Identified by Infection with a Herpes Simplex Virus 1 ICP34.5 Mutant
Maria Korom, Kristine M. Wylie,* Hong Wang, Katie L. Davis, Meher S. Sangabathula,* Gregory S. DeLassus, Lynda A. Morrison
Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, Missouri, USA
The cellular prion protein (PrP) often plays a cytoprotective role by regulating autophagy in response to cell stress. The stress of infection with intracellular pathogens can stimulate autophagy, and autophagic degradation of pathogens can reduce their repli-cation and thus help protect the infected cells. PrP also restricts replirepli-cation of several viruses, but whether this activity is related to an effect on autophagy is not known. Herpes simplex virus 1 (HSV-1) effectively counteracts autophagy through binding of its ICP34.5 protein to the cellular proautophagy protein beclin-1. Autophagy can reduce replication of an HSV-1 mutant,⌬68H, which is incapable of binding beclin-1. We found that deletion of PrP in mice complements the attenuation of⌬68H, restoring its capacity to replicate in the central nervous system (CNS) to wild-type virus levels after intracranial or corneal infection. Cul-tured primary astrocytes but not neurons derived from PrPⴚ/ⴚmice also complemented the attenuation of⌬68H, enabling
⌬68H to replicate at levels equivalent to wild-type virus. Ultrastructural analysis showed that normal astrocytes exhibited an increase in the number of autophagosomes after infection with⌬68H compared with wild-type virus, but PrPⴚ/ⴚastrocytes
failed to induce autophagy in response to⌬68H infection. Redistribution of EGFP-LC3 into punctae occurred more frequently in normal astrocytes infected with⌬68H than with wild-type virus, but not in PrPⴚ/ⴚastrocytes, corroborating the ultrastruc-tural analysis results. Our results demonstrate that PrP is critical for inducing autophagy in astrocytes in response to HSV-1 in-fection and suggest that PrP positively regulates autophagy in the mouse CNS.
T
he cellular prion protein (PrP) is a glycophosphatidylinositol(GPI)-anchored glycoprotein that is conserved among
verte-brates. PrP is highly expressed on mouse neurons (1–4) and to
lower levels on many other cell types, including astrocytes (1,5)
and hematopoietic cells (2,6–8). The majority of PrP localizes to
the plasma membrane (9,10), but some is also found in the
cyto-plasm (11,12). Cellular PrP can be converted to a
protease-resis-tant aggregating isoform (PrPSc) (13,14). Transmission of PrPSc
with subsequent conversion of cellular PrP to the pathogenic ag-gregating form causes invariably fatal, neurodegenerative prion diseases, such as scrapie, bovine spongiform encephalopathy, and
Creutzfeld-Jakob disease (15,16). PrPScaggregates accumulate in
the cytoplasm of the infected cell, but it is unclear whether PrPSc
-infected cells die due to the toxicity of the intracellular aggregates or because a cytoprotective function of normal cellular PrP is lost
upon conversion to PrPSc(15–17). The severity of PrPSc-related
diseases and the unusual mechanism of disease transmission have generated intense interest in the physiologic role(s) of PrP in cel-lular metabolism and defense. Studies have suggested PrP involve-ment in such diverse biological processes as embryogenesis, cellu-lar proliferation, activation, differentiation, migration, adhesion, copper uptake, and formation of cytoplasmic protrusions and
nanotubes (10,18–23). The evolutionary conservation and
ubiq-uitous expression of PrP suggest its importance in cellular pro-cesses, yet surprisingly, mice and cows lacking PrP are remarkably
normal (24,25), with only a variety of subtle defects observed
(10).
PrP is also implicated in regulating the cellular response to stressors, including misfolded proteins, oxidative damage, and
nutrient starvation (10,19,26). These stressors induce autophagy
as a cell survival mechanism (27). Autophagy, specifically
mac-roautophagy, defines a cellular process by which cytoplasmic components become engulfed in a double membrane structure and are eventually degraded upon fusion with lysosomes.
Accom-panying formation of the double membrane vesicles, the proteo-lytically processed form of microtubule-associated protein 1 light chain 3 (LC3-I) is modified to the LC3-II form by lipidation and
becomes associated with the autophagosome membrane (28,29).
The autophagy pathway functions constitutively to degrade or-ganelles, long-lived cellular proteins, and lipids. Cell stress gener-ally upregulates autophagy to promote clearance of toxic protein aggregates, to recycle macromolecules for repair, or to maintain homeostasis. A functional link between cellular PrP and the au-tophagy response to cell stress has been suggested by the results of
several studies (30–32). Most often, PrP is observed to protect cells
by negatively regulating autophagy. For example, in a
hippocam-pal neuronal cell line established from PrP⫺/⫺mice, serum
depri-vation increased accumulation of LC3-II and reduces viability
compared with a cell line from wild-type mice (33).
Reintroduc-tion of PrP retards LC3-I to LC3-II conversion, suggesting that PrP downregulates autophagosome formation. In addition, cellu-lar PrP inhibits induction of autophagy and
autophagy-depen-dent cell death in gliomas (30).
Encounters with intracellular pathogens often cause cell stress,
which stimulates autophagy (29). Direct autophagic degradation
of intracellular pathogens, termed xenophagy (27), reduces
repli-Received19 September 2012Accepted5 March 2013
Published ahead of print13 March 2013
Address correspondence to Lynda A. Morrison, [email protected].
* Present address: Kristine M. Wylie, The Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA; Meher S. Sangabathula, Department of Internal Medicine, Bronx-Lebanon Hospital, Bronx, New York, USA. M.K. and K.M.W. were equal contributors to this work.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.02559-12
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cation of numerous intracellular bacteria and viruses and facili-tates major histocompatibility complex class II (MHC-II)-medi-ated presentation of pathogen peptides to elicit adaptive
immunity (34,35). These effects imply that autophagy induction
has a cytoprotective role in pathogen clearance. Cellular PrP also restricts replication of several viruses, including adenovirus 5 in
HuH7 cells (36), HIV-1 in 293T cells (37), and coxsackievirus B3
in cells derived from mouse brain (38). In addition, upregulation
of cellular PrP in mouse spleen coincides with reduced replication
of an endogenous murine retrovirus (39). Observations of PrP
upregulation in hepatitis C virus-infected hepatocytes (40),
astro-cytes infected with HIV-1 (41), and neurons of HIV-1-infected
persons suffering from neurological disorders (42) further suggest
PrP responsiveness to cellular stress associated with pathogen in-fection. Thus, PrP and autophagy each play a role in defense of the cell against pathogens, but whether they are convergent or inde-pendent activities has not been established.
Herpes simplex virus 1 (HSV-1) is a neurotropic alphaherpes-virus that has been well studied for its capacity to block autophagic
degradation of virus particles (33,43,44). The virus undergoes
lytic replication in epithelial cells and enters sensory neurons in-nervating the epithelium to spread into the nervous system. Neu-rons harbor latent virus, which periodically reactivates to reinfect the epithelium, or more rarely, spread into the central nervous system (CNS). Cold sores and potentially blinding herpes keratitis result from HSV-1 infection of the oral mucosa or corneal epithe-lium, respectively. Devastating encephalitis may also occur in adults and newborns. HSV-1 encodes a multifunctional neuro-virulence factor, ICP34.5, that counteracts IRF-3 activation of the
beta interferon (IFN-) gene (45) and opposes the activity of
pro-tein kinase RNA-activated (PKR) (46). During infection with
HSV-1 ICP34.5 mutants that lack the capacity to counteract PKR, activated PKR phosphorylates the eukayotic translation initiation
factor 2␣(eIF2␣) to arrest cap-dependent protein translation (47,
48). ICP34.5 produced by wild-type virus interacts with protein
phosphatase 1␣(PP1␣) (49) and eIF2␣(50), redirecting PP1␣to
dephosphorylate eIF2␣and thus allowing translation of viral
pro-teins (51). Activated PKR also mediates the induction of
au-tophagy (44), and HSV-1 ICP34.5 counteracts autophagy (44,52)
by binding to the proautophagy protein beclin-1 (33). HSV-1
variants unable to synthesize ICP34.5 are highly neuroattenuated
in mice (53,54), because they cannot prevent translational shutoff
and autophagy (33). An HSV-1 variant missing only the ICP34.5
beclin-1 binding domain is also neuroattenuated, suggesting that the capacity to counteract autophagy in the CNS contributes to
neurovirulence (33).
We speculated that PrP might also reduce pathogen replication by regulation of autophagy. Interestingly, although in many stud-ies PrP restricts pathogen replication, mice lacking PrP are re-ported to be less susceptible to HSV-1 than normal mice after
subcutaneous inoculation into the ear pinna (55,56). This
re-duced susceptibility correlates with slightly lower virus titers in the
nervous system and lymph nodes of PrP⫺/⫺mice than in normal
mice (55,56). The reason for the reduced titers and pathogenicity
of wild-type HSV-1 in PrP⫺/⫺mice was not determined, and it
may be difficult to identify because the differences were small. As with many viruses, HSV-1 masterfully controls cellular antiviral responses to optimize its replication. Thus, HSV-1 mutant viruses that cannot counteract specific host antiviral activities have at least two advantages over wild-type HSV-1 as tools for identifying a
function of PrP during virus infection. First, PrP may affect rep-lication of mutant viruses more than wild-type virus because mu-tant viruses lacking the means to block an antiviral activity may be
more susceptible to the antiviral effect in normal versus PrP⫺/⫺
mice. Second, the defects in some HSV-1 mutants have been well characterized, and thus success or failure of the mutants to repli-cate and cause disease in a host may reveal antiviral activities of specific host proteins. In this study, we utilized HSV-1 ICP34.5 mutants to investigate a role for normal cellular PrP in the antivi-ral defense exerted through induction of autophagy.
MATERIALS AND METHODS
Cell lines and viruses.Vero cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 3% newborn calf serum, 3% bovine growth serum, 1%L-glutamine, and 1% penicillin-streptomycin. Wild-type HSV-1 strain 17,␥34.5 mutant viruses⌬␥34.5 and⌬68H, and⌬68H rescue virus⌬68HR were provided by Diane Alexander and David Leib. The ICP34.5 null mutant⌬␥34.5 has a stop linker inserted into the BstEII site in␥34.5, resulting in the termination of ICP34.5 translation after the first 30 amino acids (57).⌬␥34.5 lacks both PP1␣and beclin-1 binding capabilities. The ICP34.5 mutant⌬68H (43) lacks amino acids 68 to 87, which are required for interaction with beclin-1 (58), but it retains the PP1␣interaction domain (43). The rescue viruses⌬␥34.5R and⌬68HR were generated by homologous recombination of⌬␥34.5 or⌬68H DNA, respectively, with plasmid containing the wild-type␥34.5 open reading frame. HSV-1 strain dl1403, provided by Nigel Stow, has a 2-kb deletion in the␣0 gene, which encodes ICP0 (59).⌬TK contains a 361-bp deletion between the BglII-SmaI restriction enzyme sites in the thymidine kinase (TK) gene. To construct⌬TK, the BglII-to-SmaI fragment was excised from the TK gene of HSV-1 strain KOS in plasmid p101086.7 (D. Yager and D. Coen, unpublished data). Blunt ends were created with DNA poly-merase I Klenow fragment, and the plasmid was recircularized to create p101086.7⌬TK. Next, p101086.7⌬TK was cotransfected with strain 17syn⫹infectious DNA, and TK-deficient viruses were positively selected by the addition of 100M acyclovir and plaque purified to homogeneity. HSV-1 strain 17-6B, which was provided by David Leib, expresses -ga-lactosidase under the control of the ICP6 promoter. Viruses were propa-gated in Vero cells, and titers were determined by standard plaque assay (60).
Mice.All animals were handled in strict accordance with good animal practices as defined by institutional and U.S. Public Health Service guide-lines, and all animal work was approved by the University Animal Care and Use Committee. Mice were housed and bred in the Saint Louis Uni-versity School of Medicine Department of Comparative Medicine and were used at 6 to 7 weeks of age. The PrP⫺/⫺mice used in this study have
a deletion in the coding exon of the PrP gene, but flanking regions of the gene remain intact (24). C57BL/6J (B6) mice heterozygous for deletion in the PrP allele were provided by David Harris. B6 PrP⫺/⫺mice were
com-pared to normal littermate controls. PrP⫺/⫺mice on a C57BL/6J⫻129
SvEv (B6x129) background were obtained from the European Mouse Mutant Archive. Wild-type B6⫻129 F1 mice were purchased from Taconic.
Mouse infection.To study virus replicative capacity in the CNS, anes-thetized mice were infected intracranially (i.c.) in the right cortex with 1⫻ 103PFU/mouse in a 10-l volume, using a 30-gauge needle (61). Brains
and brainstems were dissected 3 days postinfection (p.i.) and homoge-nized for determination of virus titers by standard plaque assay. For cor-neal infection, 2⫻105or 2⫻106PFU of virus in a 5-l volume was
applied to each lightly scarified cornea of anesthetized mice (62,63). Virus replication in the corneal epithelium was assessed at 4 h and days 1 through 4 p.i. The trigeminal ganglia, brainstem, and brain were dissected 4 days p.i. and homogenized, and virus titers in tissue samples were de-termined.
Histology.B6 and PrP⫺/⫺mice were infected by the corneal route
with 17-6B and dissected 4 days p.i. The cranium was removed, and the
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brain, brainstem, and trigeminal ganglion roots were dissected together and fixed by submersion in phosphate-buffered saline (PBS) containing 4% formalin at room temperature (RT) for 1 h. After rinsing with PBS, tissues were transferred to 5-bromo-4-chloro-3-indolyl--D -galactopyra-noside (X-Gal) staining solution (1 mg/ml X-Gal diluted in PBS contain-ing 5 mM potassium ferrocyanide, 5 mM postassium ferricyanide, and 2 mM magnesium chloride) and incubated at 37°C overnight. Tissues were rinsed in PBS and stored in 4% neutral buffered formalin (NBF). Photo-graphs were taken prior to sectioning and adjusted for brightness and contrast. Sections (4 m) were cut from paraffin-embedded tissues, picked up on glass slides, and dried overnight in a 36°C oven. The sections were rehydrated, and antigen retrieval was performed (120°C for 3 min). The slides were then washed and blocked for 1 h in 5% normal goat serum, 1% bovine serum albumin, and 0.25% Triton X-100 in PBS at RT. Sec-tions were incubated in glial fibrillary acidic protein (GFAP) anti-body (Abcam) overnight at 4°C, washed extensively in PBS, and incubated in goat anti-mouse Alexa Fluor 488 (Invitrogen) for 1 h at RT. Sections were washed, and coverslips were mounted with Fluoro-Gel II containing 4=,6-diamidino-2-phenylindole (DAPI; EMS).
Primary cell cultures.Neuron cultures were established from the brainstems of embryonic day 14 (E14) to E15 embryos as described pre-viously (64,65). Primary neuron cultures were used 6 to 8 days after seeding. To establish primary astrocyte-rich (91 to 98%) glial cell cultures (referred to as astrocyte cultures), 1- to 3-day-old mice were sacrificed and the brainstems were isolated. Meninges were removed completely, and brainstems were thoroughly rinsed in PBS before digesting the tissue in trypsin for 15 min at room temperature. Cells were dissociated by pi-petting. Trypsin was neutralized with the addition of serum-containing medium, and cells were passed through a 70-m cell strainer. Cells were pelleted, and pellets were resuspended in glial cell medium (DMEM sup-plemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1% nonessential amino acids, and 5 mM HEPES) and seeded into 100-mm dishes coated with laminin. After 24 h, the medium was replaced. Cells were cultured until confluent, replacing half the medium every 2 to 3 days. Before experiments, cells were shaken at 250 rpm on an orbital shaker overnight to remove microglia. Adherent cells were trypsinized and seeded into culture dishes. To determine the purity of the astrocyte cul-tures, cells were stained with anti-CD11b–peridinin chlorophyll protein (BD Biosciences) to detect microglia, or cells were permeabilized with Cytofix/Cytoperm (BD Biosciences) and stained with anti-GFAP (Dako) and an Alexa Fluor-conjugated secondary antibody (Invitrogen) to detect astrocytes. Samples were analyzed by flow cytometry using the LSRII flow cytometer (BD Biosciences) and FlowJo software (TreeStar). To assess virus replication in cultured cells, confluent monolayers were infected at a high multiplicity of infection (MOI) (10) or low MOI (0.01). Virus titers were determined by standard plaque assay.
Real-time reverse transcriptase PCR.For real-time reverse transcrip-tase PCR assays, mice were left untreated or were infected by the corneal route with 2⫻106PFU of⌬68H or⌬68HR. brainstems were dissected 4
days p.i. and placed in TRI reagent (Molecular Research Center). Alter-natively, TRI reagent was added to cultures of primary astrocytes 12 h postinfection. RNA was prepared according to the manufacturer’s in-structions and treated with Turbo DNase (Ambion). cDNA was synthe-sized using the Transcriptor first strand cDNA synthesis kit (Roche) and random hexamers. cDNA was used as a template for real-time PCR with primers to amplify IFN-from mouse brain (forward primer, 5=-TCC CTA TGG AGA TGA CGG AG-3=; reverse primer, 5=-ACC CAG TGC TGG AGA AAT TG-3=) and from astrocytes (forward primer, 5=-CAG CTC CAA GAA AGG ACG AAC-3=; reverse primer, 5=-GGC AGT GTA ACT CTT CTG CAT-3=). Signal was normalized to that of 18S rRNA (forward primer, 5=-GTA ACC CGT TGA ACC CCA TT-3=; reverse primer, 5=-CCA TCC AAT CGG TAG TAG CG-3=). The signal for unin-fected samples was set to 1, and the relative quantity for signal in inunin-fected samples was calculated based on the cycle threshold (CT), using the⌬⌬CT
method (66,67).
Western blot assays.To assess PKR levels, lysates of cultured primary astrocytes were analyzed by Western blotting with a rabbit polyclonal antibody against PKR (1:5,000; Santa Cruz Biotechnology). Signal was normalized to total eIF2␣(1:5,000; Santa Cruz Biotechnology). To assess PKR activity, astrocyte cultures were infected with ⌬␥34.5, ⌬68H,
⌬␥34.5R, or⌬68HR at an MOI of 10 for 12 h, at which time cells were lysed and samples were analyzed by Western blotting with rabbit poly-clonal antibodies against total eIF2␣and phosphorylated eIF2␣(1:5,000; Biosource). To assess LC3-II accumulation, astrocytes were starved or infected at an MOI of 10 for 6 or 12 h in the absence or presence of 50M chloroquine. Cell lysates were analyzed by Western blotting by probing with polyclonal rabbit antibodies against total eIF2␣and LC3 (Novus) and horseradish peroxidase-conjugated anti-rabbit secondary antibody (KPL). Images were captured using AlphaImager software, and band den-sities were quantified using ImageJ software.
Ultrastructural analysis.Astrocyte cultures were infected with⌬68H or⌬68HR at an MOI of 10 in glial cell medium for 12 h. Alternatively, astrocytes were incubated in glial cell medium (untreated) or serum-free DMEM (starved) for 6 h. Cells were fixed, and quantitative electron mi-croscopy analysis of autophagosomes was carried out by the Washington University School of Medicine Molecular Microbiology Imaging Facility as previously described (52).
EGFP-LC3 aggregation assay.Primary astrocytes (1⫻106per
cul-ture) were mixed with 1g of pEGFP-LC3 plasmid (kind gift of Skip Virgin) and 100l nucleofection solution (0.7 mM ATP disodium salt, 11.6 mM MgCl2·6H2O, 67.5 mM K2HPO4, 14 mM NaHCO3, 2.2 mM
glucose in distilled H2O; pH 7.4). The cell/nucleofection solution mix was
electroporated in an Amaxa Nucleofector II electroporator (Lonza) set at T-20. The cells were then transferred to six-well plates containing glass coverslips. Twenty-four hours posttransfection, cells were examined by fluorescence microscopy to assess transfection efficiency. Only cultures with more than 80% transfected cells were used for subsequent experi-ments. Transfected cultures were mock infected or infected at an MOI of 10 with HSV-1⌬68H or⌬68HR. Alternatively, cells were starved by wash-ing twice with PBS and then culturwash-ing in HBSS. Cells were fixed with 4% paraformaldehyde 6, 12, or 24 h postinfection/starvation. Coverslips were then immunostained with a 1:200 dilution of anti-HSV-1 rabbit poly-clonal antibody (Dako) followed by a 1:2,000 dilution of anti-rabbit Alexa 594 secondary antibody (Invitrogen) and mounted with Pro-Long Gold containing DAPI (Invitrogen). Mock, starved, or infected cells (those that stained with anti-HSV-1) were photographed at 60⫻magnification with a Kalman value of 3 by using a confocal microscope (FV1000; Olympus). Photographs of cytoplasmic portions of cells were scored by a masked observer to determine the proportion containing enhanced green fluores-cent protein-LC3 (EGFP-LC3) punctae. Because starved cells are elongate and infected cells are round, only images of cell cytoplasmic regions were evaluated, so that conclusions could not be drawn by the masked observer based on the shape of the cell.
Statisical analyses.Statistical significances of differences in virus titers were determined using one-way analysis of variance (ANOVA) with Bon-ferroni correction for multiple groups or the Studentttest when compar-ing two groups. Statistical significances of differences in autophagosome numbers were determined using the Studentttest. Significance of differ-ences in proportions of cells containing EGFP-LC3 punctae were deter-mined by using the Fisher exact test. Areas under the curves were com-pared using the Wilcoxon matched pairs test.
RESULTS
⌬68H replicates to wild-type virus levels in the CNS of PrPⴚ/ⴚ mice but not of normal mice.PrP is highly expressed in cell types
in the nervous system that are targets for HSV-1 infection (58,
68–71). Under the stress of virus infection, if autophagy is
en-hanced in the absence of PrP, then replication of the beclin-1
binding mutant ⌬68H, which cannot counteract autophagy,
should be strongly suppressed compared with wild-type virus.
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When we examined virus titers in the nervous systems of normal
B6 and PrP⫺/⫺mice 3 days after i.c. inoculation of the cortex, we
found⌬68H replicated to levels 600-fold lower than its marker
rescue virus⌬68HR in the brains and brainstems of normal mice
(Fig. 1A).⌬68HR replicated equivalently to HSV-1 strain 17, from
which it and⌬68H were derived (Fig. 1AandB), implying that the
phenotype of⌬68H relates to its incapacity to bind beclin-1 rather
than to an adventitious mutation. Unexpectedly, the mutant virus replicated equivalently to the wild-type viruses in the brains and
brainstems of PrP⫺/⫺mice (Fig. 1B). Thus, PrP has an antiviral
role that attenuates replication of⌬68H in the CNS.
Next, virus replication in the nervous system was tested after infection by a peripheral route. HSV infections of the eye spread from the cornea into peripheral sensory neurons of the trigeminal
ganglia and then to the CNS via the brainstem (71,72). After virus
inoculation on the corneal surface,⌬68H replicated equivalently
to⌬68HR in the corneas of normal B6 (Fig. 2A) and PrP⫺/⫺
(Fig. 2B) mice through 4 days p.i., demonstrating that⌬68H is not attenuated for replication in the corneal epithelium, and thus equivalent amounts of virus were available to enter into sensory nerve endings innervating the cornea. In the trigeminal ganglia,
⌬68H replicated significantly less well than⌬68HR in both
nor-mal B6 (Fig. 2C) and PrP⫺/⫺mice (Fig. 2D) at 4 days p.i.,
indicat-ing that⌬68H is attenuated in the trigeminal ganglia and PrP does
not influence this attenuation. In the brainstems of normal B6
mice,⌬68H replicated to lower levels than⌬68HR (Fig. 2C). Viral
titers in the brain were too low to evaluate an effect of PrP.⌬68H
and⌬68HR replicated equivalently, however, in brainstems and
brains of PrP⫺/⫺mice (Fig. 2D).
Using normal and PrP⫺/⫺B6⫻129 mice, we tested our
obser-vations in a different host genetic background. After corneal
in-fection, titers of⌬68H were lower than⌬68HR in the trigeminal
ganglia and brainstems of normal mice (Fig. 3A). In mice lacking
PrP,⌬68H was still attenuated in the trigeminal ganglia (Fig. 3B),
but it recovered the capacity to replicate equivalently with⌬68HR
in the brainstems and also caused as much lethality (Fig. 4),
al-though⌬68H titers still lagged behind⌬68HR in the brain (Fig.
3B). The marker rescue virus⌬68HR also replicated in CNS
tis-sues equivalently to HSV-1 strain 17 after corneal inoculation (Fig. 3AandB), providing further evidence that⌬68HR is a true
rescue virus, and therefore the specific mutation in⌬68H confers
its phenotype. Lastly, HSV-1 strains with attenuating mutations in the genes encoding ICP0 (an immediate-early gene product) and TK (an early gene product) did not recover the capacity to
repli-cate to wild-type virus levels in PrP⫺/⫺mice (data not shown),
indicating that the result is specific to the mutation in ICP34.5. Taken together, these data indicate that PrP has an antiviral role in the CNS after direct i.c. inoculation and, specifically, in the brain-stem after corneal inoculation.
PrPⴚ/ⴚand normal mice express equivalent levels of IFN- transcript in infected brainstems. HSV-1 mutants lacking ICP34.5 are attenuated for replication and pathogenicity because
they cannot effectively counteract PKR (33,46), which stimulates
beclin-1-dependent autophagy (44). An ICP34.5 beclin-1 binding
mutant, then, recovers the capacity to replicate to wild-type virus
levels in PKR⫺/⫺mice (33). PKR synthesis is upregulated in
re-sponse to IFN-␣//␥(73); thus, PKR levels would be lower if less
IFN-␣//␥synthesis occurred upon virus infection in the
brains-tems of infected PrP⫺/⫺mice compared with normal B6 mice, and
⌬68H would replicate more like⌬68HR. We found that IFN-␣1,
IFN-␣4, and IFN-␥transcripts were not induced in the brainstems
of normal and PrP⫺/⫺mice at 4 days after corneal infection with
⌬68H (data not shown).⌬68H-infected mice had higher levels of
IFN-transcript relative to uninfected mice; however, the relative
amount induced in normal and PrP⫺/⫺mice was equivalent (Fig.
5). These data indicate that the difference in replication of⌬68H
in the CNS of normal and PrP⫺/⫺mice cannot be explained by a
defect in type I IFN induction.
The replication phenotypes observed in the brainstems of normal and PrPⴚ/ⴚmice are reproduced in primary astrocyte cultures.An appropriate in vitroculture system to discern the
antiviral function of PrP would recapitulate thein vivophenotype
of⌬68H. Specifically,⌬68H would replicate to lower levels than
⌬68HR in normal cells, and replication of the two viruses would
be equivalent in PrP⫺/⫺cells. HSV-1 is neurotropic, but cultured
primary neurons derived from the cortex or brainstem are not a
usefulin vitromodel because⌬68H is not attenuated for
replica-tion compared with⌬68HR after infection at either a high or low
MOI (data not shown). Astrocytes are abundant in the CNS and
are productively infected by HSV-1 (58,69,71). In addition, the
trigeminal route entry zone was previously characterized as an astrocyte-rich region infected by HSV-1 after corneal inoculation
(71,74). Infection of mice by the corneal route with 17-6B, an
HSV-1 strain that expresses-galactosidase (-Gal), revealed
re-gions of blue staining in the brainstem, including the trigeminal
root entry zone (Fig. 6A). Many of the-Gal-expressing cells
FIG 1⌬68H replicates equivalently with⌬68HR in the CNS of B6 PrP⫺/⫺ mice after i.c. infection. Normal, wild-type (WT) B6 (A) and PrP⫺/⫺mice on a B6 background (B) were infected with 1⫻103PFU of HSV-1 strain 17, ⌬68HR, or⌬68H. Virus titers were determined in the brain and brainstem 3 days p.i. Dashed lines indicate the limit of detection. Data represent the com-bined results of three independent experiments for a total of 10 to 13 mice per
group. **,P⬍0.001; ns, not significant.
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[image:4.585.73.254.67.342.2](Fig. 6B) costained with antibody to GFAP (Fig. 6C). In primary astrocyte cultures derived from brainstems of normal B6 mice, we
found that⌬68H replicated to titers about 100-fold lower than
those of⌬68HR at 24 and 48 h after infection at a low MOI
(Fig. 7A), and remained attenuated by about 10-fold at 18 and 24
h after infection at a high MOI (Fig. 7B). These results identify
astrocytes as a primary cell culture system in which replication of
⌬68H is attenuated compared with⌬68HR, similar to the
atten-uation of⌬68H compared with⌬68HR in the brainstems of
nor-mal mice. In astrocyte cultures derived from PrP⫺/⫺mice,
repli-cation of⌬68H remained attenuated by 50-fold compared with
⌬68HR after infection at a low MOI (Fig. 7C). However, titers of
⌬68H were equivalent to those of ⌬68HR after infection of
PrP⫺/⫺astrocytes at a high MOI (Fig. 7D), analogous to the
re-covery of⌬68H in the brainstems of PrP⫺/⫺mice. These results
indicate that astrocytes are a relevant cell type in which to examine
in vitrothe role of PrP in attenuating replication of⌬68H
com-pared with wild-type virus. Furthermore, because⌬68H cannot
counteract autophagy, these data suggest that astrocytes from
PrP⫺/⫺mice may be defective in autophagyin vitroandin vivo.
PKR levels and activity are not reduced in primary astrocyte cultures from PrPⴚ/ⴚmice compared with astrocytes from nor-mal mice.PKR-mediated phosphorylation of eIF2␣triggers the induction of autophagy in response to infection with HSV-1
ICP34.5-deficient mutants that cannot antagonize PKR (47).
Therefore, if the amount of PKR or PKR-mediated
phosphoryla-tion of eIF2␣is reduced in PrP⫺/⫺astrocytes compared with
nor-mal astrocytes, autophagy will not be efficiently induced in
re-sponse to virus infection. However, we found equivalent levels of
PKR in normal B6 and PrP⫺/⫺astrocytes (Fig. 8A). PKR activity
also was unaffected by the absence of PrP. Specifically, as expected,
wild-type viruses⌬␥34.5R and⌬68HR suppressed
phosphory-lated eIF2␣below the level in mock-infected cells, as did⌬68H,
which lacks only the beclin-1 binding domain, and markedly
more phosphorylated eIF2␣ accumulated in normal astrocytes
infected with⌬␥34.5 (Fig. 8B). More phosphorylated eIF2␣also
accumulated in PrP⫺/⫺astrocytes infected with⌬␥34.5 compared
to both mock-infected cells and cells infected with⌬68H or
wild-type viruses (Fig. 8B). These observations provide evidence that
the PKR pathway is intact in PrP⫺/⫺astrocytes, indicating that a
defect in PKR levels or activity in PrP⫺/⫺astrocytes is not the
reason that⌬68H recovers the capacity to replicate equivalently to
⌬68HR in these cells.
Primary astrocyte cultures from PrPⴚ/ⴚmice are defective in their capacity to induce autophagy in response to virus infec-tion.⌬68H is defective in its capacity to counteract
beclin-1-me-diated induction of autophagy (33,43) and is attenuated for
rep-lication in mice compared with its marker rescue virus,⌬68HR
(33). In PrP⫺/⫺mice and astrocytes,⌬68H recovers the capacity
to replicate to wild-type virus levels, suggesting PrP⫺/⫺mice and
astrocytes could have a defect in autophagy. Therefore, we used electron microscopy to examine the accumulation of
autophago-somes in normal (Fig. 9A) and PrP⫺/⫺(Fig. 9B) astrocytes in
response to infection with⌬68H and⌬68HR. As expected,
nor-mal astrocytes infected with⌬68H had more autophagosomes per
cell profile than those infected with⌬68HR. Quantification of
FIG 2⌬68H replicates equivalently to⌬68HR in the CNS of B6 PrP⫺/⫺mice after corneal infection. Normal wild-type (WT) B6 mice (A and C) and PrP⫺/⫺mice on a B6 background (B and D) were infected with 2⫻106PFU/eye of⌬68H or⌬68HR. Virus titers were determined in corneal swabs (A and B) and nervous system tissues (C and D) 4 days p.i. Data represent the combined results of two independent experiments for a total of 13 to 14 mice per group. Dashed lines indicate the limit of detection. ***,P⬍0.0001; ns, not significant.
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imaging results from three experiments revealed that the increase
in accumulation of autophagosomes was 1.8-fold (Fig. 9C).
How-ever, in PrP⫺/⫺astrocytes infected with ⌬68H or ⌬68HR, the
number of autophagosomes per cell profile was equivalent,
indi-cating autophagy is not induced in PrP⫺/⫺astrocytes in response
to infection with⌬68H (Fig. 9C). The autophagosome numbers
per cell profile in untreated normal and PrP⫺/⫺astrocytes were
equivalent (Fig. 9D), indicating that PrP⫺/⫺astrocytes do not
have a defect in basal autophagy. In addition to HSV infection, other stresses, such as starvation, induce autophagy. Therefore, we compared the accumulation of autophagosomes in normal and
PrP⫺/⫺astrocytes in serum-containing medium and serum-free
medium. After 6 h of starvation of normal astrocytes, the numbers of autophagosomes per cell profile increased nearly 2-fold, but the
number of autophagosomes in starved PrP⫺/⫺ astrocytes
re-mained the same as in untreated cells (Fig. 9D). Together, these
data indicated that PrP⫺/⫺astrocytes are not defective in basal
autophagy, but they fail to mount an autophagic response to virus infection or brief starvation.
Upon induction of autophagy, LC3 undergoes lipidation and
associates stably with the autophagosome membrane (28).
Con-sequently, EGFP-LC3 reorganizes from a diffuse distribution in
normal cells to punctae in cells undergoing autophagy (28). In
primary astrocytes transfected with an EGFP-LC3-expressing plasmid, EGFP-LC3 had a fine grainy appearance throughout the
cytoplasm (Fig. 10A). EGFP-LC3 in normal astrocytes coalesced
into numerous cytoplasmic punctae after starvation (Fig. 10B).
This reorganization of EGFP-LC3 into punctae was observed in the majority of cultured normal astrocytes infected for 24 h with
⌬68H, but not in those infected with⌬68HR (Fig. 10C),
confirm-ing the inability of the beclin-1 bindconfirm-ing mutant to block induction
of autophagy. PrP⫺/⫺astrocytes, however, showed no increase in
accumulation of EGFP-LC3 punctae upon infection with⌬68H
FIG 3⌬68H replicates equivalently with⌬68HR in the CNS after corneal infection of B6⫻129 PrP⫺/⫺mice. Normal wild-type (WT) B6⫻129 mice (A) and PrP⫺/⫺mice on a B6⫻129 background (B) were infected with 2⫻105 PFU/eye of⌬68H,⌬68HR, or wild-type HSV-1 strain 17. Virus titers were determined in nervous system tissues 4 days p.i. Each group consisted of 6 to 7 mice compiled from two independent experiments. Dashed lines indicate the limit of detection. **,P⬍0.001; *,P⬍0.05; ns, not significant.
FIG 4⌬68H causes as much lethality as⌬68HR after corneal infection of B6⫻ 129 PrP⫺/⫺mice. Normal wild-type (WT) B6⫻129 mice (A) and PrP⫺/⫺ mice on a B6⫻129 background (B) were infected with 2⫻105PFU/eye of ⌬68H or⌬68HR (10 mice per group), and mice were monitored daily for survival. Data represent the combined results of two independent experi-ments.
FIG 5IFN-transcript increases equivalently in the brainstems of normal B6 and PrP⫺/⫺mice after⌬68H infection. Wild-type (WT) B6 mice and PrP⫺/⫺ mice were infected with 2⫻106PFU/eye of⌬68H or were left uninfected. The levels of IFN-transcript in infected mice were determined by using real-time reverse transcriptase PCR and are represented relative to the level found in uninfected mice (set as 1). Each bar corresponds to the mean⫾standard deviation from 2 to 3 mice per group, and the data are representative of 3 independent experiments.
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[image:6.585.330.513.64.342.2] [image:6.585.328.511.528.648.2]compared with⌬68HR (Fig. 10C). Interestingly, EGFP-LC3
ag-gregated in PrP⫺/⫺astrocytes after 12 h of starvation (Fig. 10D),
indicating that autophagy could be induced in PrP⫺/⫺astrocytes
under conditions of prolonged serum deprivation. EGFP-LC3
ag-gregation occurred in a smaller proportion of PrP⫺/⫺astrocytes
after 6 h of starvation (69% versus 81% in normal astrocytes) (data not shown). These results provide independent confirma-tion that cellular PrP has a role in response to infecconfirma-tion, as revealed with an HSV-1 mutant that cannot bind beclin-1.
Lipidation of LC3-I generates the LC3-II form that associates with the autophagosome membrane. Subsequent degradation of the contents of mature autophagosomes leads to loss of LC3-II. Treatment of cells with an inhibitor of autophagosome matura-tion prevents LC3-II degradamatura-tion and permits an accounting of
the level of autophagic flux in cells. We therefore starved or
in-fected normal and PrP⫺/⫺astrocytes in the presence or absence of
chloroquine (Fig. 11). Endogenous LC3-II was barely detectable
by Western blotting in starved normal astrocytes, and only a low
level of LC3-II could be observed in infected cells (Fig. 11A).
How-ever, a prominent LC3-II band was observed in starved,
chloro-quine-treated astrocytes (Fig. 11A), indicating a high level of
au-tophagic flux. Accumulation of LC3-II in the presence of chloroquine was also observed in infected normal astrocytes (Fig. 11A), with 1.5-fold more LC3-II accumulating in cells
in-fected with⌬68H than in those infected with⌬68HR (Fig. 11B). In
PrP⫺/⫺astrocytes, LC3-II accumulated in the presence of
chloro-quine to the greatest extent in cells undergoing starvation (Fig. 11CandD). Approximately equivalent amounts of LC3-II
FIG 6Astrocytes infected in the brainstem 4 days after corneal infection. Wild-type B6 mice and PrP⫺/⫺mice were infected with 2⫻106PFU/eye of 17-6B. Brain tissue (including the brainstem and trigeminal ganglion roots) was dissected 4 days postinfection, fixed, and incubated in X-Gal, followed by sectioning and staining with anti-GFAP and fluorescein isothiocyanate-labeled secondary antibodies. (A) Representative image of intact X-Gal-stained brainstem (ventral view) from a PrP⫺/⫺mouse. The box encloses the trigeminal root entry zone. (B and C) A tissue section under bright-field microscopy (B) or confocal fluorescence microscopy (C), showing cells that costained for-Gal and GFAP, respectively. Bar, 20m.
FIG 7Primary astrocyte cultures model the replication phenotypes of⌬68H observed in normal B6 and PrP⫺/⫺mice. Astrocyte cultures derived from wild-type (WT) B6 mice (A and B) or PrP⫺/⫺mice (C and D) were infected at a low MOI (0.01) (A and C) or high MOI (10) (B and D) with⌬68HR or⌬68H. Each data point represents the mean⫾standard error of the mean of duplicate samples. The data are representative of two independent experiments and were confirmed by two additional experiments in which replication was measured only at 24 h p.i. (low MOI) or 18 h p.i. (high MOI). Independently derived astrocyte cultures with a purity of 90 to 99% astrocytes were used for each experiment. **,Pⱕ0.0075; *,P⬍0.05; ns, not significant.
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[image:7.585.113.475.67.171.2]were found in chloroquine-treated PrP⫺/⫺ astrocytes infected
with ⌬68H or ⌬68HR (Fig. 11CandD). A similar pattern of
LC3-II accumulation was also observed at 6 h postinfection (data not shown). These data are consistent with the previous assays of autophagosome enumeration and EGFP-LC3 aggregation,
al-though the difference in response of normal astrocytes to⌬68H
versus⌬68HR was slightly less pronounced.
IFN-mRNA is upregulated in⌬68H-infected astrocyte cul-tures independently of PrP.We had observed that replication of
⌬68H was attenuated compared with⌬68HR after low-MOI
in-fection of both normal and PrP⫺/⫺primary astrocyte cultures
(Fig. 7AandB), indicating that a PrP-independent mechanism must regulate the capacity of the mutant virus to replicate under conditions of low-MOI infection. Because cellular production of
type I IFN can suppress virus growth, we assessed IFN-mRNA
by real-time PCR. Both normal and PrP⫺/⫺astrocyte cultures
infected with⌬68H contained 3-fold more IFN-mRNA than
those infected with⌬68HR (Fig. 12). Preliminary results,
how-ever, suggest that addition of IFN--neutralizing antibody to
cul-tures does not improve virus growth (data not shown). Thus,
PrP⫺/⫺astrocytes are not defective for type I IFN induction, and
⌬68H attenuation likely involves a PrP-independent, IFN-
-in-dependent mechanism.
DISCUSSION
Cells have evolved numerous strategies to identify the threat posed by a viral pathogen and produce autocrine and paracrine re-sponses designed to curtail virus replication and spread. These responses may vary in type or strength between different cell and tissue types. Viruses, in turn, have developed strategies to success-fully neutralize host antiviral defenses and replicate robustly. Mu-tant viruses that lack one of these countermeasures are attenuated by the host response and so can be used to identify an antiviral function of a host protein in the response pathway. Here we used
the HSV-1 mutant⌬68H to identify autophagy as a cellular
anti-viral response regulated by cellular PrP.⌬68H is attenuated in the
nervous systems of normal mice because it encodes an ICP34.5 protein that cannot bind to and inhibit the proautophagy protein beclin-1, a function associated with neurovirulence. We found
that deletion of PrP in mice complements the defect in⌬68H,
restoring the mutant virus’ capacity to replicate in the CNS to wild-type virus levels after either intracranial or corneal routes of
infection. Thus, the⌬68H mutant revealed a role for PrP in the
host response to infection that wild-type virus masks through the autophagy-suppressing activity of its ICP34.5 protein. The
im-proved replication and virulence of a beclin-1 binding mutant in
PrP⫺/⫺mice suggests that the mice have a defect in autophagy in
the CNS.
The influence of PrP on the antiviral activity exerted by
au-tophagyin vivoappears to vary depending on the tissue examined.
After corneal infection,⌬68H and⌬68HR replicate equally well in
the ocular epithelium of normal mice through 4 days
postinfec-tion, as was previously observed (75), and also in PrP⫺/⫺mice.
This observation indicates that the inability of⌬68H to bind
be-clin-1 and inhibit autophagy is inconsequential to HSV replica-tion in corneal tissue over this time period. Fortuitously, this
ob-servation also indicates that equal amounts of⌬68H and⌬68HR
are available to enter the trigeminal ganglion. In trigeminal
gan-glia of either normal or PrP⫺/⫺mice, ⌬68H replicates poorly
compared with⌬68HR, suggesting that autophagy is induced in
the peripheral nervous system but that PrP has no discernible
regulatory effect in this site. Replication of⌬68H is restored to the
level of⌬68HR in the brainstems of PrP⫺/⫺mice after intracranial
inoculation, and also after corneal inoculation, despite the lower
input of⌬68H from the trigeminal ganglia. Although B6⫻129
PrP⫺/⫺ mice are generally more susceptible to infection with
HSV-1 than normal mice, the lethality of⌬68H matched that of
⌬68HR, possibly due to similar levels of replication in the
brain-stem, a region critical to basal functions, such as respiration. Thus, the negative regulatory effect that PrP exerts on virus replication
in vivoand presumably its stimulation of the autophagy pathway is observed specifically in the CNS.
Cultured primary astrocytes are a relevant and useful cell type in which to examine the role of cellular PrP in the antiviral
re-sponse to HSV infection. After corneal infection with a
-Gal-expressing HSV, we observed colocalization of-Gal with GFAP⫹
cells in brainstems, including dense staining in the astrocyte-rich trigeminal root entry zone. In cultures of brainstem-derived pri-mary astrocytes, the genetic absence of cellular PrP complements
the defect in⌬68H as it did in the brainstemin vivo, enabling
⌬68H to replicate equivalently with its rescue virus. Interestingly,
although cellular PrP is highly expressed in neurons and PrPscis
associated with neurodegeneration, primary astrocytes
recapitu-late thein vivovirus replication findings but primary neurons
from either the cortex or brainstem do not (K. Wylie, unpublished observation). Our data do not preclude the possibility that cellular PrP also regulates autophagy in neurons in response to HSV in-fection, but such a role was not revealed in our system. Whether
the restoration of⌬68H replication in PrP⫺/⫺brainstems reflects
FIG 8PKR levels and activity are intact in astrocytes from PrP⫺/⫺mice. Primary astrocyte cultures from wild-type (WT) B6 and PrP⫺/⫺mice were assessed for PKR levels (A) and PKR activity (B) by Western blotting. Proteins were separated by SDS-PAGE and detected by Western blotting with anti-PKR antibody in lysates of uninfected cells (A) or anti-phosphorylated eIF2␣antibody in lysates of uninfected cells or cells infected for 12 h with the indicated virus (B). Total eIF2␣was used as a loading control. The data are representative of two separate experiments in which independently derived astrocyte cultures were used.
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[image:8.585.137.450.65.165.2]increased replication in astrocytes will be a subject of future inves-tigation.
Virus infection can be considered a form of cell stress, and cell stress induces autophagy. Based on ultrastructural analysis, nor-mal astrocytes exhibited an increase in the number of
autophago-somes after infection with⌬68H compared with wild-type virus,
but PrP⫺/⫺astrocytes did not. Brief starvation also resulted in an
increase in the number of autophagosomes in normal but not
PrP⫺/⫺astrocytes. By using aggregation of EGFP-LC3 as an
inde-pendent measure of autophagosome formation, we again
ob-served that normal astrocytes but not PrP⫺/⫺astrocytes mounted
a significantly greater autophagic response to infection with⌬68H
than⌬68HR, indicating that wild-type virus maintains its control
over autophagy even after a prolonged period of time and PrP stimulates autophagy in response to the stress of virus infection.
Interestingly, however, PrP⫺/⫺astrocytes are not unresponsive to
all types of cell stress, because starvation increased formation of EGFP-LC3 aggregates with time. A third analysis of endogenous LC3-II accumulation was unable to detect consistent differences in LC3-I conversion to LC3-II, but addition of chloroquine to prevent LC3-II degradation demonstrated that more autophagic
flux occurs in normal astrocytes responding to⌬68H than to
⌬68HR. The smaller differences in endogenous LC3-II
accumu-lation between groups may have been due to the relative insensi-tivity of the Western blot technique. Different types of cells, ex-perimental stress, and methods of analysis could in part explain why some authors have noted a proautophagy role for PrP and others have not. For example, the authors of a previous study who used starved, transformed cell lines derived from hippocampal
neurons concluded that PrP was an inhibitor of autophagy (31).
In contrast, we used low-passage-number primary brainstem as-trocytes undergoing HSV-1 infection. The strength of our study is
that we have bothin vitro andin vivodata which support the
FIG 9PrP regulates autophagy in cultured primary astrocytes. (A and B) Representative electron microscopic images of autophagosomes from normal primary astrocytes (A) and PrP⫺/⫺ primary astrocytes (B) infected with
⌬68HR are shown. (C and D) Autophagosomes were quantified from the images from infected cells (C) or untreated or serum-starved cells (D). Each data point represents the combined results (means⫾standard deviations) of three independent experiments using independently derived astrocyte cul-tures. For each condition in each experiment, autophagosomes were counted in 50 cell profiles (C) or 25 cell profiles (D). **,P⫽0.009; *,P⫽0.034; ns, not significant. Bar, 200 nm.
FIG 10PrP regulates LC3 distribution in cultured primary astrocytes. Cul-tures of primary astrocytes derived from brainstems of wild-type (WT) B6 or PrP⫺/⫺mice were transfected with a plasmid expressing EGFP-LC3 and 24 h later were mock treated, starved, or infected. (A and B) Photographs were taken of cytoplasmic portions of cells and scored by a masked observer. Rep-resentative images of unstressed cells (diffuse GFP appearance) (A) or stressed cells (B) (containing GFP punctae) are shown. Magnification,⫻60. (C and D) Graphs showing the proportion of cells scored as stressed in astrocyte cultures infected with⌬68HR or⌬68H for 24 h (C) or starved for 12 h or left untreated (D). The combined results of three independent experiments for a total of 45 to 79 images per cell type and condition are shown. ***,P⬍0.0001; ns, not significant.
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[image:9.585.330.511.65.398.2] [image:9.585.61.262.67.426.2]conclusion that PrP is a positive regulator of autophagy, at least in
response to virus infection. Interestingly, Alais et al. (11) recently
demonstrated that PrP restricts HIV-1 production in 293T cells by binding to the viral genomic RNA and reducing its translation rate. Thus, PrP may constitute a multifunctional antiviral defense mechanism.
Infection of cultured primary astrocytes at a high MOI revealed
a PrP-dependent effect on⌬68H replication. The attenuation of
⌬68H correlated with a difference in induction of autophagy
rather than PKR levels or accumulation of phosphorylated eIF2␣
(Fig. 8). Infection of primary astrocyte cultures at a high MOI is
likely representative ofin vivoinfection of the brainstem, because
clusters of HSV-infected cells occur in discrete foci in the
brains-tems 4 days after corneal infection (Fig. 6) (71,74), suggesting
local release of large numbers of virions. eIF2␣phosphorylation
triggers the induction of autophagy (52), yet while eIF2␣
phos-phorylation is robust in PrP⫺/⫺astrocytes (Fig. 8), these cells
failed to induce autophagy in response to infection. Thus, cellular PrP regulates the stress-induced autophagic response by a mech-anism that occurs either independent of or downstream of the
phosphorylation of eIF2␣. PrP could have a role as a sensor of
cellular stress imposed by viruses or short periods of starvation, as a signaling molecule, or as an effector of enhanced autophago-some formation or maturation. Our data support the idea that the regulation of autophagy in response to cellular stress is nuanced and involves yet-undefined pathways. The mechanism of cellular
PrP induction of autophagy in response to⌬68H infection is
cur-rently being investigated.
While we have focused on the role of PrP in induction of au-tophagy, some observations on the autophagic response of cul-tured primary astrocytes to HSV-1 infection deserve mention. As-trocytes may utilize autophagy in several strategies to promote
cellular and host defense, which HSV-1 ICP34.5 governs to
differ-ent extdiffer-ents, depending on the cell type infected (33,43,44,57,76,
77). First, in electron micrographs we frequently observed virus
particles adjacent to phagophores and inside completed autopha-gosomes, suggesting that xenophagic degradation of virus parti-cles may be a defense mechanism of astrocytes that would also reduce virus release to neighboring neurons. Second, astrocytes express MHC class II and efficiently present antigens in the CNS
(78). The induction of autophagy may stimulate a vigorous CD4 T
cell response by catabolizing viral proteins in autophagosomes for
MHC-II-mediated presentation (35, 57). Third, we observed
IFN-production by primary astrocytes in response to HSV
in-fection, which may further enhance autophagy or may itself be an
important cellular antiviral defense (79). Macroautophagy
regu-lates the type I IFN response to virus infection in dendritic cells
(80). Conceivably,⌬68H permits enhanced signaling for type I
IFN production in infected astrocytes because it cannot inhibit autophagy. Autophagosome maturation as well as
IRF3-depen-dent type I IFN production requires TBK1 (81), and HSV-1
ICP34.5 binds TBK1 via a domain that partially overlaps the
be-clin-1 binding domain (45). Greater production of IFN-by
pri-mary astrocytes infected with⌬68H than⌬68HR suggests possible
loss of TBK1 binding function in addition to beclin-1 binding, although this observation is at odds with the unrelieved
attenua-tion of⌬68H in IRF3⫺/⫺mice (75). Further investigation into the
cause and effect of enhanced IFN-production in⌬68H-infected
astrocytes will therefore be required.In toto, our data are
consis-tent with a model in which astrocytes in the mouse CNS induce autophagy to inhibit virus replication and increase adaptive im-mune responses, helping to protect adjacent neurons from infec-tion. Participation of PrP in these defense mechanisms deserves further scrutiny.
In light of recent observations indicating cell-type-specific dif-ferences in reliance on xenophagy versus type I IFN to defend
against HSV-1 infection (79), it will be interesting to assess the role
of these two host defense mechanisms in primary brainstem as-trocytes. A paper published during revision of this report theo-rized that mitotic and nonmitotic cell types differ in their reliance
eIF2α
LC3-I LC3-II
Mock Starved Starved ∆68H
∆68H
∆68HR
∆68HR WT
PrP
-/-CQ - - + - + - +
A B
C D
CQ - - + - + - + eIF2α LC3-I LC3-II 0 5 10 15 20 WT De ns ity (L C3 -II /e IF 2 α ) MockStarved Starved + ∆68H
∆68H +∆68HR
∆68HR +
0 5 10 15 20 PrP
-/-D en sity (L C 3-II/e IF 2 α ) Mock
StarvedStarved + ∆68H∆68H +∆68HR ∆68HR +
FIG 11PrP regulates endogenous LC3-II accumulation in cultured primary astrocytes. Cultures of primary brainstem astrocytes were untreated or starved or infected at an MOI of 10 for 12 h in the absence or presence of chloroquine. (A and C) LC3-I and LC3-II were detected by Western blotting of normal B6 astrocytes (A) or PrP⫺/⫺astrocytes (C). (B and D) Quantification of Western blot results, showing relative band densities for LC3-II normalized to eIF2␣for normal astrocytes (B) and PrP⫺/⫺astrocytes (D). Results of one representative experiment (of the two performed) are shown.
FIG 12IFN-transcript increases equivalently after⌬68H infection in astro-cytes cultured from normal B6 or PrP⫺/⫺mice. Cultures of primary astrocytes from the brainstems of wild-type (WT) B6 mice and PrP⫺/⫺mice were in-fected at an MOI of 10 with⌬68H or⌬68HR or left uninfected. The levels of IFN-transcript were determined 12 h postinfection by using real-time re-verse transcriptase PCR and are presented relative to the levels found in unin-fected cells (set at 1). Each bar represents the combined results (mean⫾ stan-dard deviation) of triplicate cultures from two independent experiments. ***,
P⬍0.0001.
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[image:10.585.330.510.64.191.2] [image:10.585.43.285.65.265.2]on autophagy to restrict HSV-1 replication (79). Notably,
replica-tion of⌬68H is suppressed compared with⌬68HR in cultured
primary astrocytes from normal mice but not in other mitotic cell
types, namely, mouse embryo fibroblasts (43) and neuroblastoma
cells (33). Thus, primary astrocyte cultures may be a useful cell
type in which to evaluate the effects of cell division versus resi-dence in the nervous system on the autophagic response to HSV and to infection with other pathogens or types of stress, as well as participation of PrP in this response.
An understanding of the function(s) of PrP is critical for in-sight into the pathogenesis of prion diseases. PrP is not essential for autophagy but regulates the induction of autophagy over basal levels in response to HSV-1 infection. A nonessential role of PrP was predicted, because otherwise we would expect the deletion of PrP to be a lethal mutation at embryonic or postnatal stages due to the importance of autophagic degradation in embryogenesis and
maintenance of cell health (82,83). Furthermore, acquired defects
or haplo-insufficiency in essential components of the autophagy pathway, such as ATG7 or beclin-1, lead to neurodegeneration
and a higher incidence of cancer (83,84), but PrP⫺/⫺mice are
generally normal (24). Our observations suggesting that PrP
pos-itively regulates autophagy in the mouse CNS in response to in-fection imply that cell death observed in the CNS during prion diseases may be in part caused by loss of a protective function of
normal cellular PrP upon its conversion to PrPsc.
ACKNOWLEDGMENTS
We thank Diane Alexander and David Leib for providing wild-type HSV-1 strain 17,⌬68HR, and 17-6B viruses and the ICP34.5 mutants
⌬68H and⌬␥34.5. Nigel Stow graciously provided the ICP0 mutant dl1403. We also thank David Harris for providing (B6) PrP⫺/⫺mice, Skip
Virgin for the plasmid expressing EGFP-LC3, and Mike Green for expert technical assistance. We are grateful to Wandy Beatty for assistance with electron microscopy and autophagosome enumeration and to Barbara Nagel and Jan Ryerse for assistance with histology.
This work was supported by PHS award R21AI085206, a Saint Louis University Presidential Fellowship, and Institutional funds.
REFERENCES
1.Brown HR, Goller NL, Rudelli RD, Merz GS, Wolfe GC, Wisniewski HM, Robakis NK.1990. The mRNA encoding the scrapie agent protein is present in a variety of non-neuronal cells. Acta Neuropathol.80:1– 6. 2.Ford MJ, Burton LJ, Morris RJ, Hall SM.2002. Selective expression of
prion protein in peripheral tissues of the adult mouse. Neuroscience113: 177–192.
3.Manson J, West JD, Thomson V, McBride P, Kaufman MH, Hope J.
1992. The prion protein gene: a role in mouse embryogenesis? Develop-ment115:117–122.
4.Piccardo P, Safar J, Ceroni M, Gajdusek DC, Gibbs CJ, Jr. 1990. Immunohistochemical localization of prion protein in spongiform en-cephalopathies and normal brain tissue. Neurology40:518 –522. 5.Moser M, Colello RJ, Pott U, Oesch B.1995. Developmental expression
of the prion protein gene in glial cells. Neuron14:509 –517.
6.Dodelet VC, Cashman NR.1998. Prion protein expression in human leukocyte differentiation. Blood91:1556 –1561.
7.Durig J, Giese A, Schulz-Schaeffer W, Rosenthal C, Schmucker U, Bieschke J, Duhrsen U, Kretzschmar HA.2000. Differential constitutive and activation-dependent expression of prion protein in human periph-eral blood leucocytes. Br. J. Haematol.108:488 – 495.
8.Liu T, Li R, Wong BS, Liu D, Pan T, Petersen RB, Gambetti P, Sy MS.
2001. Normal cellular prion protein is preferentially expressed on sub-populations of murine hemopoietic cells. J. Immunol.166:3733–3742. 9.Chakrabarti O, Ashok A, Hegde RS.2009. Prion protein biosynthesis
and its emerging role in neurodegeneration. Trends Biochem. Sci.34:287– 295.
10. Linden R, Martins VR, Prado MA, Cammarota M, Izquierdo I, Bren-tani RR.2008. Physiology of the prion protein. Physiol. Rev.88:673–728. 11. Alais S, Soto-Rifo R, Balter V, Gruffat H, Manet E, Schaeffer L, Darlix JL, Cimarelli A, Raposo G, Ohlmann T, Leblanc P.2012. Functional mechanisms of the cellular prion protein (PrPC) associated anti-HIV-1 properties. Cell. Mol. Life Sci.69:1331–1352.
12. Miesbauer M, Rambold AS, Winklhofer KF, Tatzelt J.2010. Targeting of the prion protein to the cytosol: mechanisms and consequences. Curr. Issues Mol. Biol.12:109 –118.
13. Caughey B, Raymond GJ.1991. The scrapie-associated form of PrP is made from a cell surface precursor that is both protease- and phospho-lipase-sensitive. J. Biol. Chem.266:18217–18223.
14. Prusiner SB.1982. Novel proteinaceous infectious particles cause scrapie. Science216:136 –144.
15. Aguzzi A, Calella AM.2009. Prions: protein aggregation and infectious diseases. Physiol. Rev.89:1105–1152.
16. Kovacs GG, Budka H.2008. Prion diseases: from protein to cell pathol-ogy. Am. J. Pathol.172:555–565.
17. Westergard L, Christensen HM, Harris DA.2007. The cellular prion protein (PrPC): its physiological function and role in disease. Biochim. Biophys. Acta1772:629 – 644.
18. Isaacs JD, Jackson GS, Altmann DM.2006. The role of the cellular prion protein in the immune system. Clin. Exp. Immunol.146:1– 8.
19. Mehrpour M, Codogno P.2010. Prion protein: from physiology to can-cer biology. Cancan-cer Lett.290:1–23.
20. Miyazawa K, Emmerling K, Manuelidis L.2010. Proliferative arrest of neural cells induces prion protein synthesis, nanotube formation, and cell-to-cell contacts. J. Cell. Biochem.111:239 –247.
21. Mouillet-Richard S, Ermonval M, Chebassier C, Laplanche JL, Leh-mann S, Launay JM, KellerLeh-mann O.2000. Signal transduction through prion protein. Science289:1925–1928.
22. Schrock Y, Solis GP, Stuermer CA.2009. Regulation of focal adhesion formation and filopodia extension by the cellular prion protein (PrPC). FEBS Lett.583:389 –393.
23. Zhang CC, Steele AD, Lindquist S, Lodish HF.2006. Prion protein is expressed on long-term repopulating hematopoietic stem cells and is im-portant for their self-renewal. Proc. Natl. Acad. Sci. U. S. A.103:2184 – 2189.
24. Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp HP, DeArmond SJ, Prusiner SB, Aguet M, Weissmann C.1992. Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature
356:577–582.
25. Richt JA, Kasinathan P, Hamir AN, Castilla J, Sathiyaseelan T, Vargas F, Sathiyaseelan J, Wu H, Matsushita H, Koster J, Kato S, Ishida I, Soto C, Robl JM, Kuroiwa Y.2007. Production of cattle lacking prion protein. Nat. Biotechnol.25:132–138.
26. Ano Y, Sakudo A, Onodera T. 2012. Role of microglia in oxidative toxicity associated with encephalomyocarditis virus infection in the cen-tral nervous system. Int. J. Mol. Sci.13:7365–7374.
27. Levine B.2005. Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell120:159 –162.
28. Kabeya Y, Mizushima N, Ueno T, Yamamoto A, Kirisako T, Noda T, Kominami E, Ohsumi Y, Yoshimori T.2000. LC3, a mammalian homo-logue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J.19:5720 –5728.
29. Orvedahl A, Levine B. 2009. Eating the enemy within: autophagy in infectious diseases. Cell Death Differ.16:57– 69.
30. Barbieri G, Palumbo S, Gabrusiewicz K, Azzalin A, Marchesi N, Spedito A, Biggiogera M, Sbalchiero E, Mazzini G, Miracco C, Pirtoli L, Ka-minska B, Comincini S.2011. Silencing of cellular prion protein (PrPC) expression by DNA-antisense oligonucleotides induces autophagy-dependent cell death in glioma cells. Autophagy7:840 – 853.
31. Oh JM, Shin HY, Park SJ, Kim BH, Choi JK, Choi EK, Carp RI, Kim YS.
2008. The involvement of cellular prion protein in the autophagy pathway in neuronal cells. Mol. Cell. Neurosci.39:238 –247.
32. Park Y, Kim W, Kim AY, Choi HJ, Choi JK, Park N, Koh EK, Seo J, Koh YH.2011. Normal prion protein in Drosophila enhances the toxicity of pathogenic polyglutamine proteins and alters susceptibility to oxidative and autophagy signaling modulators. Biochem. Biophys. Res. Commun.
404:638 – 645.
33. Orvedahl A, Alexander D, Talloczy Z, Sun Q, Wei Y, Zhang W, Burns D, Leib DA, Levine B.2007. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe1:23–35.
