carrasco, CBM, Madrid, Spain) was propagated in
Vero cells and purified as previously described (Carrascosa
et al., 1982). The titers of the purified virus and HSV-1 sam-
ples were determined by plaque assays. SK-N-MC cells,
seeded in their corresponding media without serum, were
exposed to HSV-1 at 378C for 1 hr. Mock infections were
performed using a virus-free suspension. Unbound virus was
removed, and cells were incubated in medium with 2% serum
at 378C. The multiplicity of infection (moi) was 1 or 10 pla-
que-forming units (pfu) per cell for 5 and 18 hr as indicated
for each experiment.
HSV-1 DNA Quantification
The concentration of HSV-1 DNA was quantified by
real-time quantitative PCR as previously described (Burgos
et al., 2003) using an ABI Prism 7900HT SD system (Applied
Biosystems, Foster City, CA). The quantification of human
genomic DNA was performed using an Assay-On-Demand
probe specific for the GAPDH housekeeping gene (Applied
Byosystems; item No. Hs99999905_m1). The quantification
results were expressed as viral DNA copy numbers per nano-
gram of genomic DNA. All experiments were performed in
triplicate.
Analysis of Cell Viability
Cell viability was evaluated by using the 3-(4,5-dime-
thylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT)
assay. Briefly, the M-96 plates were seeded at a rate of 30 000
cells/well and, after exposure to the different stimuli, incu-
bated with 0.5 mg ml
21
MTT for 3 hr at 378C. The MTT/
formazan released from the cells during overnight incubation
at 378C with 100 ll extraction buffer (20% sodium dodecyl
sulfate [SDS], 50% formamide adjusted to pH 4.7 with 0.02%
acetic acid and 0.025 N HCl) was determined. Optical den-
sities were measured at 570 nm using an automated model
680 (Bio-Rad, Hercules, CA) microplate reader.
HSV-1 Induces Tau Phosphorylation at VRCs 1021
Journal of Neuroscience Research
Immunoblot and Immunofluorescence Analyses
For the immunoblot assays, cells were infected with
HSV-1 in the presence or absence of heparin and PAA and
lysed in tau buffer (20 mM HEPES, pH 7.4, 100 mM NaCl,
5 mM EDTA, and 1% Triton X-100) containing protease
inhibitors (protease inhibitor cocktail; Roche) and phosphatase
inhibitors (1 mM sodium orthovanadate, 100 mM NaF, and
100 nM okadaic acid) and incubated for 30 min at 48C. This
buffer was used because it minimizes tau dephosphorylation
during cell lysis. Cell lysates were mixed with 23 Laemmli
buffer, sonicated, and heated for 5 min at 1008C. After elec-
trophoretic separation, the gels were blotted and stained with
primary antibodies. A peroxidase-coupled antibody was used
as secondary antibody. The detection method used, enhanced
chemiluminescence (Amersham Biosciences, Arlington
Heights, IL), was performed according to the manufacturer’s
instructions.
For the immunofluorescence assays, SK-N-MC cells
were grown on coverslips and infected with HSV-1 in the
presence or absence of lithium chloride (5–20 mM), H-89 (5
lM), roscovitine (25 lM), GW8510 (5 lM), heparin (10 lg
ml
21
), and PAA (400 lg ml
21
). The cells were then fixed
with paraformaldehyde (4%) and incubated with the primary
antibodies. Texas red- and FITC-labeled secondary antibodies
were used. 4
0
,6-Diamidino-2-phenylindole (DAPI) was added
10 min before the end of the procedure to visualize nuclei.
Cells were examined with a Bio-Rad confocal microradiance
microscope or a Zeiss Axiovert 135 fluorescence microscope
equipped with a 3100 oil-immersion objective (Neofluor)
and filters optimized for triple-label experiments (FITC, Texas
red, and DAPI fluorescence). Pictures were taken with a digi-
tal camera Spot RT slider (Diagnostic) using the MetaMorph
imaging system software package. Images were processed in
Adobe Photoshop CS3.
Generation of Adenoviral Vectors and Gene Silencing
Assays
The shRNA sequence targeting the tau gene (shTau)
corresponded to coding region 17–37 relative to the first nu-
cleotide of the start codon (GenBank accession No.
NM_016835). An adenoviral vector expressing shTau was
constructed by using the BLOCK-iT adenoviral RNAi
expression system (Invitrogen) according to the manufacturer’s
instructions. HEK 293A cells were then transfected with pAd-
shTau, and the supernatants containing adenoviruses express-
ing shTau (Ad-shTau) were amplified by infecting a larger
number of HEK 293A cells. Adenoviral titers were deter-
mined by plaque assays using the HEK 293A cell line. An ad-
enovirus expressing an shRNA to lamin A/C (Ad-shlamin),
included in the BLOCK-iT adenoviral RNAi expression sys-
tem, was used as a negative control (Harborth et al., 2001).
For gene silencing assays, SK-N-MC cells were transduced
with adenovirus expressing shRNA at an moi of 15 pfu per
cell for 18 hr. Seventy-two hours after transduction, cells
were infected with HSV-1 as previously described, or their
proteins were extracted for the examination of knockdown by
Western blot analysis.
RESULTS
Tau Protein Is Hyperphosphorylated in HSV-1-
Infected Neuroblastoma SK-N-MC Cells
The effect of HSV-1 infection on the phosphoryla-
tion state of tau in human neuroblastoma SK-N-MC
cells was examined by immunoblotting using the 7.51
antibody and the phosphorylation-sensitive antibodies
PG5, PHF1, and TAU-1. Immunoblots of cells infected
with HSV-1 at an moi of 10 pfu per cell for 18 hr
showed a marked increase of the immunoreactivity of
the PG5 and PHF1 antibodies, which recognize phos-
phorylated epitopes of tau. Infection did not affect over-
all tau levels, as revealed by using the 7.51 antibody,
which reacts with a phosphorylation-independent epi-
tope. Moreover, the enhanced tau phosphorylation
induced by HSV-1 was consistent with a loss of TAU-1
immunoreactivity, which recognizes a tau epitope only
when it is not phosphorylated (Fig. 1A).
To determine whether this enhancement of tau
phosphorylation took place in HSV-1-infected cells, we
went on to perform a series of immunofluorescence
double-labeling assays. SK-N-MC cells were infected at
an moi of 10 pfu per cell for 18 hr. The cells were then
fixed and visualized by fluorescence microscopy. In con-
trol cells, PG5 and PHF1 antibodies stained the cyto-
plasm weakly (Fig. 1B), whereas, in infected cells, this
pattern was much diminished, and PG5 and PHF1 label-
ing was restricted to defined structures. The increase in
the PG5 and PHF1 immunoreactivity occurred only in
HSV-1-infected cells, which were visualized by an anti-
body that recognizes several proteins of the HSV-1 par-
ticle (Fig. 1C). PG5 staining was observed in almost all
HSV-1-infected cells, whereas PHF1 immunoreactivity
was found exclusively in a group of infected cells (97%
vs. 38%, at an moi of 10 pfu per cell). To analyze the
effect of time of infection and viral dose on tau phos-
phorylation, time-course and dose-response experiments
were performed. We found that PG5 immunoreactivity
was detected as soon as 5 hr postinfection and increased
in a time-dependent manner. The increment of phos-
phorylated tau was also dependent on the viral dose
(Fig. 1D). Analysis of viral titer in the medium of
infected cells (at 18 hr postinfection; Fig. 1E) and viral
DNA levels (at earlier times of infection; Fig. 1F)
showed a strong correlation between the viral yields and
the amount of phosphorylated tau.
Because PG5 and PHF1 recognize different tau
species by Western blot, the staining patterns of PHF1
and Tau phosphorylated at Ser
409
(PG5 epitope) were
examined. The high degree of colocalization of PG5 and
PHF1 signals confirmed the accumulation of phospho-
rylated tau in the same structures (Fig. 1G). Treatment
with heparin, an inhibitor of HSV-1 entry that greatly
reduces infection, led to a decreased number of PG5-
and PHF1-positive cells, indicating that tau hyperphos-
phorylation was specifically provoked by HSV-1 infec-
tion (data not shown). These results agree with those
obtained by immunoblotting. Taken together, these
1022 A
´
lvarez et al.
Journal of Neuroscience Research
Fig. 1. HSV-1 induces accumulation of phosphorylated tau at sites of
viral DNA replication in SK-N-MC neuroblastoma cells. Cells,
mock-infected and HSV-1-infected, were analyzed using anti-tau
antibodies. A: Immunoblots of total lysates show that HSV-1 infec-
tion induces an increase in tau phosphorylation. The blot is represen-
tative of six independent experiments. As a control for equal loading,
a tubulin blot is shown. Densitometric analysis of different tau anti-
body signals is presented below the blots. The numbers represent
absolute optical density values normalized to the expression of tubu-
lin.. Immunofluorescence analysis of mock-infected (B) and HSV-1-
infected (C) cells shows that HSV-1 infection induces a different
localization of phosphorylated tau. Immunofluorescence tests using an
antibody that recognizes HSV-1 show that tau phosphorylation in
PG5 and PHF1 epitopes occurs in HSV-1-infected cells (arrows).
DAPI staining reveals that HSV-1 induces the generation of black
patches corresponding to chromatin margination. The merged images
show that the structures stained by PG5 and PHF1 antibodies occupy
the holes of marginated chromatin. D: Analysis by Western blotting
of phosphorylated tau in HSV-1-infected cells at different times and
moi. HSV-1 infection induces an increase in PG5 immunoreactivity
dependent on the time of infection and viral dose. The blot is repre-
sentative of three independent experiments. Densitometric analysis of
PG5 signals is presented below the blots. The numbers represent
absolute optical density values normalized to the expression of tubu-
lin. E: SK-N-MC cells were infected with HSV-1 at different moi.
At 18 hr postinfection, viral titers were determined by plaque assay.
F: Quantification of viral DNA by real-time quantitative PCR for
HSV-1-infected SK-N-MC cells for different times. Data are mean
6 SD for triplicate samples from one of three independent experi-
ments (E,F). G: Colocalization of phosphorylated tau species stained
by PG5 and PHF antibodies in HSV-1-infected cells. The merged
image shows that the structures stained by both antibodies colocalized
in the nucleus and occupy the holes of marginated chromatin
(arrows). H: Confocal microscopic images of HSV-1-infected cells.
Confocal analysis of SK-N-MC cells infected with HSV-1 shows
aggregation of phosphorylated tau resembling the staining pattern
generated by the protein components of VRCs. I: Colocalization of
phosphorylated tau and ICP4 in HSV-1-infected cells. The merged
image shows that the structures stained by pS409 and ICP4 antibod-
ies are colocalized in the nucleus and occupy the holes of marginated
chromatin (arrows). Scale bars 5 10 lm.
HSV-1 Induces Tau Phosphorylation at VRCs 1023
Journal of Neuroscience Research
results suggest that tau, mainly hyperphosphorylated tau,
is concentrated in particular structures. With the aim of
studying these structures, we performed confocal micro-
scopic experiments. SK-N-MC cells were infected at an
moi of 10 pfu per cell for 18 hr, fixed, and stained with
PG5 and PHF1 antibodies. PG5 and PHF1 images
revealed that hyperphosphorylated tau appeared, forming
amorphous patches of fluorescence. However, PG5
staining showed ring-like structures and more concen-
trated patches than PHF1 staining, the pattern of which
showed dotted structures (Fig. 1H). Despite these differ-
ences, both patterns were reminiscent of the distribution
of different VRCs proteins, suggesting a nuclear localiza-
tion for the structures containing hyperphosphorylated
tau.
Phosphorylated Tau Protein Shows Nuclear
Localization at Sites of Viral DNA Replication in
HSV-1-Infected SK-N-MC Cells
That the distribution of host cell DNA within the
nucleus, visualized by DAPI staining, is altered after
HSV-1 infection is a well-documented feature of chro-
matin margination (Wilcock and Lane, 1991). The
nucleolus became indistinct, and large dark patches
appeared within the DAPI staining. Hence, to confirm
that the structures defined by phosphorylated tau were
located in the nucleus, we analyzed the staining pattern
of PG5, PHF1, and 7.51 antibodies and DAPI. Figure
1C shows that the structures stained by the PG5 and
PHF1 antibodies were arranged as discrete foci within
the nuclei of HSV-1-infected cells. Furthermore, these
structures filled the large dark patches that appeared in
the nuclei of infected cells (Fig. 1C, merged). Numerous
reports have shown that dark areas in the nuclei of
infected cells coincide with the localization of several
protein components of VRCs. Moreover, the 7.51 anti-
body, which recognizes tau regardless of its phosphoryla-
tion state, revealed a nuclear localization of tau in
infected cells. This localization pattern is similar to that
defined by the PG5 and PHF1 antibodies (Fig. 1C).
Collectively, these data indicate that hyperphosphory-
lated tau accumulates within the nuclei of HSV-1-
infected cells. Finally, to demonstrate further the local-
Fig. 2. Nuclear structures of phosphorylated tau are dependent on
viral DNA replication. PAA-treated SK-N-MC cells were infected
with HSV-1 and subjected to immunoblotting and immunofluores-
cence analysis. A: In the presence of PAA, phosphorylated tau shows
a dotted staining pattern in the nuclei of HSV-1-infected cells as
revealed PG5 and PHF1 antibodies. DAPI staining indicates no chro-
matin margination in the nuclei of infected cells. Cells showing this
pattern are marked by arrows. B: Immunoblots of total lysates show
that PAA reduces PG5 immunoreactivity in HSV-1-infected cells.
The effect of heparin is also shown. As a control for equal loading, a
tubulin blot is shown. The ratio of PG5 to tubulin is presented
below the blots. The blot is representative of four independent
experiments. C: PAA treatment of HSV-1 infected cells provokes the
dispersion of ICP4 staining characteristic of prereplicative sites. DAPI
staining reveals that there is no chromatin margination in the nuclei
of infected cells treated with PAA. D: Confocal analysis employing
anti-ICP4 and pS409 antibodies. The merged image shows the low
rate of colocalization between ICP4 and phosphorylated tau in the
presence of PAA. Scale bars 5 10 lm.
1024 A
´
lvarez et al.
Journal of Neuroscience Research
ization of tau at VRCs, the staining patterns of ICP4
and tau phosphorylated at Ser
409
(PG5 epitope) were
then examined by double-immunofluorescence assays in
SK-N-MC cells infected at an moi of 10 pfu per cell for
18 hr. ICP4 is an HSV-1 immediate early protein
involved in the viral transcription and replication proc-
esses that take place at the VRCs (Smith et al., 1993).
HSV-1 infection caused the colocalization of ICP4 and
phosphorylated tau in the nucleus (Fig. 1I). Taken to-
gether, these results indicate that hyperphosphorylated
tau is localized at sites where viral DNA replication
occurs in HSV-1-infected SK-N-MC cells.
When cells are infected in the presence of PAA,
there is a redistribution of the nuclear and viral proteins
contained in the VRCs (de Bruyn Kops and Knipe,
1994). Under these conditions, VRCs are lost, and small
structures appear showing a dotted distribution, termed
prereplicative sites. These structures contain several viral
and cellular proteins that make up the VRCs (Rice
et al., 1994). We therefore proposed to analyze the dis-
tribution of hyperphosphorylated tau in SK-N-MC cells
infected with HSV-1 at an moi of 10 pfu per cell for 18
hr and treated with PAA. When viral DNA replication
was inhibited, the fluorescent nuclear patches determined
by hyperphosphorylated tau were lost, and a new, diffuse
and dotted pattern appeared (Fig. 2A). Then, immuno-
blotting experiments using PG5 antibody were under-
taken to determine whether PAA and heparin affected
the accumulation of phosphorylated tau induced by
HSV-1 in SK-N-MC cells. PAA and heparin treatments
did not affect PG5 and PHF1 immunoreactivity in
mock-infected cells. In HSV-1-infected cells, PAA led
to decreased immunoreactivity of PG5 antibody,
whereas treatment with heparin completely abolished the
PG5 immunoreactivity induced by HSV-1 infection
(Fig. 2B). Consistently with these results, the staining
pattern of ICP4 protein was changed and showed a nu-
clear distribution corresponding to the prereplicative sites
(Fig. 2C). To exclude a cross-reactivity of phosphoryl-
ated tau with ICP4, the colocalization of both proteins
was monitored by confocal microscopy in the presence
of PAA. Although both proteins showed a diffuse nu-
clear distribution in infected cells, colocalization was not
common (Fig. 2D). These data demonstrate that the
concentrated nuclear structures defined by hyperphos-
phorylated tau are dependent on viral DNA replication,
confirming that hyperphosphorylated tau is accumulated
in the VRCs.
Cyclin-Dependent Kinase Inhibitors Reverse the
Phosphorylation and Nuclear Localization of Tau
Protein Induced by HSV-1
To investigate the kinases involved in the hyper-
phosphorylation of tau induced by HSV-1, SK-N-MC
cells were treated with inhibitors of several kinases that
phosphorylate tau before infection with HSV-1. First,
we tested whether PKA was involved in this process,
because PG5 is a monoclonal antibody that recognizes
the PKA-dependent phosphorylation of Ser
409
in tau
(Jicha et al., 1999). H89, an inhibitor of PKA, did not
inhibit tau hyperphosphorylation in HSV-1-infected cells
(Fig. 3B). GSK-3b has recently been reported to be
involved in HIV-1-induced tau phosphorylation and
neurotoxicity (Dou et al., 2003). Thus, to establish
whether this kinase played a role in the phosphorylation
of tau provoked by HSV-1, we treated HSV-1-infected
cells with lithium chloride, a specific GSK-3b inhibitor,
and found no effects on the formation of nuclear struc-
tures of hyperphosphorylated tau, as revealed by staining
with the PG5 antibody (Fig. 3B). Searching for other
candidates, we evaluated the involvement of cyclin-de-
pendent kinases (cdks) in this process. Thus, treatment
with roscovitine, a commonly used cdk inhibitor that
inhibits cdk1, -2, -5, and -7 most potently, almost com-
pletely abolished the increase in tau phosphorylation at
the PG5 epitope and the nuclear distribution of hyper-
phosphorylated tau in infected SK-N-MC cells, as
revealed by immunofluorescence analysis (Fig. 3B).
DAPI staining showed that roscovitine also inhibited the
chromatin margination induced by HSV-1, indicating
that viral replication was blocked. Roscovitine has been
described as blocking HSV-1 replication as efficiently as
the viral DNA polymerase inhibitor PAA (Schang et al.,
1998). However, PAA did not completely inhibit tau
phosphorylation in HSV-1-infected SK-N-MC cells
(Fig. 2A,B), suggesting that the blockade of tau phos-
phorylation caused by roscovitine is a consequence of
inhibition of cdk activity. These results were reproduced
when HSV-1-infected SK-N-MC cells were treated
with GW8510, another potent inhibitor of cdks when
tested in vitro that inhibits cdk1, -2, -4, and -5 (Fig.
3B). All these inhibitors did not affect PG5 immunore-
activity in mock-infected cells (Fig. 3A). Identical results
were also obtained with the PHF1 antibody (data not
shown). To determine the specificity of the reported
data, the effects of kinase inhibitors on cell viability were
tested by using the MTT reduction assay. All these
inhibitors had no effect on viability of mock and HSV-
1-infected cells (Fig. 3C). Interestingly, most of infected
cells were viable 18 hr after infection at an moi of 10
pfu per cell. We worked with these infection conditions
because this viral dose guaranteed that all cells were
infected, and a maximum increase of tau phosphoryla-
tion was observed. In our cellular model, HSV-1 estab-
lishes a lytic replication cycle, resulting in cell lysis at
18–24 hr. The number of dead cells increased with
time of infection, reaching 80% at 24 hr postinfection
(Fig. 3D). These results suggest that cdks are involved in
the tau phosphorylation elicited by HSV-1 and raise the
question of the possible requirement of hyperphosphory-
lated tau for viral replication.
Inhibition of Tau Expression Does Not Affect
HSV-1 Infection in SK-N-MC Cells
Base-paired 21-nucleotide siRNAs with overhang-
ing 3
0
ends mediate efficient sequence-specific mRNA
HSV-1 Induces Tau Phosphorylation at VRCs 1025
Journal of Neuroscience Research
degradation. This tool is thought to be most useful for
the study of cellular proteins involved in viral infection
(Qin et al., 2003). In an attempt to determine whether
tau is functionally involved in the HSV-1 infection
cycle, tau expression was silenced using the adenovirus
vector Ad-shTau. When SK-N-MC cells were trans-
duced with Ad-shTau, the levels of all tau isoforms were
markedly reduced 72 hr after transduction, as revealed
by Western blotting of total cell lysates (Fig. 4A). With
the aim of verifying that accumulation of phosphorylated
tau induced by HSV-1 infection was not a cross-reactiv-
ity event, we analyzed tau levels by immunoblotting in
HSV-1-infected cells transduced with Ad-shTau. A
strong reduction (>90%) of total and phosphorylated tau
was observed in infected cells treated with Ad-shTau
(Fig. 4B). Moreover, in Ad-shTau-transduced cells
visualized by fluorescence microscopy, the nuclear local-
ization of hyperphosphorylated tau seen at the VRCs in
HSV-1-infected cells was not apparent (data not shown).
These results are consistent with the downregulation of
tau induced by Ad-shTau and confirm that phosphoryla-
tion-dependent tau antibodies do not react with phos-
phoepitopes that may be shared with viral proteins. To
examine the effect of blocking tau expression on HSV-1
growth, the expression levels of viral protein VP16 and
the infectious titer of HSV-1 were determined in SK-N-
Fig. 3. Cdk inhibitors block the hyperphosphorylation of tau protein
induced by HSV-1. SK-N-MC cells were treated with H-89, lith-
ium, roscovitine, or GW8510 and then mock infected or infected
with HSV-1 at an moi of 10 pfu per cell for 18 hr. Immunofluores-
cence studies were then performed using PG5 and anti-HSV-1 anti-
bodies. A: Exposure to the drugs did not affect the pattern of tau
phosphorylated at PG5 epitope in mock-infected SK-N-MC cells. B:
SK-N-MC cells treated with H-89 and lithium show nuclear struc-
tures stained with the PG5 antibody and chromatin margination.
However, roscovitine and GW8510 almost completely abolished the
formation of nuclear structures containing hyperphosphorylated tau
and inhibited the chromatin margination induced by HSV-1. C: Cell
viability of mock and HSV-1-infected cells subjected to 18 hr of
treatment with different kinase inhibitors was monitored by using the
MTT reduction assay. D: Cell viability of SK-N-MC cells infected
with HSV-1 at an moi of 10 pfu per cell for different times was
determined by using the MTT reduction assay. Values are expressed
relative to the optical density of untreated mock-infected cells. Data
are mean 6 SD for triplicate samples from one of three independent
experiments (C,D). Scale bars 5 10 lm.
1026 A
´
lvarez et al.
Journal of Neuroscience Research
MC cells. No differences in the amount of VP16
detected by immunoblotting in Ad-shTau-transduced
cells were observed compared with nontransduced and
Ad-shLam-transduced cells at 18 hr postinfection (Fig.
4C). At the same time postinfection, the HSV-1 titer
was measured by plaque assay, and the replication of
HSV-1 was unchanged in Ad-shTau-transduced cells
(Fig. 4D). Taken together, these data indicate that the
absence of tau had no effect on viral growth.
DISCUSSION
Here we show that HSV-1 is capable of intensely
modifying the phosphorylation state of tau, an MAP
involved in the pathogenesis of AD and other neurode-
generative disorders (Avila et al., 2002) and that infec-
tion with this virus leads to the accumulation of phos-
phorylated tau at replication sites in the nucleus. This
accumulation is dependent on the time of infection and
viral dose. The phosphorylation state of three different
phosphorylation-sensitive epitopes of tau protein (PHF1,
PG5, and TAU-1) were examined, all of which are
characteristic of the PHFs present in the brains of
patients with AD. Interestingly, two recent reports indi-
cate that HSV-1 triggers phosphorylation of PHF1 and
TAU-1 epitopes in neural cell models (Zambrano et al.,
2008; Wozniak et al., 2009). To rule out cross-reactivity
of phosphorylated tau with a viral protein, the presence
of nuclear structures stained by tau antibodies was moni-
tored in the absence of tau. Inhibiting the expression of
tau resulted in a dramatic reduction of this staining pat-
tern, without affecting the expression level of viral pro-
teins. Thus, tau knockdown experiments demonstrated
that these nuclear structures are composed of phospho-
rylated tau. Tau protein was first discovered as an MAP,
but it has also been detected in the ribosomes and nu-
cleus, specifically in the nucleolar regions of dividing
cells (Thurston et al., 1997). Although the presence of
tau in the nuclei of neurons has been reported (Brady
et al., 1995; Sultan et al., 2011), the function of neuro-
nal nuclear tau remains unclear. In our cell model,
HSV-1 infection provokes a specific increase in hyper-
phosphorylated tau in clearly defined nuclear regions
corresponding to the compartments where replication
and transcription of viral DNA takes place. This selective
distribution of hyperphosphorylated tau strongly suggests
a functional role for the protein in the nucleus of HSV-
1-infected cells. Tau hyperphosphorylation induced by
HSV-1 may thus determine the recruitment of tau to
the nucleus or may alter tau activity to promote the viral
replication/transcription processes in neuronal cells, with
the consequence of neurodegeneration. The present
results regarding Ad-shTau-transduced neuronal cells
indicate that tau is not essential for neuronal infection by
HSV-1 in vitro. This might be because redundant pro-
tein functions present in the VRCs may complement
the tau deficiency, thus masking any phenotype. Consis-
tently with this hypothesis, several proteins localized in
VRCs are not absolutely required for HSV-1 growth
(Taylor and Knipe, 2004). Alternatively, cellular proteins
may be targeted to damaged viral DNA that arises dur-
ing replication (Mohni et al., 2010). In this respect, a
role for tau in neuronal DNA protection has been
recently reported. Moreover, the tau capacity to protect
neurons from DNA damage was correlated with an
increased binding of tau with genomic DNA (Sultan
et al., 2011). However, it cannot be excluded that tau
plays an active role in the HSV-1 infection of the brain.
Murine models of HSV-1 infection using TAU knock-
out mice could be useful for evaluating the role of tau
in in vivo infections.
Understanding the role of tau hyperphosphoryla-
tion in HSV-1-infected cells requires the identification
of the kinase(s) responsible. Numerous pieces of evi-
dence indicate that PKA, GSK-3b, and cdks are
involved in tau hyperphosphorylation associated with
AD. Moreover, a recent report shows that HSV-1 causes
Fig. 4. Inhibition of tau expression had no effect on viral growth.
SK-N-MC cells were transduced with Ad-shLam and Ad-shTau
(moi of 15 pfu per cell) for 72 hr before infection with HSV-1 at an
moi of 10 pfu per cell. A: Ad-shTau led to a marked decrease in tau
expression in mock-infected cells. Tau expression was analyzed by
immunoblotting with the 7.51 antibody. As a control for equal load-
ing, a tubulin blot is shown. B: Ad-shTau induced a strong decrease
of total tau and phosphorylated tau at PG5 and PHF1 epitopes in
HSV-1-infected cells. Tau levels were monitored by immunoblotting
using PG5, PHF, and Tau5 antibodies. A tubulin blot is shown as a
control for equal loading. C: The accumulation of viral protein
VP16 was analyzed by immunoblotting. Ad-shTau does not affect
VP16 levels in HSV-1-infected cells. The blot shown is representa-
tive of four independent experiments. As a control for equal loading,
a tubulin blot is also provided. D: Viral titers, determined by plaque
assay, showed that Ad-shTau had no effect on virus replication. Data
are mean 6 SD for triplicate samples from one of four independent
experiments.
HSV-1 Induces Tau Phosphorylation at VRCs 1027
Journal of Neuroscience Research
tau phosphorylation in several epitopes and PKA and
GSK-3b kinases could be involved in these phosphoryla-
tion events (Wozniak et al., 2009). However, in our
model, specific inhibitors of GSK-3b and PKA have no
effect on HSV-1-induced tau phosphorylation. In con-
trast, roscovitine and GW8510, two inhibitors of several
cdks, are able to reverse the phosphorylation and nuclear
localization of tau induced by HSV-1 and to inhibit viral
replication in neuronal cells. It has been reported that
roscovitine inhibits HSV-1 replication by targeting cell
proteins (Schang et al., 2002). However, the blockade of
tau phosphorylation by roscovitine is not secondary to the
inhibition of viral DNA replication but a direct conse-
quence of the abolishing of cdk activity, because tau phos-
phorylation induced by HSV-1 is not fully prevented by
PAA. In addition, cdc-2 and the neuronal cdc-2-like ki-
nase cdk5 are able to phosphorylate tau (Delobel et al.,
2002; Hamdane et al., 2003). Deregulation of cdk5 pro-
motes neurodegeneration and may contribute to the
pathogenesis of AD (Patrick et al., 1999; Cruz et al., 2003;
Noble et al., 2003). Importantly, a recent report has shown
that GW8510 does not inhibit mitotic cdks in intact cells
(Johnson et al., 2005). In this setting, it is tempting to
speculate that HSV-1 could contribute to the pathogenesis
of AD through the deregulation of cdk activity, especially
cdk5. We are presently engaged in studies designed to
identify the relevant kinase(s) responsible for tau hyper-
phosphorylation in HSV-1-infected neuronal cells.
Mounting evidence suggests that HSV-1 might be
involved in the pathogenesis of AD. It has been reported
that HSV-1 infection is an important risk factor for AD in
carriers of the apoE-e4 allele of the APOE gene (Itzhaki
et al., 1997). Previous work by our team has shown that
human apoE4 accelerates HSV-1 infection in the brain
(Burgos et al., 2003) and that HSV-1 infection induces an
intense increase in intracellular amyloid-b peptide, which
accumulates in autophagosomes (Santana et al., 2011).
Recent data link HSV-1 infection directly to the aberrant
brain features of AD (for review see Wozniak and Itzhaki,
2010). Furthermore, several lines of evidence supporting
the infectious hypothesis of AD have appeared in the last
2 years: HSV-1 deeply interferes with APP processing in
neuronal cells, resulting in the accumulation of amyloid-b
peptides and other neurotoxic fragments (De Chiara
et al., 2010), and HSV-1 particles interact with APP,
enhancing viral transport and disrupting APP cell distribu-
tion and processing (Cheng et al., 2011). Moreover, epi-
demiological studies have shown that a set of AD-linked
gene variants may predispose to an increased susceptibility
for HSV-1 and other viral infections of the brain (Porcel-
lini et al., 2010) and that HSV reactivation was highly
correlated with incident AD (Letenneur et al., 2008).
Taken together, these findings suggest that brain infection
by HSV-1 may trigger a cascade of events, including tau
hyperphosphorylation and amyloid-b peptide accumula-
tion, which could contribute to the massive neurodegen-
eration characteristic of AD.
In summary, the present findings indicate that the
phosphorylation state and the intracellular distribution of
tau are strongly affected in HSV-1-infected neuronal
cells. Tau hyperphosphorylation induced by HSV-1 may
therefore contribute to the neuronal death observed in
neurodegenerative processes linked to HSV-1 infection.
ACKNOWLEDGMENTS
The institutional grant awarded by the Fundacio´n
Ramo´n Areces to the Centro de Biologı ´a Molecular
Severo Ochoa is gratefully acknowledged. We thank L.
Carrasco for providing the HSV-1 KOS strain, C.M.
Wischik and P. Davis for providing the anti-tau antibod-
ies, J. Avila for continuous encouragement and help, and
Isabel Sastre for technical assistance.
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