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Experimental Eye Research 123 (2014) 8e15

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Experimental Eye Research

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Inhibitors of signal peptide peptidase (SPP) affect HSV-1 infectivityin vitro and in vivo

Sariah J. Allen, Kevin R. Mott, Homayon Ghiasi*

Center for Neurobiology & Vaccine Development, Ophthalmology Research, Department of Surgery, Los Angeles, CA

a r t i c l e i n f o

Article history:Received 14 February 2014Accepted in revised form 3 April 2014Available online 24 April 2014

Keywords:virus replicationglycoprotein K (gK)(Z-LL)2 ketonenucleus

* Corresponding author. Center for NeurobiologyD2066, 8700 Beverly Blvd., Los Angeles, CA 90048, U

E-mail address: ghiasih@cshs.org (H. Ghiasi).

http://dx.doi.org/10.1016/j.exer.2014.04.0040014-4835/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Recently we have shown that the highly conserved herpes simplex virus glycoprotein K (gK) binds tosignal peptide peptidase (SPP), also known as minor histocompatibility antigen H13. In this study wehave demonstrated for the first time that inhibitors of SPP, such as L685,458, (Z-LL)2 ketone, aspirin,ibuprofen and DAPT, significantly reduced HSV-1 replication in tissue culture. Inhibition of SPP activityvia (Z-LL)2 ketone significantly reduced viral transcripts in the nucleus of infected cells. Finally, whenadministered during primary infection, (Z-LL)2 ketone inhibitor reduced HSV-1 replication in the eyes ofocularly infected mice. Thus, blocking SPP activity may represent a clinically effective and expedientapproach to the reduction of viral replication and the resulting pathology.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

HSV-1 encodes at least 85 genes and 12 of these genes code forglycoproteins (Barnett et al., 1992; Ghiasi et al., 1998; McGeochet al., 1988; Spear, 2004; Spear et al., 2000), one of which isglycoprotein K (gK) (Ghiasi et al., 1994b; Hutchinson et al., 1992;McGeoch et al., 1988). gK is encoded by the UL53 open readingframe and is a highly hydrophobic 338-amino-acid protein with apredictedmolecular mass of 37-kDa (McGeoch et al., 1988). gK fromHSV-1 and HSV-2 are both 338 amino acids long with approxi-mately 84% amino acid homology (Dolan et al., 1998; McGeochet al., 1991, 1988). gK has a cleavable 30-amino-acid amino-terminal signal sequence and is N-glycosylated on amino acids 48and 58 (Debroy et al., 1985; McGeoch et al., 1988; Ramaswamy andHolland, 1992). In HSV-1-infected cells gK is expressed as a 39 kDahigh-mannose precursor polypeptide, designated precursor gK,which is further glycosylated to produce a 41 kDa mature glyco-protein (Hutchinson et al., 1992).

When we expressed gK using a recombinant baculovirus sys-tem, four gK-related baculovirus-expressed polypeptides of 29-,35-, 38- and 40-kDa were detected (Ghiasi et al., 1994b). The 35-,38-, and 40-kDa species were susceptible to tunicamycin treatmentrevealing that they were N-linked glycosylated, the 35-kDa protein

and Vaccine Development,SA. Tel.: þ1 310 248 8582.

represented the cleaved and partially glycosylated peptide, whilethe 29-kDa protein represented the cleaved unglycosylated protein.gK translated in vitro has a molecular mass of 36-kDa with three(Mo and Holland, 1997; Ramaswamy and Holland, 1992) to four(Foster et al., 2003) predicted membrane-spanning regions.

Studies using insertion/deletion mutants have shown theimportance of gK in virion morphogenesis and egress (Foster andKousoulas, 1999; Hutchinson and Johnson, 1995; Hutchinsonet al., 1995). gK is also required for virus replication (Foster andKousoulas, 1999; Hutchinson and Johnson, 1995), a concept thatis supported by the observation that gK-deficient virus can only bepropagated on complementing cells which express gK (Foster andKousoulas, 1999; Hutchinson and Johnson, 1995). Although gK isnot involved in virus attachment or penetration, it is involved invirus entry as entry substantially slower in the absence of gK(Foster and Kousoulas, 1999; Hutchinson and Johnson, 1995;Jambunathan et al., 2011). Recently we have shown that the virusreplication function of gK is dependent on signal peptide peptidase(SPP) (Allen et al., 2014). SPP, also known as minor histocompati-bility antigen H13, is a member of the intramembrane cleavingproteases family. SPP cleaves peptide bonds within the plane of thelipid bilayer (Lemberg and Martoglio, 2002; Weihofen et al., 2002)and is highly conserved between human and mouse (Golde et al.,2009). SPP localizes predominantly to the endoplasmic reticulumand exists in different forms depending on its glycosylation status(Grigorenko et al., 2002). Unlike other family members, SPP ap-pears to achieve enzyme activity in the absence of protein cofactors(Sato et al., 2006; Weihofen et al., 2002). SPP has been linked to

S.J. Allen et al. / Experimental Eye Research 123 (2014) 8e15 9

pathogenic conditions such as Alzheimer’s disease (Esler et al.,2002), certain cancers (Taniguchi et al., 2003), and HCV infection(McLauchlan et al., 2002; Okamoto et al., 2004).

Recently we have shown that SPP dominant negative mutantsand shRNA against SPP significantly reduced HSV-1 replicationin vitro (Allen et al., 2014). In addition to the use of dominantnegative mutants and shRNA (Okamoto et al., 2004), blocking theinteraction of viral protein with SPP using SPP inhibitors has beensuggested as an alternative anti-viral treatment (Dovey et al., 2001;Lanz et al., 2003; Li et al., 2000; Seiffert et al., 2000; Targett-Adamset al., 2006). Thus, in this study we used a panel of different SPPinhibitors to evaluate their potential to block or reduce HSV-1infectivity in vitro and in vivo and we have shown for the firsttime that: 1) inhibitors of SPP enzyme catalysis significantlyreduced HSV-1 replication in vitro by blocking the transcription ofviral DNA in the nucleus of infected cells; and 2) SPP is required forvirus infectivity in vivo. These results highlight the importance ofSPP in HSV-1 infectivity in vitro and in vivo. Thus, blocking SPPactivity may represent an alternative approach to the reduction ofviral replication and the resulting pathology.

2. Materials and methods

Virus, cells, and mice

Triple plaque-purified HSV-1 strain McKrae was grown in RS(rabbit skin) cell monolayers as described previously (Ghiasi et al.,1994b). RS cells were grown in minimal essential medium (MEM)plus 5% fetal calf serum (FCS). Female BALB/c and C57BL/6 mice (6-weeks of age) were purchased from The Jackson Laboratories. Allanimal procedures adhered to the Association for Research inVision and Ophthalmology (ARVO) statement for the Use of Ani-mals in Ophthalmic and Vision Research and according to institu-tional animal care and use guidelines.

SPP chemical inhibitors

There are several chemicals that have been shown to inhibit theproteolytic activity of SPP. We selected the following SPP inhibitorsfor analysis of their effects on HSV-1 replication: A) L685,458 (1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydroxy-5-phenylpentyl) carbamicAcid tert-butyl Ester) (Tocris Bioscience, Ellisville, MO); B) (Z-LL)2ketone (Calbiochem, San Diego, CA); C) DAPT (N-[(3,5-Difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester) (Tocris Bioscience, Ellisville, MO); D) Aspirin(Sigma Aldrich, Saint Louis, MO); and E) Ibuprofen (Sigma Aldrich).We tested different concentrations of each inhibitor and choseconcentrations specific for SPP inhibition which caused no toxicityin Vero, HeLa or RS cell lines as determined by tryptan blue stainingand direct observation of cytotoxicity from 0 to 48 hours (h) post-treatment. Concentrations of inhibitors used were as follows: 1 mmL685,458; 20.0 mm (Z-LL)2 ketone; 150 mm DAPT; 250 mm aspirin;and 100 mm ibuprofen. Ibuprofen was diluted in ethanol, while therest of the inhibitors were diluted in DMSO. No toxicity was re-ported with the use of these compounds in mice, rats, or tissuecultures (Dev et al., 2006; Dovey et al., 2001; Jeong et al., 2011; Liet al., 2009; Shearman et al., 2000; Weihofen et al., 2000; Zaraet al., 2013). In addition, all of the compounds that we have usedin this study have been shown to have high membrane perme-ability (Gutknecht, 1992; Kang et al., 2004; Marks and Berg, 2008;Weihofen et al., 2000).

RS cells were pre-treated with each inhibitor 2 h prior to HSV-1infection with 0.1 or 1.0 PFU/cell of HSV-1 strain McKrae for 1 h at37 �C. Cells were then washed 3 times with 1� PBS and medium

plus inhibitor was added back to the infected cells. Infected cellswere harvested at 12, 24 and 36 h post-infection (PI) and virus titersdetermined by standard plaque assay on RS cells. In addition, as anegative control for the above experiments the effect of each in-hibitor on HSV-1 replication was measured by direct incubationwith different concentrations of each inhibitor with HSV-1. Briefly,HSV-1 was pre-treated with each inhibitor for 1 h at 37 �C. Afterincubation, virus was added to RS cells without any inhibitor for48 h and titers were determined by standard plaque assay.

In vivo administration of inhibitors

Mice received 100 mg of (Z-LL)2 ketone or DAPT as an eye drop in5 ml of DMSO 1 h before ocular infection and at 2, 4, 6 and 8 h PI. (Z-LL)2 ketone administration was repeated 5 times daily for 4consecutive days. Sham control mice were treated similarly using5 ml of DMSO alone. For ocular infection, mice were infected in botheyes without scarification or anesthesia by placing eye drops con-taining 2 � 104 PFU of HSV-1 strain McKrae in 2 ml of tissue culturemedium. Eyes were swabbed once daily with a Dacron swab(Spectrum type 1) prior to administering the (Z-LL)2 ketone. Theswab was transferred to a culture tube containing 1 ml of medium,frozen, thawed, and virus titers determined by standard plaqueassay on RS cells as above.

Cell fractionation

RS cells were cultured inMEM containing 5% FCS. The day beforethe experiment, approximately 8 � 108 cells were plated on 100-mm tissue culture dishes and cultured overnight in regular cul-ture medium or medium containing 20 mm (Z-LL)2 ketone. Thefollowing day themediumwas replacedwith freshmediumwith orwithout (Z-LL)2 ketone and the cells were infected with 0.1 PFU/cellof HSV-1 strain McKrae. At 1 h PI, cells were washed to remove freevirus and fresh mediumwas added with or without (Z-LL)2 ketone.At 2, 4, and 12 h PI, cells were harvested and partitioned into nu-clear and cytoplasmic fractions with subsequent isolation of totalRNA using the Protein and RNA Isolation System (PARIS Kit AM1921,Life Technologies, Grand Island, NY) as per manufacturer protocol.

Gene expression analyses

Quantitative real-time PCR (qRT-PCR) was performed as wehave described previously (Allen et al., 2011). The differences in theexpression levels of mRNAs were evaluated using custom-madeTaqMan gene expression primers against ICP0, gB and gK withoptimized primer and probe concentrations (Life Technologies,Grand Island, NY). Primer probe sets consisted of two unlabeledPCR primers and the FAM� dye-labeled TaqMan MGB probeformulated into a single mixture. Additionally, all cellular ampli-cons included an introneexon junction to eliminate signal fromgenomic DNA contamination. The assays used in this study were asfollows: 1) gB specific primers (forward, 50-AACGCGACGCA-CATCAAG-30; Reverse e 50-CTGGTACGCGATCAGAAAGC-30; andProbe e 50-FAM-CAGCCGCAGTACTACC-30) with amplicon length of72 bp; 2) ICP0 specific primers (forward, 50-CGGA-CACGGAACTGTTCGA-30; reverse, 50-CGCCCCCGCAACTG-30; andprobe, 50-FAM-CCCCATCCACGCCCTG-30) with amplicon length of111 bp; and 3) gK specific primers (forward, 50-GGCCACC-TACCTCTTGAACTAC-30; reverse primer, 50-CAGGCGGG-TAATTTTCGTGTAG-30; and probe, 50-FAM-CAGGCCGCATCGTATC-30)with amplicon length of 82 bp. As an internal control, a set ofGAPDH primers from Applied Biosystems (ASSAY I.D.m999999.15_G1 e Amplicon Length ¼ 107 bp) was used.

Fig. 1. SPP Inhibitors reduce HSV-1 replication in infected cells. Confluent RS cells were incubated with SPP inhibitors 2 h prior to infection with 0.1 PFU/cell of HSV-1 strainMcKrae. Infected cells were harvested 12, 24 and 36 h PI and titered on fresh RS cells. Virus yield in the presence of each inhibitor is shown for A) Aspirin; B) Ibuprofen; C) (Z-LL)2; D)L685,458, and E) DAPT. Each point represents the mean titer � SEM from three independent experiments. “*”; Indicates significantly different from no inhibitor control group(p < 0.01, Student’s t-test).

S.J. Allen et al. / Experimental Eye Research 123 (2014) 8e1510

The relative copy numbers for ICP0, gB, and gK mRNAs werecalculated using standard curves generated from the plasmidspGem-ICP0, pAc-gB1, and pAC-gK1. In all experiments, GAPDHwas used for normalization of transcripts. The inhibitors had noeffect on GAPDH mRNA expression. qRT-PCR was performed

using an ABI ViiA7 sequence detection system (Applied Bio-systems). The threshold cycle values, which represent the PCRcycles at which there is a noticeable increase in the reporterfluorescence above baseline, were determined using ViiA7 RUOsoftware.

Fig. 2. (Z-LL)2 ketone reduces viral gene expression in the nucleus of infectedcells. RS cells were incubated with (Z-LL)2 ketone as in Fig. 1 above. Infected cellswere harvested 2, 4 and 12 h PI and separated into nuclear and cytoplasmicfractions followed by RNA extraction and cDNA synthesis. Expression of ICP0, gBand gK in the nuclear fraction were measured using qRT-PCR and presented as copynumber per time point. Each point represents the mean � SEM from 3 experi-ments. Panels: A) ICP0 in nuclear fraction; B) gB in nuclear fraction; and C) gK in

S.J. Allen et al. / Experimental Eye Research 123 (2014) 8e15 11

Statistical analysis

Student’s t-tests were performed using the computer programInstat (GraphPad, San Diego). Results were considered statisticallysignificant when the p value was <0.05.

3. Results

SPP inhibitors reduce HSV-1 replication in vitro

Recently we have shown that both SPP shRNA and SPP dominantnegative mutants reduced virus replication in vitro (Allen et al.,2014). Many forms of g-secretase inhibitors interfere with SPP ac-tivity and are of potential interest as therapeutics for Alzheimer’sdisease (Wolfe, 2009). The majority of commercially available SPPinhibitors, including the ones that we have chosen, have beenstudied extensively in cell lines (Bihel et al., 2004; Das et al., 2003;Esler et al., 2002;Weggen et al., 2001). Based on the reported safetyand efficacy studies (Okamoto et al., 2008; Weihofen et al., 2003),we have selected aspirin, ibuprofen, (Z-LL)2 ketone, L685,458, andDAPT to test our hypothesis that SPP inhibitors would reduce HSV-1replication similar to the SPP shRNA and SPP dominant negativesthat we reported recently (Allen et al., 2014).

We tested different concentrations of each inhibitor and choseconcentrations which caused no toxicity in HeLa, Vero or RS celllines as determined by trypan blue staining and direct observationof cytotoxicity from 0 to 48 h post-treatment. To determine theeffect of SPP inhibitors on virus replication in vitro, RS cells wereincubatedwith inhibitor before and after infectionwith 0.1 PFU/cellof HSV-1 strainMcKrae and titer was determined by plaque assay atvarious times PI. Virus yield in the presence of aspirin (Fig. 1A),ibuprofen (Fig. 1B), (Z-LL)2 ketone (Fig. 1C), L685,458 (Fig. 1D), andDAPT (Fig. 1E) were reduced as compared to mock-treated controlcells. Our results also suggest that ibuprofen had the greatest effecton reducing virus replication (Fig. 1B). Similar results were alsoobtained using 1 PFU/cell of HSV-1 (data not shown). In addition,HSV-1 was incubated alone with each inhibitor to verify that theobserved effects were not due to inactivation of the virus by theinhibitor. As expected direct incubation of HSV-1 with each inhib-itor showed no side effect on virus titer (not shown). Thus, theseresults demonstrate that HSV-1 replication requires functional SPPin vitro and that chemical inhibitors are able to reduce HSV-1replication in vitro. Similar to our finding, previously it wasshown that both (Z-LL)2 ketone and L-685,458 effectively inhibitedmalaria parasite invasion as well as growth in human erythrocytes(Li et al., 2009).

Viral gene expression is reduced in the nucleus of infected cells in thepresence of SPP inhibitor

The transcription of viral DNA takes place in the nucleus ofinfected cells and our in vitro results suggest that SPP inhibitorsreduced virus replication in infected RS cells (Fig. 1). To determine ifthis significant reduction in virus replication specifically involvedviral gene expression, we sought to determine if SPP inhibitionaltered transcription of viral genes in the nucleus of infected cells.As (Z-LL)2 ketone was the most specific SPP inhibitor in our panel(Nyborg et al., 2006; Okamoto et al., 2008), we infected RS cells inthe presence and absence of (Z-LL)2 ketone. At various times PI,infected cells were fractionated into nuclear and cytoplasmic frac-tions. qRT-PCR was performed on total RNA isolated from each

nuclear fraction. “*”; Indicates significantly different from no inhibitor controlgroup (p < 0.01, Student’s t-test).

S.J. Allen et al. / Experimental Eye Research 123 (2014) 8e1512

fraction as described in Materials and Methods. We detected sig-nificant reductions in ICP0 (Fig. 2A), gB (Fig. 2B), and gK (Fig. 2C)expressions in the presence of (Z-LL)2 ketone compared withmock-treated control cells. Since ICP0 is a transcriptional regulator ofgene expression its reduced expression may also reduce gB and gKexpressions. However, this reduction in gB and gK expressions isprobably independent of ICP0, as our published results suggest thatinhibition of SPP directly suppresses HSV-1 replication by blockingthe binding of gK to SPP (Allen et al., 2014). In contrast to the dif-ferences that we observed in expression of viral transcripts in thenuclear fraction of infected cells in the presence of (Z-LL)2 ketone,expression of ICP0 (Fig. 3A), gB (Fig. 3B), and gK (Fig. 3C) mRNAs inthe cytoplasmic fraction of infected cells were not reduced in thepresence of (Z-LL)2 ketone compared with mock-treated controlcells. Interestingly, the levels of ICP0 (Fig. 3A) and gK (Fig. 3C) butnot gB (Fig. 3B) increased by 12 h PI in the presence of inhibitorcompared with control group.

The results indicate that selective cytoplasmic accumulation ofsome of the viral transcripts correlates with blocking SPP synthesis.Thus, our results with regards to the cytoplasmic fraction suggestthat the net mRNA transport to the cytoplasm was not adverselyaffected at the time points tested in our study. Taken together, ourresults show that HSV-1 gene expression is impaired in the nucleusbut not cytoplasm of infected cells when SPP activity is inhibited.

SPP inhibitor reduces virus replication in vivo

Collectively, our in vitro results suggest that SPP inhibitorsreduced virus replication in infected RS cells (Fig. 1). We next testedwhether the most specific SPP inhibitor, (Z-LL)2 ketone would alsoreduce HSV-1 replication in vivo. (Z-LL)2 ketonewas given to C57BL/6 mice (100 mg/eye as an eye drop, 5�/day for 4 consecutive days),starting 1 h before ocular infection with HSV-1 strain McKrae.Control mice received DMSO as an eye drop. (Z-LL)2 ketonesignificantly decreased virus replication in the eyes of infectedmiceon days 1e5 PI (Fig. 3A) (p< 0.001, Student’s t-test compared to thesham control). Similar results were obtained with BALB/c micetreated with (Z-LL)2 ketone compared with sham control mice onboth days 1 and 3 PI (Fig. 3B). Thus, consistent with our in vitroresults (Fig. 1), SPP inhibition by (Z-LL)2 ketone treatmentdecreased viral replication in tears of ocularly infected mice. Fig. 4

Fig. 3. (Z-LL)2 ketone does not reduce viral gene expression in the cytoplasm ofinfected cells. RS cells were incubated with (Z-LL)2 ketone as in Fig. 2 above. Infectedcells were harvested 2, 4 and 12 h PI and separated into nuclear and cytoplasmicfractions followed by RNA extraction and cDNA synthesis. Expression of ICP0, gB andgK in the cytoplasmic fraction were measured as in Fig. 2 above. Each point representsthe mean � SEM from 3 experiments. Panels: A) ICP0 in cytoplasmic fraction; B) gB incytoplasmic fraction; and C) gK in cytoplasmic fraction.

4. Discussion

HSV-1-induced corneal scarring (CS), also broadly referred to asherpes stromal keratitis, can lead to blindness and HSV-1 is theleading cause of corneal blindness due to an infectious agent indeveloped countries (Barron et al., 1994; Dawson, 1984; Hill, 1987;Liesegang, 1999, 2001; Wilhelmus et al., 1996). In the U.S.,approximately 30,000 people suffer recurrent ocular HSV episodesannually, requiring doctor visits, medication, and in severe cases,corneal transplants. It is estimated that 70e90% of American adultshave antibodies to HSV-1 and/or HSV-2 and about 25% of theseindividuals have clinical symptoms upon routine clinical inquiry(Barron et al., 1994; Dawson, 1984; Hill, 1987; Liesegang, 1999,2001; Wilhelmus et al., 1996) with HSV-1 being responsible for>90% of ocular HSV infections. A significant proportion (15e50%) ofprimary genital herpes is caused by HSV-1 and recent studiesindicate that the proportion of clinical first episode genital herpesdue to HSV-1 is increasing (Auslander et al., 2005; Roberts et al.,2003; Singh et al., 2005). It was recently reported that the globalincidence of HSV keratitis is roughly 1.5 million, including 40,000new cases of severe monocular visual impairment or blindnesseach year (Farooq and Shukla, 2012).

Fig. 4. SPP inhibitor (Z-LL)2 ketone reduces virus replication in vivo. One hourbefore ocular infection with 2 � 104 PFU per eye of HSV-1 strain McKrae, C57BL/6 orBALB/c mice received 100 ug/eye of (Z-LL)2 ketone in DMSO as eye drops. Sham controlmice received DMSO alone. (Z-LL)2 ketone treatments were repeated as described inMaterials and Methods. In C57BL/6 mice, tear swabs were collected from infected eyeson days 1e5 PI, while in BALB/c infected mice swabs were collected on day 1 and day 3PI. Virus titers were determined by standard plaque assay. Each point represents themean titer from 20 eyes from two independent experiments. Panels: A) C57BL/6 mice;and B) BALB/c mice. “*”; Indicates significantly different from sham control group(p < 0.001, Student’s t-test).

S.J. Allen et al. / Experimental Eye Research 123 (2014) 8e15 13

It is well established that HSV-1-induced CS, and thus HSV-1-induced corneal blindness, are the result of immune responsestriggered by the virus (Brandt, 2005; Dix, 2002; Hendricks andTumpey, 1990; Ksander and Hendricks, 1987; Metcalf andKaufman, 1976; Thomas and Rouse, 1997). However, the exactidentity of the immune responses, including the fine specificity ofthe potentially harmful T cell effectors expressing classic TCRabantigen receptors that lead to CS, remains an area of intense con-troversy (Banerjee et al., 2002; Huster et al., 2002; Zhao et al., 1998).

At present time there is no vaccine available to control HSV-1infection and spread. In the past two decades several large clin-ical HSV vaccine trials were performed but both vaccine studiesfailed to reach their goals (Awasthi and Friedman, 2014; Farooq andShukla, 2012). Despite the seriousness of recurrent ocular herpes,no drug has been FDA approved for prevention of ocular re-currences. In addition, the wide use of acyclovir and its homolog inthe treatment of both HSV-1 and HSV-2 has raised concern over thedevelopment of resistant subtypes of HSV (Antoine et al., 2013;Farooq and Shukla, 2012). Previously it was shown that there is ahigh prevalence of acyclovir-resistant HSV-1 isolates in patientswith HSK (Duan et al., 2008). Thus, the absence of a viable vaccineagainst the virus and the increase in incidence of resistance toacyclovir and its homolog adds to the critical need for the devel-opment of alternative approaches for the prevention and control ofserious HSV-1-induced ocular diseases.

Recently we have shown that HSV-1 gK binds to SPP and thatblocking of SPP through dominant negative mutants or SPP shRNAreduced viral replication in vitro (Allen et al., 2014). Our publishedstudy also demonstrated that gK is the only HSV-1 gene that bindsto SPP in vitro. There are several chemicals that have been shown toinhibit the function of SPP. Many forms of g-secretase inhibitorsinterfere with SPP activity and are of potential interest as thera-peutics in Alzheimer’s disease, HCV, pestivirus and malarial infec-tion (Dovey et al., 2001; Heimann et al., 2006; Lanz et al., 2003; Liet al., 2009; Okamoto et al., 2008; Seiffert et al., 2000; Weihofenet al., 2003; Wolfe, 2009). The g-secretase inhibitor LY-450139 isone of the most potent inhibitors and is currently in phase II clinicaltrials and it appears to be well tolerated in human Alzheimer’spatients (Siemers et al., 2006). While this inhibitor is the only onecurrently being tested in humans, other SPP inhibitors, such as LY-411,575 (Eli Lilly), BMS-299,897 (Bristol-Meyers Squibb) and DAPT(Eli Lilly) have been studied for their effect in vivo (Best et al., 2005;Dovey et al., 2001; Siemers et al., 2005, 2006). In this study we haveshown for the first time that SPP chemical inhibitors are able toblock HSV-1 infectivity in vitro. Similarly our published resultsclearly demonstrated that SPP and gK can colocalize and bind withone another and blocking this interaction with shRNA reduced vi-rus replication in vitro (Allen et al., 2014). Thus, our results suggestthat inhibition of SPP activity reduced virus infectivity. Since gK isinvolved in exacerbation of CS and eye disease in mice, rabbits andhumans (Ghiasi et al., 1997, 1994a,b; Mott et al., 2009; Mott et al.,2007a; Mott et al., 2007b; Osorio et al., 2007), this HSV-1 depen-dence on SPP may be considered as a specific therapeutic target forthe prevention of corneal infection in patients at risk. Overall, theglobal result of these studies suggests that these inhibitors could bean alternative and expedient anti-viral treatment for control ofocular HSV-1 infection.

Acknowledgments

This work was supported by Public Health Service grant 1 RO1EY13615.

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