Human Cytomegalovirus Infection Causes ... - Journal of Virology

JOURNAL OF VIROLOGY, Apr. 2010, p. 3528–3541 0022-538X/10/$12.00 doi:10.1128/JVI.02161-09 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 84, No. 7

Human Cytomegalovirus Infection Causes Premature and Abnormal Differentiation of Human Neural Progenitor Cells䌤† Min Hua Luo,1,2‡ Holger Hannemann,1‡ Amit S. Kulkarni,1 Philip H. Schwartz,3 John M. O’Dowd,1 and Elizabeth A. Fortunato1* Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, Idaho 83844-30521; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, People’s Republic of China2; and National Human Neural Stem Cell Resource, Children’s Hospital of Orange County Research Institute, Orange, California 928683 Received 12 October 2009/Accepted 29 December 2009

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, largely manifested as central nervous system (CNS) disorders. The principal site of manifestations in the mouse model is the fetal brain’s neural progenitor cell (NPC)-rich subventricular zone. Our previous human NPC studies found these cells to be fully permissive for HCMV and a useful in vitro model system. In continuing work, we observed that under culture conditions favoring maintenance of multipotency, infection caused NPCs to quickly and abnormally differentiate. This phenotypic change required active viral transcription. Whole-genome expression analysis found rapid downregulation of genes that maintain multipotency and establish NPCs’ neural identity. Quantitative PCR, Western blot, and immunofluorescence assays confirmed that the mRNA and protein levels of four hallmark NPC proteins (nestin, doublecortin, sex-determining homeobox 2, and glial fibrillary acidic protein) were decreased by HCMV infection. The decreases required active viral replication and were due, at least in part, to proteasomal degradation. Our results suggest that HCMV infection causes in utero CNS defects by inducing both premature and abnormal differentiation of NPCs. ducing more severe outcomes (3, 46). However, the mechanism of HCMV pathogenesis in the developing CNS remains poorly understood. Studies of HCMV in human subjects have obvious limitations; therefore, model systems of both in vitro and in vivo HCMV infections have been devised to provide insights into infection of the developing brain. Congenital infection studies have been performed principally with the mouse model. Studies of mice revealed that very early embryos were nonpermissive to mouse cytomegalovirus (MCMV) infection, as judged by the absence of viral gene expression following blastocyst (25) or zygote (71) injection. Mouse embryonic stem (ES) cells were also nonpermissive to MCMV infection, but cells differentiated from these ES cells were susceptible and permissive (37). Mouse multipotent CNS stem cells (neural stem/progenitor cells [NPCs]) isolated from the ventricular/periventricular zones of both late-stage embryonic mouse and adult mouse brains were permissive for infection. It was reported that MCMV infection inhibited mouse NPC proliferation and differentiation. Neuronal differentiation appeared to be inhibited more severely than glial differentiation (28). Radial glial cells were the main targets of MCMV during infection in the neonatal (postnatal day 1 [P1] to P3) mouse (49, 73). These glial cells are thought to be the earliest neural stem cells and play an important role in guiding neuron migration (30). Immunostained brain slice cultures indicated that virus-susceptible cells were located in the subventricular zone and cortical marginal regions (areas positive for NPCs) (10, 27). Shinmura et al. (59) found that injection of MCMV into the cerebral ventricles of mouse embryos caused a profound disturbance of neuronal migration and a marked loss of neurons. They proposed that this disturbance might be a cause of

Congenital human cytomegalovirus (HCMV) infection is a leading cause of birth defects, primarily affecting the central nervous system (CNS). Primary infection during pregnancy poses a 30 to 40% risk of intrauterine transmission, with severe adverse outcomes more likely if the infection occurs within the first half of gestation (46). Each year, approximately 1% of all newborns are congenitally infected with HCMV. Approximately 5 to 10% of these infants manifest signs of serious neurological defects at birth, including deafness, mental retardation, blindness, microencephaly, hydrocephalus, and cerebral calcification (2, 4, 65). In addition, 10 to 15% of congenitally infected infants who are asymptomatic at birth subsequently develop brain disorders such as sensorineural hearing loss (12, 47, 52). Moreover, accumulating evidence suggests that more subtle changes in human brain development, such as autism and language development, may be related to congenital HCMV infection (68, 76, 77). Although HCMV can infect a wide range of tissues in vivo (61), the fetal brain is the principal site of the deleterious manifestations of infection. It has been suggested that the severity of neuropathological changes and clinical outcomes may be associated with the stage of CNS development at which congenital infection occurs, with early-gestation infections pro-

* Corresponding author. Mailing address: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844-3052. Phone: (208) 885-6966. Fax: (208) 885-6518. E-mail: [email protected]. † Supplemental material for this article may be found at http://jvi .asm.org/. ‡ Holger Hannemann and Min Hua Luo contributed equally to this work. 䌤 Published ahead of print on 13 January 2010. 3528

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HCMV INFECTION DISRUPTS NPC DIFFERENTIATION

microencephaly due to CMV infection. These mouse studies found that progenitor cells, as well as glial cells and neurons, were permissive to CMV infection. Recent advancements in human NPC isolation and culture (56) allow for the characterization of HCMV infection in this medically relevant human system. Previous studies from our group and others have shown that human NPCs are fully permissive for HCMV infection (11, 35, 38, 43, 44). Our studies found that the timing of viral gene expression and the titers of infectious virions produced in human NPCs were similar, although somewhat protracted, to those for permissive fibroblasts. These studies also showed that astroglia and neurons derived from cultured human NPCs were permissive for infection (35). In the current study, genome-wide expression analysis found downregulation of mRNA levels of several genes important for maintaining NPC multipotency and establishing their neural identity. Quantitative PCR (qPCR), Western blot, and immunofluorescence (IF) analyses performed at various times postinfection (p.i.) on four NPC marker proteins, namely, nestin (NES), doublecortin (DCX), sex-determining homeobox 2 (SOX2), and glial fibrillary acidic protein (GFAP), confirmed the array results. Inhibition of HCMV replication delayed protein level declines. Additionally, proteasomal inhibition delayed and decreased NPC marker protein declines. These results suggested that HCMV infection disrupted NPCs’ multipotency and, in the absence of exogenous differentiation signals, induced their differentiation toward a nonneuronal lineage, implying a mechanism for the CNS manifestations of HCMV pathogenesis. MATERIALS AND METHODS Cell culture. The human NPC line SC27 was obtained postmortem from the brain of a premature neonate (estimated gestational age, 23 to 24 weeks). The neonate died of natural causes unrelated to HCMV infection (56; unpublished results). NPCs were cultured as described previously (35). Briefly, NPCs were cultured in Dulbecco’s modified Eagle’s medium-F12 medium (DMEM-F12) containing L-glutamine (2 mM) (Glutamax; Gibco/BRL), penicillin-streptomycin (100 U/ml and 100 ␮g/ml), gentamicin (50 ␮g/ml), amphotericin B (2 ␮g/ml) (Fungizone; Gibco/BRL), 10% BIT9500 (5 mg/ml bovine serum albumin, 5 ␮g/ml recombinant human insulin, 100 ␮g/ml human transferrin; Stem Cell Technologies), human basic fibroblast growth factor (bFGF; Prospec) (20 ng/ ml), and human epithelial growth factor (EGF; Prospec) (20 ng/ml). NPCs were cultured as neurospheres by seeding cells into uncoated tissue culture dishes. Under these conditions, NPCs agglomerate into balls of free-floating cells. NPCs were cultured as adherent monolayers by seeding them onto fibronectin-coated dishes. For cell culture, the medium was refreshed by half every other day. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2. Virus infection of adherent NPCs. HCMV strain Towne (ATCC VR977) was used for all infections and was propagated and titrated on a monolayer of human foreskin fibroblasts as described previously (70). Prior to virus infection, NPC monolayer cells were seeded onto poly-D-lysine-coated dishes or uncoated dishes containing poly-D-lysine-coated coverslips. Cells were seeded, allowed to attach overnight, infected at a multiplicity of infection (MOI) of 3, and incubated with virus for 2 h to allow for adsorption. The inoculum was then removed and cells were refed (35). Medium was refreshed by half every other day as necessary. Cells and coverslips were harvested at the indicated times p.i. UV-inactivated HCMV was prepared and used as described previously (15). NPC neurosphere infection. NPC neurospheres were cultured as described previously (35, 51). To estimate the cell number before infection, an averagesized neurosphere was dissociated by trypsinization and cells were counted on a hemocytometer. NPC neurospheres were infected at an MOI of ⬃3 with virus or UV-inactivated virus, as described above. Cell and neurosphere morphology changes were monitored at the indicated times p.i. Experiments were performed at least twice. Drug treatments. 9-[(1,3-Dihydroxy-2-propoxy)methyl] guanine (ganciclovir [GCV]) (450 ␮M) was used to inhibit virus replication. Mock infection (with

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vehicle), mock infection plus GCV, virus infection (with vehicle), and virus infection plus GCV were included in all experiments. GCV was added at the time of infection and every 24 h thereafter; the medium was changed by half every other day. Cells and coverslips were harvested at the indicated times p.i. MG132, a proteasome inhibitor, was used to inhibit proteasome activity (78). MG132 was added at a final concentration of 12.5 ␮M at 24 h p.i. and incubated for 24 h prior to harvest at 48 h p.i. (78); analogously, 12.5 ␮M MG132 was added at 48 h p.i. and incubated for 48 h prior to harvest at 96 h p.i. Live-cell morphology changes. Cell morphology changes were monitored on an inverted Nikon TMS-F microscope (using a 20⫻ objective). Mock, virus, and UV-inactivated virus infections were monitored in parallel. A Nikon Coolpix 5400 camera was used to obtain the images. Experiments were performed at least twice, and representative images are shown. The magnification for all live-cell images presented is ⫻500. Western blotting. Cells were harvested at the indicated times p.i. by being rinsed with phosphate-buffered saline (PBS), trypsinized, and pelleted. Cell pellets were resuspended in ice-cold PBS, counted, and pelleted. Cell pellets were snap-frozen in liquid nitrogen and stored at ⫺80°C until the time course was completed. Cell lysates were prepared as described previously (34). Lysates were derived from 1 ⫻ 105 cells for analysis of viral proteins and 1.6 ⫻ 105 cells for cellular proteins, with the exception of SOX2, for which a total of 2.4 ⫻ 105 cell equivalents was used per lane. Lysates were electrophoresed using SDS-PAGE and transferred to a Protran membrane (Schleicher & Schuell BioScience). All Western blot experiments were performed twice, with representative images shown. IP of ubiquitinated/sumoylated DCX. Modifications of DCX were examined using immunoprecipitation (IP) of cell lysates from mock-infected or virusinfected NPCs, with or without 12.5 ␮M MG132 treatment. Briefly, 2.3 ⫻ 106 NPCs were lysed in 100 ␮l of denaturing buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 1% SDS, 12.5 ␮M MG132, and 1 mM dithiothreitol [DTT]), followed by direct boiling for 10 min to avoid coimmunoprecipitation of other modified proteins. The lysates were then diluted 10-fold in 50 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 12.5 ␮M MG132, 100 mM NaF, 0.2 mM sodium orthovanadate, 10 ␮g/ml leupeptin, and 10 ␮g/ml aprotinin, followed by incubation with protein A/G Magbeads (Genscript) for 1 h at 4°C on a rotary mixer to preclear the lysates. The precleared lysates were incubated with anti-DCX antibody (Ab) (2 ␮g) and allowed to rock gently at 4°C overnight. The next day, immunocomplexes were recovered by incubation with protein A/G Magbeads for 3 h at 4°C followed by four extensive washes with 1 ml of 250 mM NaCl, 0.1% NP-40, 12.5 ␮M MG132, 50 mM HEPES, pH 7.0, 5 mM EDTA, and 500 mM DTT. After each wash, the tubes were placed on a Dynal magnetic particle concentrator. The proteins were eluted by being heated at 95°C for 30 min in 2⫻ Laemmli sample buffer. Eluted proteins were used for immunoblotting with either anti-ubiquitin (rabbit; Biomol), anti-Sumo-1, or anti-Sumo-2/3 Abs (both rabbit; Cell Signaling). Dephosphorylation of proteins with SAP. Phosphorylation of DCX was examined using cell lysates from mock-infected or virus-infected NPCs, with or without MG132 treatment. Samples were run in duplicate in SDS-PAGE gels and transferred to Protran membranes. Prior to membrane blocking, half of the membrane, which contained duplicate samples, was cut and separately treated in the presence of 150 units shrimp alkaline phosphatase (SAP) (Fermentas), while the other half of the membrane was left untreated. The treated membrane was sealed in a plastic bag to maximize the exposure to SAP and incubated at 37°C overnight. The next day, both the SAP-treated and untreated membranes were probed for the presence of the ⬃82-kDa DCX band, using an anti-phospho-DCX (Ser334) Ab (Cell Signaling Technology). IF analysis. The IF protocol used in this study was described previously (35). Coverslips were mounted in glycerol containing para-phenylenediamine after staining to inhibit photobleaching. Nuclei were counterstained with Hoechst dye. The images were obtained using a Nikon Eclipse E800 fluorescence microscope equipped with a Nikon DXM camera and Metavue software. Abs. Viral antigens (Ags) were detected after Western blotting with anti-IE1/2 (Ch16.0; IgG1), anti-UL44 (IgG1), anti-pp65 (IgG1) (all from Rumbaugh-Goodwin Institute for Cancer Research, Inc.), and anti-gB (IgG2b; a kind gift from Bill Britt) monoclonal Abs (MAbs). Viral proteins were detected by IF analysis, using anti-IE1 (IgG2A; a kind gift from B. Britt) or anti-UL44 MAb. Cellular proteins were detected by Western blotting with anti-NES (IgG1; Chemicon), anti-DCX, anti-SOX2 (both goat polyclonal IgGs; Santa Cruz Biotechnology), anti-GFAP (IgG2b; Fitzgerald Industries International, Inc.), and anti-actin (clone ACT05; Neomarkers) Abs. Secondary Abs used for Western blotting were horseradish peroxidase-conjugated sheep anti-mouse or sheep anti-goat Abs (Amersham Bioscience). IF experiments used tetramethyl rhodamine isocyanate

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(TRITC)-conjugated anti-mouse IgG1 (Southern Biotech) and Alexa Fluor 488conjugated goat anti-mouse IgG2a (Invitrogen) Abs. qPCR. NPCs were cultured, infected (as described above), and harvested at the indicated times p.i. A total of 5 ⫻ 105 cells/sample was used for total RNA extraction. Total RNA was extracted from cells by using an SV Total RNA isolation kit (Promega). RNA was reverse transcribed with Superscript II (Invitrogen) or ImProm-II (Promega) reverse transcriptase. qPCR was performed on an ABI Prism 7900 instrument (Applied Biosystems), using Power SYBR green PCR master mix (Applied Biosystems) in 15-␮l reaction mixtures for 40 PCR cycles. Calculations were based on absolute starting quantities, using reactions specific for glucose-6-phosphate dehydrogenase (G6PD) as normalization controls (60). Each experiment consisted of three technical replicates, and their averaged values were used for downstream calculations. Experiments were performed on two separate occasions, and their averages are shown in the bar charts. Error bars represent ranges. Molecular cloning. All plasmids used in qPCR experiments were generated with a pCR8 Topo cloning kit (Invitrogen). Taq polymerase was used to amplify the insert fragments used as standards in qPCR experiments. Oligonucleotide sequences are available upon request. Whole-genome expression analysis. For gene expression analysis, adherent monolayer cells were either mock or virus infected. Mock-infected cells were harvested at 12 h p.i., and virus-infected cells were harvested at 4, 12, and 24 h p.i. Virus and mock infections were performed in duplicate, with each individual sample harvested separately, thereby providing biological replicates for analysis. RNA was extracted using an SV Total RNA isolation kit (Promega). First- and second-strand cDNAs were synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen) and poly(T) primers containing a sequence recognized by T7 RNA polymerase. The resulting cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction, using a BioArray high-yield RNA transcript labeling kit (T7; Enzo Diagnostics). Fifteen micrograms of the resulting biotin-tagged cRNA was fragmented to strands of 35 to 200 bases according to prescribed protocols (Affymetrix). Subsequently, 10 ␮g of this fragmented target cRNA was hybridized (45°C with rotation for 16 h in an Affymetrix GeneChip 640 hybridization oven) to probe sets present on an Affymetrix HG-U133 2.0 Plus array. The GeneChip arrays were washed and then stained (streptavidin-phycoerythrin [SAPE]) on an Affymetrix 450 fluidic station, followed by scanning on an Affymetrix GeneChip 3000 7G scanner. Whole-genome expression data analysis. Raw fluorescence data were normalized using MAS5 software. All features classified as absent in all samples by the MAS5 algorithm were omitted from further analysis. A second call of presence/ absence was calculated by MAS5 for each feature, based on the individual P values. The present/absent calls for the biological replicates were combined, requiring both replicates to be present for the feature to be considered present. The signal strengths (SSs) of the two biological replicates were averaged. A threshold signal strength (TSS) was determined for samples obtained from mock infection and virus infection, at 4 h p.i., at 12 h p.i., and at 24 h p.i. The TSS was set at the absolute SS value at which 5% of the absent features exceeded this number (mock ⫽ 220, 4 h ⫽ 222, 12 h ⫽ 234, and 24 h ⫽ 236). Gene regulation was classified as “on,” “off,” or “fold regulated” (see Table S1 in the supplemental material). Genes classified as turned on by HCMV were absent and below the mock TSS (m-TSS) in the mock sample and present and above the time point TSS (tp-TSS) in the virus-infected sample. Genes classified as turned off by HCMV were present and above the m-TSS in the mock sample and absent and below the tp-TSS in the virus-infected sample. Fold-regulated genes were those present in both the mock and viral samples for which both features were above their respective TSSs. All fold regulation comparisons were for mock samples versus virus samples. To further narrow the fold-regulated group, the distribution of the features meeting the above parameters was examined, and a fold regulation significance factor was determined for each time point. The significance factor was set to the value along the distribution curve where the curve flattened to a gradual asymptotic slope (see Fig. 2A). The significance factors used were as follows: for 4 h, threefold; for 12 h, sixfold; and for 24 h, eightfold. Additionally, genes were also considered fold regulated if they were present in both mock and viral samples but below their respective TSSs if the mock sample/virus sample comparison exceeded the significance factor when the appropriate TSS value was substituted. The fold-regulated entry reported in the table for these genes is either mock/tp-TSS or m-TSS/virus. Any gene represented by more than one feature after the above culling was included in Table S1 in the supplemental material and was listed using the most regulated feature (since oligonucleotides for features with less regulation might be located in untranscribed regions or in alternatively spliced exons). A gene represented by one (or more) feature(s) being turned on or off but which was also fold regulated was included in the fold-regulated category. Classification of

J. VIROL. gene product function used the latest annotation information from the Affymetrix website. Microarray data accession number. Original MAS5 data for the whole-genome expression analysis were deposited in the Gene Expression Omnibus (GEO) database under accession number GSE19345.

RESULTS HCMV infection alters NPC attachment and migration behaviors. Standard culture protocols call for NPCs to be grown either as free-floating neurospheres over uncoated surfaces (Fig. 1A) or as monolayers on fibronectin-coated surfaces (not shown). When transferred to fibronectin-coated plates, neurospheres attached to the coated surface and cells subsequently spread onto the substrate (Fig. 1B) (14, 51, 56). In our previous studies, we observed that when NPC neurospheres underwent glial differentiation by replacement of mitogens (EGF and bFGF) with fetal bovine serum, they attached tightly to uncoated surfaces and cells began to spread across the surface within 2 h (35; our unpublished data). We also observed that NPCs cultured as monolayers reverted to neurospheres when they were transferred to uncoated surfaces in the absence of a differentiating stimulus. This reversion to neurospheres illustrates the propensity of these cells to resume their normal behavior patterns in the absence of outside influences. During our current studies, we observed a corollary, but much more intriguing, effect in addition to the attachment behavior noted above. In parallel with other ongoing NPC monolayer infections, NPC neurospheres in uncoated dishes were infected with HCMV. These neurospheres were grown under conditions designed to maintain multipotency, but following HCMV infection they spontaneously attached to the uncoated plates, suggesting that differentiation had been initiated. Between 4 and 12 h p.i., individual cells began migrating from the spheres (Fig. 1C). Cells withdrew back into spheres at 18 h p.i. and exhibited the normal cell-rounding phenotype of permissively infected cells (8). By 96 h p.i., these infected NPCs began to spread again. During the second spread, the NPCs’ morphology resembled that of HCMV-infected fibroblasts at late (L) times p.i. (8). All infected NPC neurospheres were disassociated by 120 h p.i., consistent with the observation of others (72). UV-inactivated virus infections of neurospheres did not lead to attachment or migration (Fig. 1D). These experiments found that HCMV infection altered NPC neurosphere behavior in culture and that de novo viral protein expression was necessary to induce the altered attachment characteristics. Furthermore, the HCMV-infected NPCs’ attachment behavior was similar to that of NPCs under glial differentiation conditions (35; our unpublished data), which suggested a change in gene expression profiles. HCMV-induced alterations of NPC neurosphere attachment and migration are associated with a loss of multipotency. The similarities in attachment characteristics between the NPCs undergoing glial differentiation and the infected neurospheres prompted us to perform whole-genome expression analysis of infected NPCs, paying particular attention to selfrenewal and differentiation characteristics. A minor obstacle presented itself in our investigation of the HCMV-infected NPC neurosphere attachment behavior. The multilayer structure of free-floating neurospheres renders simultaneous infection of all component cells impossible. Unfortunately, asyn-

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FIG. 1. HCMV infection caused NPC neurospheres to attach and spread on uncoated surfaces, emulating differentiation. Neurospheres were cultured and infected as described in Materials and Methods. (A) Uninfected neurospheres in uncoated dishes. (B) Normal cell migration at 24 h postplating (hpp) on fibronectin-coated dishes. (C) Time course of morphology changes induced by HCMV infection of neurospheres. (D) Infection using UV-irradiated virus did not induce attachment of neurospheres. Magnification for all live-cell images ⫽ ⫻500.

chronous infections confound all timing-dependent results. We reasoned that the altered attachment behavior had been initiated solely by the accessible cells, since no other environmental changes were made. Therefore, we used monolayer cultures

for all further experiments. NPCs were infected at an MOI of 3 and harvested at 4, 12, and 24 h p.i. for mRNA analysis. These data sets were compared to those for mock-infected NPCs harvested at 12 h p.i. Although not as prevalent as those

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FIG. 2. HCMV infection altered mRNA expression levels of multiple cellular genes. Whole-genome expression analyses were performed at 4, 12, and 24 h p.i. as described in Materials and Methods. (A) Numbers of significant genes were plotted versus fold regulation to determine cutoffs for each time point, as described in Materials and Methods. (B) Genes of known function determined to be significantly up- or downregulated at 4, 12, or 24 h p.i. were categorized into functional groupings. The genes in the Neural Related category are included among the eight other functional groups, in addition to being separated for emphasis. TS, transcription factors.

at later time points, significant changes in mRNA levels began to occur as early as 4 h p.i., as outlined below and depicted in Fig. 2, Table 1, and Table S1 in the supplemental material. For NPCs, we found that HCMV infection caused changes in the regulation of 721 genes; 477 were downregulated and 244 were upregulated at 4, 12, or 24 h p.i. (see Table S1 in the supplemental material). The functions of 492 of these genes are currently known, and of these, 322 were downregulated and 170 were upregulated (percentages noted below represent fractions of genes of known function). Functional groupings revealed genes whose products are involved in cell cycle control, cell signaling, and metabolism. Ion channels, secreted proteins, cell surface receptors, transcription factors, and structural proteins were also represented in the array analysis. Representative genes from each of these classes are given in Table 1, and their distribution among different functional groups is shown in Fig. 2B. It is known that cellular transit through the G1/S checkpoint is essential for the optimal replication of HCMV (as reviewed in reference 54), and therefore, it was not surprising that many cell cycle control proteins were upregulated (or turned on) by infection of NPCs. These included the genes encoding cyclin E, E2F1 and -2, and cdt1, which were also influenced by infection of permissive fibro-

blasts (62). The cell cycle genes downregulated by infection of NPCs included that for the cell signaling protein NEDD9, which is involved in neural crest cell migration (1). The gene expression analysis revealed a number of upregulated cell surface receptors and signaling genes involved in immune functions or stress responses, including CD55, which accelerates the decay of complement proteins and prevents immune system damage to the host cells (41). A more significant number of genes were downregulated. A noticeable number of glycosyl transferases and other adhesion molecule modifying enzymes localizing to the Golgi apparatus (ST6GALNAC4 and -5, ST8SIA3, ST8SIA4, and GCNT1) were downregulated. In addition, seven different collagens (including COL1A1 and -A2 and COL6A1) and five adhesion proteins (PCDH10, NRP1 and -2, NFASC, and NLGN4X) had significantly reduced mRNA levels in virus-infected cells. These changes to the cells’ secretion patterns, many of which began as early as 4 h p.i., may have contributed to the altered adhesion characteristics of HCMV-infected NPC neurospheres seen at this early time point. Interestingly, ⬃17% of all known genes were neuron related. We found neuron-related genes among every class of genes present. These were particularly prevalent in the receptor

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TABLE 1. Infection of NPCs affects expression of cellular genes in numerous functional groupingsa Gene

CCNE1 CDC25A CDT1 CENPE E2F1 HDAC9 MCM5 NEDD9 AVIL DCX EIF4G3 FANCA NDP NPY2R RAD51 RTN4 UBE1L UBE2J2 CCK COL1A1 COL1A2 LAMA4 NFASC NRG1 SYN2 CACNB2 CHRNA9 KCNQ2 SCN1A CLN8 FABP7 GAD2 ST8SIA4 AQP4 CD55 GRIA1 NRP1 NRXN3 NTRK2 SEMA6D TLR4 GFAP H2AFY NEFH NELL1 NES ID4 JUN MIR21 OLIG1 SOX2 SOX4 SP100

Functional classb

Gene product

Cyclin E1 Cell division cycle homolog A Chromatin licensing and DNA replication factor 1 Centromere protein E E2F transcription factor 1 Histone deacetylase 9 Minichromosome maintenance deficient 5 Neural precursor cell expressed, developmentally downregulated 9 Advillin Doublecortin Eukaryotic translation initiation factor 4 gamma 3 Fanconi anemia, complementation group A Norrie disease (pseudoglioma) Neuropeptide Y receptor Y2 RAD51 homolog (Saccharomyces cerevisiae) (RecA homolog, Escherichia coli) Reticulon 4 Ubiquitin activating enzyme E1 like Ubiquitin conjugating enzyme E2, J2 Cholecystokinin Collagen, type I, alpha 1 Collagen, type I, alpha 2 Laminin, alpha 4 Neurofascin homolog (chicken) Neuregulin 1 Syapsin II Calcium channel, voltage dependent, beta 2 subunit Cholinergic receptor, nicotinic, alpha 9 Potassium voltage gated channel, KQT-like subfamily, member 2 Sodium channel, voltage gated, type I, A Ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive) Fatty acid binding protein 7, brain Glutamate decarboxylase 2 (pancreatic islets and brain) ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 Aquaporin 4 CD55 molecule, decay accelerating factor for complement Glutamate receptor, ionotropic, AMPA 1 Neuropilin 1 Neurexin 3 Neurotrophic tyrosine kinase, receptor, type 2 Sema domain, transmembrane domain (TM), and cytoplasmic domain (semaphorin) 6D Toll-like receptor 4 Glial fibrillary acidic protein H2A histone family, member Y Neurofilament, heavy polypeptide Nell-like 1 Nestin Id-related helix-loop-helix protein Id4 Jun oncogene microRNA 21 Oligodendrocyte transcription factor 1 SRY (sex determining region Y) box 2 SRY (sex determining region Y) box 4 SP100 nuclear antigen

CC CC CC CC CC CC CC CC CS CS CS CS CS CS CS CS CS CS EXO EXO EXO EXO EXO EXO EXO IC IC IC IC ME ME ME ME REC REC REC REC REC REC REC REC STR STR STR STR STR TR TR TR TR TR TR TR

Regulation 4 h p.i.

12 h p.i.

Up On On On Down

24 h p.i.

On On On On

Off Down Off On

Off Down Off On

Down On Off On Down Down Off Off Off

Down Off Off Down

On

On Down Down On Down

Off On

Down Off Down On

Down

Down*

Down* Down Down Down Off

Off On Off Off On On Down Off Off Off Down Down Off Off On Down Down Down Off On Down Down Down Off Down Off Off On On Off Down* Down Down Down Down Down* Down Off

a Representative genes from each of the functional groupings are listed. At each time p.i., whether or not each gene is turned off or on or down- or upregulated (down or up, respectively) is indicated. Note that NES was included in this table due to its importance as an NPC marker. Although it was downregulated (3-fold), it did not meet the stringent threshold requirements of the microarray analysis and was therefore not included in Table S1 in the supplemental material. Its entry, and those of the SOX2 gene at 4 and 24 h p.i. (2.5-fold and 6.5-fold, respectively) and of MIRN21 at 12 h p.i. (5-fold), are marked with asterisks. b CC, cell cycle; CS, cell signaling; EXO, secreted proteins; IC, ion channel; ME, metabolism; REC, receptor; STR, structural; TR, transcription.

category, with 52% (22/42 genes) of these genes being neuron related. Remarkably, 86% of these neuron-related receptor genes (19/22 genes) were downregulated and included those for the glutamate receptors GRIA1, -2, and -3 and the neural adhesion molecules NRP1 and -2 and NRXN3. Additional neuron-related genes that were down-

regulated included those for peripheral myelin protein 2 (PMP2) and neuron-specific ion channel subunits CACNB2, CACNG4, and CACNG5, as well as those for the transcription factors ATXN3, RUNX4, and SOX4, a pan-neuronal gene activator that acts during neurogenesis and neuronal maturation (5). These results suggest that HCMV-infected

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FIG. 3. NPC marker proteins were downregulated at the mRNA level and decreased at the protein level by HCMV infection. Monolayer NPCs were infected with HCMV and harvested at the indicated times p.i. for analysis of either the mRNA or protein level. (A) qPCR analysis of NES, DCX, SOX2, and GFAP mRNA levels from 0 to 144 h p.i. Log10 ratios of viral/mock values are given. Calculations were based on absolute starting quantities, using reactions specific for G6PD as normalization controls. The averages of two experiments are shown in the bar charts. Error bars represent the ranges. (B) NPC marker protein steady-state levels at early times p.i. (C) NPC marker protein steady-state levels at late times p.i. Actin was used as a loading control for panels B and C. M, mock infected; V, virus infected.

NPCs have a diminished capacity to differentiate in a neuronal direction. The most striking result from the gene expression analysis was the regulation of genes involved in the maintenance of the self-renewing, multipotent state of the NPC. There were large changes for several important genes, including those for (i) the transcription factor ID4, whose downregulation is associated with “precocious” differentiation (36); (ii) the structural protein GFAP and the cell signaling-related protein DCX, both of which are present in most human NPC cultures (20, 40, 45, 56, 64, 69); (iii) SOX2, a transcription factor associated with the maintenance of stem cell identity and which was recently shown to be essential for reprogramming of terminally differentiated fibroblasts to induced pluripotency (45, 64, 69); (iv) the microRNA MIR21, whose loss is associated with the subsequent loss of SOX2 (29); and (v) FABP7, a fatty acid transporter which, with the intermediate filament protein nestin (NES), is associated with neural stem cells and the developing brain (24). The large downregulation of numerous critical progenitor cell markers indicated the infected NPCs’ loss of multipotency via differentiation. Given the large number of neu-

ronal genes that were downregulated, this differentiation appeared to exclude a neuronal lineage. Multipotency gene expression is downregulated at the mRNA level and decreased at the protein level by HCMV infection. Because gene expression analysis had shown that the important NPC genes encoding DCX, SOX2, and GFAP were downregulated, their expression changes at the mRNA and protein levels were further investigated throughout the time course of infection. Additionally, NES, a protein specific for NPCs under our culture conditions (56), was included in the experiments (even though NES regulation did not meet the gene expression criteria, it was downregulated approximately threefold). qPCR analysis of these four genes showed all of their expression levels to be decreased at least 10-fold at 24 h p.i., with DCX and GFAP levels decreased ⬎100-fold (Fig. 3A). mRNA expression levels remained low throughout the duration of the experiment for all four genes. The protein levels of NES, DCX, SOX2, and GFAP were not decreased at early times p.i. (Fig. 3B), but beginning at 48 h p.i., steady-state protein levels began to decline in virus-infected cells. Each of the proteins displayed a different profile (Fig. 3C). The most

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rapid decrease in steady-state protein levels observed was for the NPC transcription factor SOX2, whose level was noticeably decreased at 48 h p.i. and undetectable from 72 h p.i. on. GFAP was reduced at 48 h p.i. and became undetectable by 120 h p.i. Interestingly, the faster-migrating form of the protein disappeared more rapidly. DCX levels declined at 72 h p.i. and were undetectable from 96 h p.i. on. NES levels were markedly decreased at 96 and 120 h p.i. and undetectable at 144 h p.i. These experiments found that HCMV infection significantly inhibited NPC marker mRNA synthesis and decreased the steady-state levels of these marker proteins, thereby compromising the multipotency and limiting the differentiation capacity of these developmentally critical cells. Downregulation of NPC multipotency genes is delayed when virus replication is inhibited. To narrow the list of viral factors responsible for the downregulation of NPC markers during HCMV infection, GCV was used to inhibit virus replication. GCV selectively and competitively inhibits the viral DNA polymerase, terminating viral replication and preventing late (L) protein synthesis (16). The following viral proteins were used as markers of the temporal progression of infection to monitor GCV’s effects: IE72 (IE1) and IE86 (IE2), the most prominent proteins of the immediate-early (IE) stage; UL44, a representative of early (E) proteins and of the development of replication centers; and two structural proteins, the tegument protein pp65 and the glycoprotein gB, employed to mark the transition to the L stage of HCMV infection. The stages and development of viral replication centers in NPCs have been described previously (35). Briefly, at 48 and 72 h p.i., a mixture of multiple small foci (indicating prereplication sites), bipolar foci, and single large foci (both indicative of advanced replication) was observed. The majority of cells developed single large replication foci by 96 h p.i. In NPCs, GCV inhibited the establishment and development of viral replication centers, similar to its effect on virus-infected fibroblasts (Fig. 4A and B) (48). All cells were positive for IE1 among both untreated cells and GCV-treated cells by 48 h p.i. (Fig. 4A). Indirect IF assay determined that untreated cells developed replication foci by 48 h p.i. In a small percentage of GCV-treated cells, multiple small foci were seen by 96 h p.i., but in the large majority of these treated cells, distinct UL44 foci did not form through 96 h p.i. (Fig. 4A). In Fig. 4A, the brightness levels of UL44 were increased in the far right panels to show that even under overexposed conditions, no foci were observed in the GCV-treated cells. Steady-state protein levels of IE1/IE2, UL44, pp65, and gB expression were determined by Western blotting (Fig. 4B). GCV significantly inhibited the expression of IE2 and UL44. pp65 and gB were completely blocked and were undetectable in GCV-treated cells during the 144-h time course. IF assay and Western blotting determined that GCV blocked viral protein expression between the IE and E stages in NPCs and, judging from the absence of replication centers, inhibited viral replication as expected. The protein levels of the NPC marker proteins NES, DCX, SOX2, and GFAP were determined for GCV-treated and untreated cells at various times p.i. (Fig. 4C). As observed for untreated infected cells (Fig. 3), the steady-state levels of all four proteins declined by 96 h p.i. compared to those in uninfected controls. However, in GCV-treated infected cells, this decline was much less dramatic. In GCV-treated infected cells,


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FIG. 4. Decreases in NPC markers at the protein level were delayed by GCV treatment. Monolayer NPCs were infected with HCMV, either with or without GCV, as described in Materials and Methods. Cells were analyzed at the indicated times p.i. (A) Cells on poly-Dlysine-coated coverslips were analyzed at the indicated time points, fixed, and stained for IE1 expression and UL44 focus formation and development. Infection without GCV is shown in the left panels, and infection with GCV is shown in the right panels. In the far right panels, brightness levels of UL44 were increased/adjusted (adj.) to show that even after overexposure, no foci were present in GCV-treated cells. At 72 and 96 h p.i., the UL44 exposure time for untreated cells (0.15 s) was half that for GCV-treated (unadjusted) cells (0.3 s) due to increasingly higher levels of UL44 in the untreated cells (as can be observed in panel B). Nuclei were counterstained with Hoechst dye (H). Bar ⫽ 5 ␮m. (B) Viral protein steady-state levels were assessed in the presence and absence of GCV. Viral IE (IE1/IE2), E (UL44), E-L (pp65), and L (gB) antigens were assessed. (C) Steady-state levels of NPC marker proteins were determined for the same time course. Actin was used as a loading control for panels B and C.

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FIG. 5. Downregulation of NPC markers at the mRNA level was delayed by GCV treatment. mRNA levels of NPC markers were determined by qPCR at the indicated times p.i. as described in the legend to Fig. 3. Each of the four proteins is represented by three shades of one color. The darkest shade of each color is the untreated viral/mock ratio; the middle shade is the GCV-treated virus/mock ratio; and the lightest shade is the UV-inactivated virus/mock ratio.

three of the four marker proteins were detectable until 144 h p.i., past the times when any of these proteins were detectable in untreated infected cells. We found that active viral replication in HCMV-infected NPCs contributed significantly to the decrease in NPC marker protein steady-state levels. qPCR was used to investigate the changes in NPC marker mRNA levels after GCV treatment (Fig. 5). The gene expression data in Fig. 3 revealed that mRNA level changes (virusinfected versus mock-infected cells) occurred by 24 h p.i., and therefore samples from virus-infected (v), virus-infected and GCV-treated (GCV), and UV-inactivated virus-infected (UV) cells were harvested at 12, 24, and 48 h p.i. and compared to mock-infected (m) cells. The last time point was included to account for any delay caused by GCV treatment. Although nearly all of the virus-infected cells (with or without GCV) stained positive for IE1 by 12 h p.i., at this time point no significant changes in the NPC markers could be measured in any comparison (v versus m, GCV versus m, and UV versus m). However, at 24 and 48 h p.i., all four NPC markers in the v/m comparison were downregulated to levels similar to those in Fig. 3 (Fig. 5, darkest shade of each color). GCV treatment of HCMV-infected cells delayed and decreased the decline in mRNA level for all four genes analyzed (Fig. 5, middle shade of each color). For example, GFAP was downregulated 34-fold at 24 h p.i. and 180-fold at 48 h p.i. in virus-infected cells but ⬍3-fold at 24 h p.i. and ⬃10-fold at 48 h p.i. in GCV-treated infected cells. Similar results were obtained for SOX2, DCX, and NES. Overall, the expression levels of the measured genes were ⬃10-fold lower at 24 and 48 h p.i. in virus-infected cells than in GCV-treated infected cells. No differences were observed in mRNA (Fig. 5, lightest shade of each color) and protein (data not shown) levels for the analyzed genes between cells infected with UV-inactivated virus and mock-infected cells at any time point. These data indicated that de novo viral transcription and protein synthesis coupled with active viral replication were required to induce the complete observed reduction in NPC marker proteins. Proteasomal inhibition slows decline in steady-state levels of NPC marker proteins. Since modification to the ubiquitin-

proteasomal machinery has been noted in HCMV-infected cells (reviewed in reference 54), mock- and virus-infected cells were treated with MG132 to determine if proteasomal degradation was involved in the decline of steady-state levels of the NPC markers. SOX2 was reduced to near the detection limit at 48 h p.i., and NES, DCX, and GFAP were reduced by 96 h p.i. (Fig. 3); therefore, MG132-treated cells and controls were harvested at these time points. The literature varies on the concentration of MG132 used to fully inhibit proteasomal activity, from a low of 0.1 ␮M to a high of 50 ␮M (17, 26). We chose an intermediate level of 12.5 ␮M MG132 for our studies. Kaspari and colleagues showed that in high-MOI infections, MG132 at concentrations above 0.5 ␮M did not inhibit IE1/ IE2 expression but did inhibit viral replication and concatemer cleavage (26). Importantly, these cells were continuously treated with MG132 for 72 h. To limit the inhibitory effects of MG132 on HCMV infection while achieving proteasomal inhibition, treatment was begun at 24 h p.i. (and cells harvested at 48 h p.i.) or at 48 h p.i. (and cells harvested at 96 h p.i.). Under these conditions, IE1 and IE2 expression was not inhibited by MG132 at 48 h p.i. or 96 h p.i. (Fig. 6A). However, there was at least partial inhibition of E protein synthesis (as measured by UL44) and complete inhibition of L protein synthesis (as measured by gB). We concluded that, as seen previously, our incubation conditions for MG132 also partially inhibited viral replication. NES protein levels were unaffected at 48 h p.i. under any conditions. However, at 96 h p.i., the HCMV-induced decrease in NES protein level was inhibited by the addition of MG132 (Fig. 6B). At 48 and 96 h p.i., DCX protein levels were slightly higher with MG132 treatment in both mock- and virus-infected samples. In addition, at both 48 and 96 h p.i., all MG132treated mock- and virus-infected cells showed a slower-migrating protein band of ⬃82 kDa. GFAP protein levels were unchanged at 48 h p.i. but were slightly elevated in both MG132-treated mock- and virus-infected cells at 96 h p.i. At both 48 h p.i. and 96 h p.i., SOX2 levels were unchanged after MG132 treatment in mock-infected cells, indicating that the proteasome was not generally involved in SOX2 degradation

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FIG. 6. Treatment of cells with MG132 partially blocked NPC marker protein degradation. Monolayer NPCs were mock or virus infected, treated with MG132 (12.5 ␮M) or left untreated as described in Materials and Methods, and harvested at the indicated times p.i. (A) Viral protein steady-state levels. (B) NPC marker protein steady-state levels. (C) DCX steady-state levels at 48 h p.i. Two exposure times for the same blot are shown. (D) IP using anti-DCX Ab and then Western blotting (WB) using anti-ubiquitin and anti-sumo-1 Abs. (E) Duplicate samples were run in a single gel and transferred to a Protran membrane, and then half the blot was treated with SAP as described in Materials and Methods prior to probing with anti-phospho-DCX Ab. Blots were then probed for actin as loading and staining controls. For panels C, D, and E, MG132 was added to the lysis and IP buffers.

under normal cellular conditions. However, from undetectable levels in untreated infected cells, SOX2 protein levels increased significantly in MG132-treated infected cells, indicating that proteasomal degradation was involved in the decline observed during infection. The more slowly migrating form of DCX, which appeared only after proteasomal inhibition, was investigated to discern whether this was a sumo- or ubiquitin-modified form of the protein. The DCX Western blots were repeated using shorter and longer exposures to discern differences. Also, MG132 was added to the sample buffer during lysate preparation to inhibit any latent proteolytic activity. The shorter exposure in Fig. 6C shows a large increase in the steady-state level of DCX in both mock- and virus-infected samples treated with the drug. A longer exposure with MG132 in the lysate revealed a laddered effect above the main DCX band, indicating modification of the protein. This effect was more prominent in the viral samples. DCX was immunoprecipitated from the lysates and run in a Western blot (Fig. 6D). These blots were probed with antiubiquitin and anti-sumo2/3 (not shown) Abs. Only very minor modifications occurred in the mock MG132-treated samples,

whereas the virus-infected cells had significant modification of both ubiquitin and all sumo moieties tested on the DCX protein. Combined with the observed effects of MG132 treatment (Fig. 6B), these IP results indicate that the decreased steadystate levels of the NPC marker proteins (particularly DCX, NES, and SOX2) were due not only to transcriptional downregulation of their respective genes but also, at least in part, to proteasomal degradation. DISCUSSION Normal brain structure and function are developmentally regulated by four different but related factors (7): (i) the proliferation of self-renewing NPCs, (ii) the differentiation of NPCs to mature neurons and glial cells, (iii) the proper migration of these neurons and glial cells (33), and (iv) the formation of proper synaptic connections. In utero HCMV infection can cause significant damage to the developing fetal brain. Our current studies found that HCMV infection in vitro can perturb at least the first two factors regulating fetal brain development. In previous studies, we observed that after changing the

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composition of the culture medium to induce differentiation to glial cells, NPCs attached to uncoated surfaces (35). During the current study, infected neurospheres attached to uncoated surfaces. Subsequently, individual cells migrated away from the spheres. This occurred without alterations to the composition of the culture medium, suggesting that HCMV infection induced premature differentiation. UV-irradiated virus infections showed that de novo viral protein expression was necessary to induce this cellular behavioral change. These results prompted our investigation of gene expression profiles to gain insight into possible causes of this differentiation-like behavior. The most remarkable change in gene expression we observed was the downregulation of the mRNAs for several NPC marker proteins, including SOX4, FABP7, ID4, MIR21, DCX, NES, SOX2, and GFAP. These proteins all play critical roles in neural differentiation and/or maintenance of the multipotent state of the NPC. qPCR and Western blotting of DCX, NES, SOX2, and GFAP confirmed the downregulation of mRNA levels and the decreases in protein levels of these four NPC markers, respectively. The four marker proteins we investigated in depth are well characterized. DCX plays an important role in neuron migration signaling during brain development and is a marker of early migratory neuroblasts. DCX haploinsufficiency can lead to various degrees of mental retardation. The extent of retardation is linked to the quantity of arrested neurons in the white matter (3, 19, 63). Loss of DCX from infected NPCs suggests an abnormally differentiated state. NES is an intermediate filament-forming protein that is downregulated during NPC differentiation (32, 57). NES is associated with cytoplasmic trafficking in NPCs and plays an important role in the distribution and organization of critical cellular factors regulating cell proliferation and differentiation (39, 53). Additionally, NES is located at 1q23 (an area that has been shown to be targeted selectively by HCMV) (42). Therefore, the downregulation of NES (and perhaps other neural genes) may be related to the selective effect of HCMV at 1q23. SOX2 is a transcription factor involved in embryonic development and stem cell self-renewal (6, 13, 66, 67). The importance of SOX2 has been highlighted in recent literature by its involvement in reprogramming terminally differentiated cells into induced pluripotent stem cells (iPSCs) (45, 64, 69). Lossof-function mutations in SOX2 can cause improper eye and brain development (22, 55, 74, 79). SOX2 downregulation in uninfected NPCs is associated with, and necessary for, both glial and neuronal differentiation. We previously found that complete downregulation of SOX2 was protracted after differentiation in vitro, taking up to 3 weeks (35). In the absence of forced differentiation, HCMV infection of NPCs rapidly downregulated SOX2, with decreases observable after 4 h p.i. in whole-genome expression analyses. This suggests that abnormal and premature differentiation was induced. Moreover, the downregulation of SOX2 mRNA was accompanied by that of the microRNA MIR21, whose regulation is related to that of SOX2 during differentiation (29). During development, GFAP is first expressed in differentiating pluripotent stem cells and marks their differentiation toward a neural precursor lineage (21, 31). GFAP is also used as a marker for differentiation of NPCs to an astroglial lineage. NPCs differentiated to astroglia displayed strong GFAP posi-

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tivity. HCMV infection did not affect GFAP expression in these differentiated astroglia (35; our unpublished data). This was in sharp contrast to our observation in undifferentiated NPCs, where GFAP mRNA levels decreased 100-fold as early as 24 h p.i., with protein declines beginning at 48 h p.i. This suggested that GFAP’s function and/or regulation was considerably different in NPCs than in astroglia, which has been suggested by others (80). In our experiments, changes to attachment and spreading characteristics observed during infection of neurospheres paralleled those seen during astroglial differentiation. However, GFAP levels in infected NPCs were markedly decreased rather than increased as in differentiated astroglia, further suggesting abnormal differentiation. The downregulation of other neuronal markers observed in the whole-genome expression experiments also indicated that these infected cells were not neuronal in nature. Thus, although premature differentiation appeared to result from HCMV infection, the resultant cells were neither astroglia nor neurons. Loss of both ID4 and SOX4 also supports the conclusion that differentiation was abnormal. MCMV infection has been shown to inhibit CNS stem cell differentiation, with neuronal differentiation being inhibited more severely than glial differentiation (28). Odeberg and collegues produced similar results with human cells (43, 44). These investigators attempted to differentiate NPCs by using standard culture medium manipulations after the onset of HCMV infection. They found that infection appeared to inhibit differentiation of NPCs down either glial or neuronal pathways, as measured by the absence of GFAP⫹ (glial marker) cells and ␤ tubulin⫹ (neuronal marker) cells at 7 days p.i. In contrast, we found rapid downregulation of GFAP, as well as other markers of multipotency and multiple neuronal lineage markers, in the absence of culture medium changes. Given the results we observed, we suspect that the infected NPCs in the earlier studies were no longer multipotent following infection and were unable to be differentiated to neurons or glia by any means. Our results found that rather than blocking an enforced differentiation trajectory, HCMV infection causes premature differentiation into an abnormal state. In an attempt to narrow the field of viral factors involved in the downregulation of the NPC markers, GCV was used to inhibit HCMV replication in NPCs. IE1 and low levels of IE2 and UL44 were detectable in GCV-treated cells, but no E-L or L protein expression was observed. GCV treatment of infected cells delayed and reduced the downregulation/decreases of NPC marker mRNA and protein levels. Importantly, UV-inactivated virus infections did not alter the expression of NPC markers at the mRNA or protein level (data not shown), indicating that de novo viral protein expression was necessary for downregulation/decreases to occur. The GCV treatment results suggested that the altered fate of infected NPCs required a full complement of viral IE and E proteins and that active viral replication perhaps also played a role in these changes. mRNA downregulation occurred more rapidly than protein declines and coincided with IE gene expression, making IE gene products likely culprits. In fact, data gathered during a suboptimal infection in which IE mRNA expression was drastically delayed showed dramatically delayed and decreased changes in NPC marker mRNA levels (data not shown). Chambers and colleagues found the following genes to be

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expressed at the IE stage: IE1, IE2, IRS1, TRS1, UL36, UL37, UL69, and UL110 (9). Particularly important for gene regulation are the transcription factors IE1, IE2, TRS1, and IRS1 and the tegument protein UL69. IE1 has been shown to interact with several cellular proteins (50), and IE2 is known to interact with several cellular transcription factors and histone deacetylases (75; reviewed in reference 54). Our future analyses will concentrate on these viral proteins, which seem likely to be responsible for downregulation of NPC markers at the mRNA level. We also investigated one of the probable mechanisms behind the decreases in NPC markers at the protein level, namely, virus-induced proteasomal degradation. Incubation of cells with MG132, a proteasome inhibitor, partially inhibited the decreases in marker proteins. Although MG132 did not affect viral IE protein expression, viral E and L protein expression was inhibited by treatment with the drug (26). Thus, in order for the complete decline in NPC marker protein levels observed without drug treatment to occur, it appeared that a full complement of viral proteins may have been required. Earlier studies showed that NES was ubiquitinated and degraded via the proteasomal pathway (53). Our IP experiments found that DCX was both ubiquitinated and sumoylated, with only minor modifications in mock-infected cells. Others have shown that viral infection induces unusual sumoylation of the cellular protein Daxx (23). These results suggest that HCMV may induce proteasomal degradation of host cellular factors to create a suitable environment for virus propagation, resulting in alterations in NPC behavior. Interestingly, in our DCX Western blots, we saw a slowermigrating form at ⬃82 kDa, which prompted us to perform the IPs shown in Fig. 6D. Although there were sumo and ubiquitin modifications on the virus-infected, MG132-treated samples, these modifications could not explain the presence of this slowermigrating form in the mock- and virus-treated samples. A more thorough analysis of the literature revealed that the addition of MG132 to cells could cause irregular phosphorylation of certain substrates (58). In addition, in researching additional DCX Abs, it became apparent that DCX can become phosphorylated after exposure to certain stimuli and that one of the phosphorylated forms migrates at ⬃82 kDa (18). We explored the possibility that this MG132-induced additional band might be a by-product of phosphorylation by treating our lysates with SAP to look for removal of this 82-kDa band. Figure 6E shows that this was indeed the case. In conclusion, we found that HCMV infection causes NPCs to differentiate prematurely and abnormally. This process appears to be mediated by the rapid downregulation of genes that maintain neural progenitor multipotency and of genes establishing these cells’ neural identity. Analysis of gene expression data also indicated an inhibition of their neuronal differentiation. Our results indicate that HCMV infection quickly induces NPC differentiation and powerfully disrupts the infected cell’s fate. ACKNOWLEDGMENTS This work was supported by NIH grants RO1-AI51463 and P20 RR015587 (COBRE program) to E.A.F. and by continuing support of the National Human Neural Stem Cell Resource by the Children’s Hospital of Orange County Research Institute (P.H.S.).

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