Herpes Simplex Virus Type 1 InducesNuclear Accumulation ofHyperphosphorylated Tau inNeuronal Cells

Gema Alvarez,1 Jesus Aldudo,1,2* Marıa Alonso,3,4 Soraya Santana,5

and Fernando Valdivieso1,2

1Departamento de Biologıa Molecular and Centro de Biologıa Molecular ‘‘Severo Ochoa’’(C.S.I.C.-U.A.M.), Madrid, Spain2Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED),Madrid, Spain3Servicio de Microbiologıa y Enfermedades Infecciosas, Hospital General Universitario Gregorio Maranon,Madrid, Spain4CIBER Enfermedades Respiratorias-CIBERES, Spain5Drug 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 statein the brains of patients with Alzheimer’s disease (AD),this protein has also been detected at other sites suchas the nucleolus. Here, we establish that HSV-1 infectiongives rise to an increase in tau phosphorylation and thathyperphosphorylated tau accumulates in the nucleus,forming defined structures in HSV-1-infected neuronalcells reminiscent of the common sites of viral DNA repli-cation. When tau expression in human neuroblastomacells 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 notrequired for HSV-1 growth in neuronal cells. Given thatHSV-1 is considered a risk factor for AD, our results sug-gest a new way in which to understand the relationshipsbetween HSV-1 infection and the pathogenic mecha-nisms leading to AD. VVC 2012 Wiley Periodicals, Inc.

Key words: HSV-1; phosphorylation; neurodegeneration;infection

Herpes simplex virus type 1 (HSV-1) infectionprovokes complex biochemical and morphologicalchanges 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 transcriptionmachinery 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 replicationinduces 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 functionalhomogeneity of the VRCs resembles functional com-partments of the nucleus, such as the nucleolus andND10 (Monier et al., 2000). It is likely that the mecha-nism responsible for VRC formation is similar to theprinciples involved in the assembly and function of thecompartmentalized 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 thisdisease, at least in the case of late-onset sporadic AD(Itzhaki et al., 1997; Pyles, 2001; Lin et al., 2002;

G. Alvarez and J. Aldudo contributed equally to this work.

Contract grant sponsor: Ministerio de Educacion y Ciencia; Contract

grant sponsor: Obra Social Caja Madrid; Contract grant sponsor: Comu-

nidad Autonoma de Madrid; Contract grant sponsor: Ministerio de Sani-

dad y Consumo (Instituto de Salud Carlos III); Contract grant sponsor:

Asociacion de Familiares de Enfermos de Alzheimer (AFAL).

*Correspondence to: Jesus Aldudo, Centro de Biologıa molecular

‘‘Severo Ochoa,’’ Universidad Autonoma de Madrid, C/Nicolas 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 beenlinked 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 theformation of amyloid plaques and neurofibrillary tangles(NFT). Hyperphosphorylation of tau seems to be anearly event preceding the formation of NFT in thebrains of AD patients (Bancher et al., 1989), and it hasbeen hypothesized that tau hyperphosphorylation mightlead to a loss of function that could eventually result inneuronal death (Lovestone and Reynolds, 1997). Tau isa predominantly neuronal microtubule-associated protein(MAP) involved in microtubule dynamics (Harada et al.,1994). Although tau was first described as an MAP, thisprotein has also been observed at other sites such asribosomes (Papasozomenos and Binder, 1987) and thenucleus (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-1infection is associated with AD, and given the importantalterations that infection causes in ribosomes (includingunusual phosphorylation of ribosomal proteins and asso-ciation of viral and cellular proteins to the ribosomalfraction; Diaz et al., 2002) and the nucleolus (includingmodification of nucleolar morphology, delocalization ofnucleolar proteins, and association of several viral pro-teins to nucleoli; Hiscox, 2002), where tau is found, wedecided to investigate the effects of HSV-1 infection onthe phosphorylation state of tau in neuronal cells. Theexperiments described here show that HSV-1 infectioninduces an intranuclear accumulation of hyperphos-phorylated tau at viral replication sites.

MATERIALS AND METHODS

Materials

The cyclin-dependent kinase inhibitors GW8510 androscovitine, the glycogen synthase kinase-3b (GSK-3b) inhib-itor lithium chloride, the viral entry inhibitor heparin, thenucleic acid stain 40,6-diamidino-2-phenylindole (DAPI), andthe viral DNA polymerase inhibitor phosphonoacetic acid(PAA) were from Sigma (St. Louis, MO). The protein kinaseA 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 recognizesan epitope containing phosphorylated Ser409, and PHF1 anti-body recognizes an epitope containing phosphorylated Ser396

and Ser404. Tau monoclonal antibodies TAU-1 (clonePC1C6; 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 Ser195, Ser198, Ser199, and Ser202. Rabbit polyclonalanti-tau (pS409) was obtained from Invitrogen (Carlsbad, CA;

catalog No. 44-760). Mouse monoclonal antitubulin antibodywas supplied by Sigma (catalog No. T5168). Antibodies thatrecognize 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 obtainedfrom the American Type Culture Collection and grown asmonolayers in minimal Eagle’s medium supplemented with10% heat-inactivated fetal calf serum (FCS), 2 mM glutamineand 50 lg ml21 gentamicin. Vero cells were passaged in Dul-becco’s modified Eagle medium supplemented with 5% FCS,2 mM glutamine, and 50 lg ml21 gentamicin. All cells weregrown at 378C in a 5% CO2 atmosphere.

HSV-1, Infection Conditions, and Plaque Assays

The wild-type HSV-1 strain Kos 1.1 (kindly providedby Dr. L. Carrasco, CBM, Madrid, Spain) was propagated inVero cells and purified as previously described (Carrascosaet 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, wereexposed to HSV-1 at 378C for 1 hr. Mock infections wereperformed using a virus-free suspension. Unbound virus wasremoved, and cells were incubated in medium with 2% serumat 378C. The multiplicity of infection (moi) was 1 or 10 pla-que-forming units (pfu) per cell for 5 and 18 hr as indicatedfor each experiment.

HSV-1 DNA Quantification

The concentration of HSV-1 DNA was quantified byreal-time quantitative PCR as previously described (Burgoset al., 2003) using an ABI Prism 7900HT SD system (AppliedBiosystems, Foster City, CA). The quantification of humangenomic DNA was performed using an Assay-On-Demandprobe specific for the GAPDH housekeeping gene (AppliedByosystems; item No. Hs99999905_m1). The quantificationresults were expressed as viral DNA copy numbers per nano-gram of genomic DNA. All experiments were performed intriplicate.

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 000cells/well and, after exposure to the different stimuli, incu-bated with 0.5 mg ml21 MTT for 3 hr at 378C. The MTT/formazan released from the cells during overnight incubationat 378C with 100 ll extraction buffer (20% sodium dodecylsulfate [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 model680 (Bio-Rad, Hercules, CA) microplate reader.

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Immunoblot and Immunofluorescence Analyses

For the immunoblot assays, cells were infected withHSV-1 in the presence or absence of heparin and PAA andlysed in tau buffer (20 mM HEPES, pH 7.4, 100 mM NaCl,5 mM EDTA, and 1% Triton X-100) containing proteaseinhibitors (protease inhibitor cocktail; Roche) and phosphataseinhibitors (1 mM sodium orthovanadate, 100 mM NaF, and100 nM okadaic acid) and incubated for 30 min at 48C. Thisbuffer was used because it minimizes tau dephosphorylationduring cell lysis. Cell lysates were mixed with 23 Laemmlibuffer, sonicated, and heated for 5 min at 1008C. After elec-trophoretic separation, the gels were blotted and stained withprimary antibodies. A peroxidase-coupled antibody was usedas secondary antibody. The detection method used, enhancedchemiluminescence (Amersham Biosciences, ArlingtonHeights, IL), was performed according to the manufacturer’sinstructions.

For the immunofluorescence assays, SK-N-MC cellswere grown on coverslips and infected with HSV-1 in thepresence or absence of lithium chloride (5–20 mM), H-89 (5lM), roscovitine (25 lM), GW8510 (5 lM), heparin (10 lgml21), and PAA (400 lg ml21). The cells were then fixedwith paraformaldehyde (4%) and incubated with the primaryantibodies. Texas red- and FITC-labeled secondary antibodieswere used. 40,6-Diamidino-2-phenylindole (DAPI) was added10 min before the end of the procedure to visualize nuclei.Cells were examined with a Bio-Rad confocal microradiancemicroscope or a Zeiss Axiovert 135 fluorescence microscopeequipped with a 3100 oil-immersion objective (Neofluor)and filters optimized for triple-label experiments (FITC, Texasred, and DAPI fluorescence). Pictures were taken with a digi-tal camera Spot RT slider (Diagnostic) using the MetaMorphimaging system software package. Images were processed inAdobe Photoshop CS3.

Generation of Adenoviral Vectors and Gene SilencingAssays

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 wasconstructed by using the BLOCK-iT adenoviral RNAiexpression system (Invitrogen) according to the manufacturer’sinstructions. HEK 293A cells were then transfected with pAd-shTau, and the supernatants containing adenoviruses express-ing shTau (Ad-shTau) were amplified by infecting a largernumber 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 transducedwith adenovirus expressing shRNA at an moi of 15 pfu percell for 18 hr. Seventy-two hours after transduction, cellswere infected with HSV-1 as previously described, or theirproteins were extracted for the examination of knockdown byWestern 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-MCcells was examined by immunoblotting using the 7.51antibody and the phosphorylation-sensitive antibodiesPG5, PHF1, and TAU-1. Immunoblots of cells infectedwith HSV-1 at an moi of 10 pfu per cell for 18 hrshowed a marked increase of the immunoreactivity ofthe 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 phosphorylationinduced by HSV-1 was consistent with a loss of TAU-1immunoreactivity, which recognizes a tau epitope onlywhen it is not phosphorylated (Fig. 1A).

To determine whether this enhancement of tauphosphorylation took place in HSV-1-infected cells, wewent on to perform a series of immunofluorescencedouble-labeling assays. SK-N-MC cells were infected atan moi of 10 pfu per cell for 18 hr. The cells were thenfixed and visualized by fluorescence microscopy. In con-trol cells, PG5 and PHF1 antibodies stained the cyto-plasm weakly (Fig. 1B), whereas, in infected cells, thispattern was much diminished, and PG5 and PHF1 label-ing was restricted to defined structures. The increase inthe PG5 and PHF1 immunoreactivity occurred only inHSV-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 allHSV-1-infected cells, whereas PHF1 immunoreactivitywas found exclusively in a group of infected cells (97%vs. 38%, at an moi of 10 pfu per cell). To analyze theeffect of time of infection and viral dose on tau phos-phorylation, time-course and dose-response experimentswere performed. We found that PG5 immunoreactivitywas detected as soon as 5 hr postinfection and increasedin 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 ofinfected cells (at 18 hr postinfection; Fig. 1E) and viralDNA levels (at earlier times of infection; Fig. 1F)showed a strong correlation between the viral yields andthe amount of phosphorylated tau.

Because PG5 and PHF1 recognize different tauspecies by Western blot, the staining patterns of PHF1and Tau phosphorylated at Ser409 (PG5 epitope) wereexamined. The high degree of colocalization of PG5 andPHF1 signals confirmed the accumulation of phospho-rylated tau in the same structures (Fig. 1G). Treatmentwith heparin, an inhibitor of HSV-1 entry that greatlyreduces 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 thoseobtained by immunoblotting. Taken together, these

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Fig. 1. HSV-1 induces accumulation of phosphorylated tau at sites ofviral DNA replication in SK-N-MC neuroblastoma cells. Cells,mock-infected and HSV-1-infected, were analyzed using anti-tauantibodies. 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 representabsolute 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 differentlocalization of phosphorylated tau. Immunofluorescence tests using anantibody that recognizes HSV-1 show that tau phosphorylation inPG5 and PHF1 epitopes occurs in HSV-1-infected cells (arrows).DAPI staining reveals that HSV-1 induces the generation of blackpatches corresponding to chromatin margination. The merged imagesshow that the structures stained by PG5 and PHF1 antibodies occupythe holes of marginated chromatin. D: Analysis by Western blottingof phosphorylated tau in HSV-1-infected cells at different times andmoi. HSV-1 infection induces an increase in PG5 immunoreactivitydependent on the time of infection and viral dose. The blot is repre-

sentative of three independent experiments. Densitometric analysis ofPG5 signals is presented below the blots. The numbers representabsolute 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 forHSV-1-infected SK-N-MC cells for different times. Data are mean6 SD for triplicate samples from one of three independent experi-ments (E,F). G: Colocalization of phosphorylated tau species stainedby PG5 and PHF antibodies in HSV-1-infected cells. The mergedimage shows that the structures stained by both antibodies colocalizedin 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 showsaggregation of phosphorylated tau resembling the staining patterngenerated by the protein components of VRCs. I: Colocalization ofphosphorylated tau and ICP4 in HSV-1-infected cells. The mergedimage shows that the structures stained by pS409 and ICP4 antibod-ies are colocalized in the nucleus and occupy the holes of marginatedchromatin (arrows). Scale bars 5 10 lm.

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results suggest that tau, mainly hyperphosphorylated tau,is concentrated in particular structures. With the aim ofstudying these structures, we performed confocal micro-scopic experiments. SK-N-MC cells were infected at anmoi of 10 pfu per cell for 18 hr, fixed, and stained withPG5 and PHF1 antibodies. PG5 and PHF1 imagesrevealed that hyperphosphorylated tau appeared, formingamorphous patches of fluorescence. However, PG5staining showed ring-like structures and more concen-trated patches than PHF1 staining, the pattern of whichshowed dotted structures (Fig. 1H). Despite these differ-ences, both patterns were reminiscent of the distributionof different VRCs proteins, suggesting a nuclear localiza-tion for the structures containing hyperphosphorylatedtau.

Phosphorylated Tau Protein Shows NuclearLocalization at Sites of Viral DNA Replication inHSV-1-Infected SK-N-MC Cells

That the distribution of host cell DNA within thenucleus, visualized by DAPI staining, is altered after

HSV-1 infection is a well-documented feature of chro-matin margination (Wilcock and Lane, 1991). Thenucleolus became indistinct, and large dark patchesappeared within the DAPI staining. Hence, to confirmthat the structures defined by phosphorylated tau werelocated in the nucleus, we analyzed the staining patternof PG5, PHF1, and 7.51 antibodies and DAPI. Figure1C shows that the structures stained by the PG5 andPHF1 antibodies were arranged as discrete foci withinthe nuclei of HSV-1-infected cells. Furthermore, thesestructures filled the large dark patches that appeared inthe nuclei of infected cells (Fig. 1C, merged). Numerousreports have shown that dark areas in the nuclei ofinfected cells coincide with the localization of severalprotein components of VRCs. Moreover, the 7.51 anti-body, which recognizes tau regardless of its phosphoryla-tion state, revealed a nuclear localization of tau ininfected cells. This localization pattern is similar to thatdefined 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 onviral DNA replication. PAA-treated SK-N-MC cells were infectedwith HSV-1 and subjected to immunoblotting and immunofluores-cence analysis. A: In the presence of PAA, phosphorylated tau showsa dotted staining pattern in the nuclei of HSV-1-infected cells asrevealed PG5 and PHF1 antibodies. DAPI staining indicates no chro-matin margination in the nuclei of infected cells. Cells showing thispattern are marked by arrows. B: Immunoblots of total lysates showthat 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 presentedbelow the blots. The blot is representative of four independentexperiments. C: PAA treatment of HSV-1 infected cells provokes thedispersion of ICP4 staining characteristic of prereplicative sites. DAPIstaining reveals that there is no chromatin margination in the nucleiof infected cells treated with PAA. D: Confocal analysis employinganti-ICP4 and pS409 antibodies. The merged image shows the lowrate of colocalization between ICP4 and phosphorylated tau in thepresence of PAA. Scale bars 5 10 lm.

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ization of tau at VRCs, the staining patterns of ICP4and tau phosphorylated at Ser409 (PG5 epitope) werethen examined by double-immunofluorescence assays inSK-N-MC cells infected at an moi of 10 pfu per cell for18 hr. ICP4 is an HSV-1 immediate early proteininvolved 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 andphosphorylated tau in the nucleus (Fig. 1I). Taken to-gether, these results indicate that hyperphosphorylatedtau is localized at sites where viral DNA replicationoccurs 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 proteinscontained in the VRCs (de Bruyn Kops and Knipe,1994). Under these conditions, VRCs are lost, and smallstructures appear showing a dotted distribution, termedprereplicative sites. These structures contain several viraland cellular proteins that make up the VRCs (Riceet al., 1994). We therefore proposed to analyze the dis-tribution of hyperphosphorylated tau in SK-N-MC cellsinfected with HSV-1 at an moi of 10 pfu per cell for 18hr and treated with PAA. When viral DNA replicationwas inhibited, the fluorescent nuclear patches determinedby hyperphosphorylated tau were lost, and a new, diffuseand dotted pattern appeared (Fig. 2A). Then, immuno-blotting experiments using PG5 antibody were under-taken to determine whether PAA and heparin affectedthe accumulation of phosphorylated tau induced byHSV-1 in SK-N-MC cells. PAA and heparin treatmentsdid not affect PG5 and PHF1 immunoreactivity inmock-infected cells. In HSV-1-infected cells, PAA ledto decreased immunoreactivity of PG5 antibody,whereas treatment with heparin completely abolished thePG5 immunoreactivity induced by HSV-1 infection(Fig. 2B). Consistently with these results, the stainingpattern 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 proteinswas monitored by confocal microscopy in the presenceof PAA. Although both proteins showed a diffuse nu-clear distribution in infected cells, colocalization was notcommon (Fig. 2D). These data demonstrate that theconcentrated nuclear structures defined by hyperphos-phorylated tau are dependent on viral DNA replication,confirming that hyperphosphorylated tau is accumulatedin the VRCs.

Cyclin-Dependent Kinase Inhibitors Reverse thePhosphorylation and Nuclear Localization of TauProtein Induced by HSV-1

To investigate the kinases involved in the hyper-phosphorylation of tau induced by HSV-1, SK-N-MCcells were treated with inhibitors of several kinases thatphosphorylate 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 Ser409 in tau(Jicha et al., 1999). H89, an inhibitor of PKA, did notinhibit tau hyperphosphorylation in HSV-1-infected cells(Fig. 3B). GSK-3b has recently been reported to beinvolved in HIV-1-induced tau phosphorylation andneurotoxicity (Dou et al., 2003). Thus, to establishwhether this kinase played a role in the phosphorylationof tau provoked by HSV-1, we treated HSV-1-infectedcells 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 stainingwith the PG5 antibody (Fig. 3B). Searching for othercandidates, we evaluated the involvement of cyclin-de-pendent kinases (cdks) in this process. Thus, treatmentwith roscovitine, a commonly used cdk inhibitor thatinhibits cdk1, -2, -5, and -7 most potently, almost com-pletely abolished the increase in tau phosphorylation atthe PG5 epitope and the nuclear distribution of hyper-phosphorylated tau in infected SK-N-MC cells, asrevealed by immunofluorescence analysis (Fig. 3B).DAPI staining showed that roscovitine also inhibited thechromatin margination induced by HSV-1, indicatingthat viral replication was blocked. Roscovitine has beendescribed as blocking HSV-1 replication as efficiently asthe viral DNA polymerase inhibitor PAA (Schang et al.,1998). However, PAA did not completely inhibit tauphosphorylation 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 ofinhibition of cdk activity. These results were reproducedwhen HSV-1-infected SK-N-MC cells were treatedwith GW8510, another potent inhibitor of cdks whentested 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 resultswere also obtained with the PHF1 antibody (data notshown). To determine the specificity of the reporteddata, the effects of kinase inhibitors on cell viability weretested by using the MTT reduction assay. All theseinhibitors had no effect on viability of mock and HSV-1-infected cells (Fig. 3C). Interestingly, most of infectedcells were viable 18 hr after infection at an moi of 10pfu per cell. We worked with these infection conditionsbecause this viral dose guaranteed that all cells wereinfected, 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 withtime of infection, reaching �80% at 24 hr postinfection(Fig. 3D). These results suggest that cdks are involved inthe tau phosphorylation elicited by HSV-1 and raise thequestion of the possible requirement of hyperphosphory-lated tau for viral replication.

Inhibition of Tau Expression Does Not AffectHSV-1 Infection in SK-N-MC Cells

Base-paired 21-nucleotide siRNAs with overhang-ing 30 ends mediate efficient sequence-specific mRNA

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degradation. This tool is thought to be most useful forthe study of cellular proteins involved in viral infection(Qin et al., 2003). In an attempt to determine whethertau is functionally involved in the HSV-1 infectioncycle, tau expression was silenced using the adenovirusvector Ad-shTau. When SK-N-MC cells were trans-duced with Ad-shTau, the levels of all tau isoforms weremarkedly reduced 72 hr after transduction, as revealedby Western blotting of total cell lysates (Fig. 4A). Withthe aim of verifying that accumulation of phosphorylatedtau induced by HSV-1 infection was not a cross-reactiv-ity event, we analyzed tau levels by immunoblotting inHSV-1-infected cells transduced with Ad-shTau. A

strong reduction (>90%) of total and phosphorylated tauwas observed in infected cells treated with Ad-shTau(Fig. 4B). Moreover, in Ad-shTau-transduced cellsvisualized by fluorescence microscopy, the nuclear local-ization of hyperphosphorylated tau seen at the VRCs inHSV-1-infected cells was not apparent (data not shown).These results are consistent with the downregulation oftau 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. Toexamine the effect of blocking tau expression on HSV-1growth, the expression levels of viral protein VP16 andthe infectious titer of HSV-1 were determined in SK-N-

Fig. 3. Cdk inhibitors block the hyperphosphorylation of tau proteininduced by HSV-1. SK-N-MC cells were treated with H-89, lith-ium, roscovitine, or GW8510 and then mock infected or infectedwith 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 tauphosphorylated 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 tauand inhibited the chromatin margination induced by HSV-1. C: Cellviability of mock and HSV-1-infected cells subjected to 18 hr oftreatment with different kinase inhibitors was monitored by using theMTT reduction assay. D: Cell viability of SK-N-MC cells infectedwith HSV-1 at an moi of 10 pfu per cell for different times wasdetermined by using the MTT reduction assay. Values are expressedrelative to the optical density of untreated mock-infected cells. Dataare mean 6 SD for triplicate samples from one of three independentexperiments (C,D). Scale bars 5 10 lm.

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MC cells. No differences in the amount of VP16detected by immunoblotting in Ad-shTau-transducedcells were observed compared with nontransduced andAd-shLam-transduced cells at 18 hr postinfection (Fig.4C). At the same time postinfection, the HSV-1 titerwas measured by plaque assay, and the replication ofHSV-1 was unchanged in Ad-shTau-transduced cells(Fig. 4D). Taken together, these data indicate that theabsence of tau had no effect on viral growth.

DISCUSSION

Here we show that HSV-1 is capable of intenselymodifying the phosphorylation state of tau, an MAPinvolved 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 andviral dose. The phosphorylation state of three differentphosphorylation-sensitive epitopes of tau protein (PHF1,PG5, and TAU-1) were examined, all of which arecharacteristic of the PHFs present in the brains ofpatients with AD. Interestingly, two recent reports indi-cate that HSV-1 triggers phosphorylation of PHF1 andTAU-1 epitopes in neural cell models (Zambrano et al.,2008; Wozniak et al., 2009). To rule out cross-reactivityof phosphorylated tau with a viral protein, the presenceof nuclear structures stained by tau antibodies was moni-tored in the absence of tau. Inhibiting the expression oftau resulted in a dramatic reduction of this staining pat-tern, without affecting the expression level of viral pro-teins. Thus, tau knockdown experiments demonstratedthat 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 dividingcells (Thurston et al., 1997). Although the presence oftau in the nuclei of neurons has been reported (Bradyet 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 regionscorresponding to the compartments where replicationand transcription of viral DNA takes place. This selectivedistribution of hyperphosphorylated tau strongly suggestsa functional role for the protein in the nucleus of HSV-1-infected cells. Tau hyperphosphorylation induced byHSV-1 may thus determine the recruitment of tau tothe nucleus or may alter tau activity to promote the viralreplication/transcription processes in neuronal cells, withthe consequence of neurodegeneration. The presentresults regarding Ad-shTau-transduced neuronal cellsindicate that tau is not essential for neuronal infection byHSV-1 in vitro. This might be because redundant pro-tein functions present in the VRCs may complementthe tau deficiency, thus masking any phenotype. Consis-tently with this hypothesis, several proteins localized inVRCs are not absolutely required for HSV-1 growth(Taylor and Knipe, 2004). Alternatively, cellular proteinsmay be targeted to damaged viral DNA that arises dur-ing replication (Mohni et al., 2010). In this respect, arole for tau in neuronal DNA protection has beenrecently reported. Moreover, the tau capacity to protectneurons from DNA damage was correlated with anincreased binding of tau with genomic DNA (Sultanet al., 2011). However, it cannot be excluded that tauplays 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 tauin in vivo infections.

Understanding the role of tau hyperphosphoryla-tion in HSV-1-infected cells requires the identificationof the kinase(s) responsible. Numerous pieces of evi-dence indicate that PKA, GSK-3b, and cdks areinvolved in tau hyperphosphorylation associated withAD. 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 anmoi of 10 pfu per cell. A: Ad-shTau led to a marked decrease in tauexpression in mock-infected cells. Tau expression was analyzed byimmunoblotting with the 7.51 antibody. As a control for equal load-ing, a tubulin blot is shown. B: Ad-shTau induced a strong decreaseof total tau and phosphorylated tau at PG5 and PHF1 epitopes inHSV-1-infected cells. Tau levels were monitored by immunoblottingusing PG5, PHF, and Tau5 antibodies. A tubulin blot is shown as acontrol for equal loading. C: The accumulation of viral proteinVP16 was analyzed by immunoblotting. Ad-shTau does not affectVP16 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 plaqueassay, showed that Ad-shTau had no effect on virus replication. Dataare mean 6 SD for triplicate samples from one of four independentexperiments.

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tau phosphorylation in several epitopes and PKA andGSK-3b kinases could be involved in these phosphoryla-tion events (Wozniak et al., 2009). However, in ourmodel, specific inhibitors of GSK-3b and PKA have noeffect on HSV-1-induced tau phosphorylation. In con-trast, roscovitine and GW8510, two inhibitors of severalcdks, are able to reverse the phosphorylation and nuclearlocalization of tau induced by HSV-1 and to inhibit viralreplication in neuronal cells. It has been reported thatroscovitine inhibits HSV-1 replication by targeting cellproteins (Schang et al., 2002). However, the blockade oftau phosphorylation by roscovitine is not secondary to theinhibition 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 byPAA. 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 thepathogenesis of AD (Patrick et al., 1999; Cruz et al., 2003;Noble et al., 2003). Importantly, a recent report has shownthat GW8510 does not inhibit mitotic cdks in intact cells(Johnson et al., 2005). In this setting, it is tempting tospeculate that HSV-1 could contribute to the pathogenesisof AD through the deregulation of cdk activity, especiallycdk5. We are presently engaged in studies designed toidentify the relevant kinase(s) responsible for tau hyper-phosphorylation in HSV-1-infected neuronal cells.

Mounting evidence suggests that HSV-1 might beinvolved in the pathogenesis of AD. It has been reportedthat HSV-1 infection is an important risk factor for AD incarriers of the apoE-e4 allele of the APOE gene (Itzhakiet al., 1997). Previous work by our team has shown thathuman apoE4 accelerates HSV-1 infection in the brain(Burgos et al., 2003) and that HSV-1 infection induces anintense increase in intracellular amyloid-b peptide, whichaccumulates in autophagosomes (Santana et al., 2011).Recent data link HSV-1 infection directly to the aberrantbrain features of AD (for review see Wozniak and Itzhaki,2010). Furthermore, several lines of evidence supportingthe infectious hypothesis of AD have appeared in the last2 years: HSV-1 deeply interferes with APP processing inneuronal cells, resulting in the accumulation of amyloid-bpeptides and other neurotoxic fragments (De Chiaraet 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-linkedgene variants may predispose to an increased susceptibilityfor HSV-1 and other viral infections of the brain (Porcel-lini et al., 2010) and that HSV reactivation was highlycorrelated with incident AD (Letenneur et al., 2008).Taken together, these findings suggest that brain infectionby HSV-1 may trigger a cascade of events, including tauhyperphosphorylation and amyloid-b peptide accumula-tion, which could contribute to the massive neurodegen-eration characteristic of AD.

In summary, the present findings indicate that thephosphorylation state and the intracellular distribution of

tau are strongly affected in HSV-1-infected neuronalcells. Tau hyperphosphorylation induced by HSV-1 maytherefore contribute to the neuronal death observed inneurodegenerative processes linked to HSV-1 infection.

ACKNOWLEDGMENTS

The institutional grant awarded by the FundacionRamon Areces to the Centro de Biologıa MolecularSevero 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, andIsabel Sastre for technical assistance.

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