Characterization of an In Vitro Model of Alphaviru
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病菌学杂志 2005年第6期
W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland
ABSTRACT
Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10- to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.
INTRODUCTION
Viral encephalitis due to infection with arthropod-borne viruses is an important cause of mortality and often results in significant long-term neurological deficits in those that survive (8, 31, 46). The enveloped, message-sense RNA virus Sindbis virus (SV) is the prototype alphavirus in the family Togaviridae and is related to viruses known to cause encephalitis and arthritis in humans (47). Neurons are the primary target cell for SV in mice (28), and the outcome of infection is determined by both host and viral factors (20, 21, 37, 50, 52). An important host determinant is the maturity of the neuronal population infected. Immature animals replicate virus in the central nervous system (CNS) to higher titers than mature animals and are highly susceptible to fatal disease, dying within 3 to 4 days after infection (20, 29, 37). Adult animals infected with the same strain of SV survive and, in the absence of a virus-specific immune response, develop persistent infection (20, 34). Immunocompetent mature animals can recover from infection and clear virus from the CNS through noncytolytic mechanisms involving anti-viral glycoprotein antibody (34) and gamma interferon (3). However, an understanding of the mechanisms underlying these virus-host interactions has been hampered by the lack of a convenient in vitro system for study.
The availability of a reproducible and convenient in vitro system is essential to understand the molecular mechanism(s) of age-dependent virus-neuron interactions and noncytolytic clearance. Several cell culture systems have been used to study aspects of SV-induced cell death and immunity-mediated virus clearance, such as neuroblastoma cells, primary cortical neuron cultures, dorsal root ganglion (DRG) neurons, and cells overexpressing the antiapoptotic protein Bcl-2 (10, 34, 41, 51, 54). However, none of these systems offers the possibility of studying large numbers of well-characterized cells while truly mimicking the responses of immature and mature neurons to infection.
To develop a cell culture system that models the interaction of SV with neurons in vivo, we have used CSM14.1 cells (11, 57), a temperature-sensitive, immortalized rat nigral neuron cell line that can be differentiated in vitro. CSM14.1 cells were derived by immortalization of primary rat embryonic day 14 mesencephalic neural cells with a retroviral vector containing tsA58, a temperature-sensitive mutant of the simian virus 40 large tumor antigen (11). We have studied undifferentiated and differentiated CSM14.1 cells for expression of neuron-specific cell markers and susceptibility to SV infection and have shown that they mimic the in vivo properties of SV infection of immature and mature neurons.
MATERIALS AND METHODS
Cell culture. The CSM14.1 cell line was obtained from Dale Bredesen (Buck Institute for Age Research, Novato, Calif.) (11, 57, 58) and was maintained and passaged in Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine (Gibco), 50 μg of gentamicin (Quality Biological, Inc, Gaithersburg, Md.) per ml, 100 U of penicillin/ml, and 100 μg of streptomycin (Gibco) per ml in a humidified 5% CO2 incubator at 31°C. For cell differentiation, CSM14.1 cells were plated in six-well plates under the permissive conditions described above. Once the cells reached confluence, they were washed once with phosphate-buffered saline, pH 7.4 (PBS), and switched to the nonpermissive culture conditions of DMEM-1% FBS and an incubation temperature of 37 to 39°C. Two different stocks of CSM14.1 cells were used in this study. One was previously infected with SV, cured of the infection, and tested negative for viral RNA by reverse transcriptase PCR (CSM14.1c), while the other had not been previously exposed to SV (CSM14.1). Essentially all experiments were performed with both stocks of cells and exhibited no differences. The types of cells used to generate the data in the figures are indicated in the figure legend for each experiment.
Baby hamster kidney 21 (BHK-21) cells grown in DMEM-10% FBS were used for production of virus stocks and for virus quantification by plaque formation.
Analysis of expression of neural proteins. For Western blot analysis of neuronal cell markers, cells were washed three times with ice-cold Tris-buffered saline (TBS; 10 mM Tris HCl [pH 7.5], 150 mM NaCl) and lysed with ice-cold lysis buffer (TBS containing 1% Igepal CA-630, 0.1% SDS, and 0.5% sodium deoxycholate; protease inhibitors were added immediately before use). Brain and liver tissues were homogenized in lysis buffer using a Fast Prep machine (Bio 101, Carlsbad, Calif.). After cellular debris was removed by centrifugation, equal amounts of lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Hybond-C Extra; Amersham, Piscataway, N.J.). Antibodies (Ab) specific for tyrosine hydroxylase (TH; polyclonal; Chemicon, Temecula, Calif.), microtubule-associated protein 2 (MAP-2; monoclonal; Sigma-Aldrich, St. Louis, Mo.), glial fibrillary acidic protein (GFAP; polyclonal; Chemicon), and actin (monoclonal; Chemicon) were used according to the manufacturers' recommendations. Levels of expression of cell markers were quantified relative to the amount of actin by using Image Gauge software (Fuji Photo Film Co., Ltd.). The intensity of each band over a specified surface area was quantified in arbitrary units determined by the software. Background intensity was subtracted from the total intensity, and the resulting difference was divided by the surface area to obtain specific intensity over area (in square millimeters). The quantified intensity per surface area of each cell marker was then divided by that of actin to get the relative level of protein expression.
For immunofluorescence staining for TH, neuron-specific nuclear protein (NeuN; monoclonal; Chemicon)-, neurofilament 68 (NF-68; polyclonal; Chemicon)-, neuron-specific enolase (NSE; polyclonal; Chemicon)-, and GFAP-stained CSM14.1 cells were plated on glass coverslips in six-well plates. At various times after the initiation of differentiation, cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized, and blocked with permeablization-blocking buffer (PBS plus 5% bovine serum albumin [BSA], 0.4% Triton X-100, and 1% normal goat serum [NGS] for TH and NeuN staining; or PBS plus 1% BSA, 0.4% Triton X-100, and 4% NGS for NF-68, NSE, and GFAP staining) and stained with Ab in dilution buffer (PBS containing 5% BSA and 1% NGS for TH and NeuN staining; PBS containing 1% BSA, 0.4% Triton X-100, and 1% NGS for NF-68 and GFAP staining; or PBS containing 1% BSA and 1% NGS for NSE staining). Ab specific for TH, NeuN, NF-68, NSE, or GFAP were used according to the manufacturers' recommendations. Cells were stained with a secondary Ab conjugated with Alexa-fluor 488 (green; Molecular Probes, Eugene, Oreg.) for TH and NeuN or with fluorescein (Pierce, Rockford, Ill.) for NF-68, NSE, and GFAP. For TH and NeuN, cellular nuclei were also stained with Hoechst dye (Molecular Probes). The fluorescent signals were visualized with a fluorescence microscope (Nikon, Melville, N.Y.).
Viruses. The 633 strain of SV, which contains glutamine at E2-55, was used (52). Recombinant SV expressing enhanced green fluorescent protein (eGFP) as a reporter gene was constructed using 633 cDNA engineered to contain a second subgenomic promoter (27). The eGFP gene was inserted into the BstEII cloning site to create 633-eGFP. Full-length viral RNA was transcribed in vitro with an SP6 promoter from an XhoI-linearized plasmid and transfected into BHK cells with Lipofectin (Invitrogen, Carlsbad, Calif.). At 24 to 48 h after transfection, supernatant fluid containing progeny virus was collected, assayed by plaque formation on BHK cells, and stored in aliquots at –80°C.
In vitro SV infection, detection of virus, and determination of cell viability. Cells plated in six-well plates were infected with SV 633-eGFP (multiplicity of infection [MOI] of 5, determined on BHK cells) diluted in DMEM-1% FBS. After 1 h, the cells were washed once with PBS and fresh medium was added. Supernatant fluids were collected at various times after infection and analyzed for virus production by plaque assay. For virus growth curves, the amount of virus was normalized to the viable cell number at the time of infection and expressed as the number of log10 PFU per cell. Cell viability was determined by trypan blue exclusion.
Immunofluorescence and Western blot analyses of SV structural proteins. For immunofluorescent staining of the SV E2 protein, infected and mock-infected CSM14.1 cells grown on glass coverslips in six-well plates were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with ice-cold acetone. After several washes with PBS, cells were stained with anti-E2 monoclonal Ab (MAb) G5 (54) for 30 min on ice, washed six times with PBS, and stained with a secondary Ab conjugated with Alexa-fluor 594 (red; Molecular Probes) for 30 min on ice. Nuclei were then stained with Hoechst dye and visualized as before.
For Western blot analysis of SV structural proteins, cells were washed three times with ice-cold PBS and lysed with ice-cold 1% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} lysis buffer (CHAPS in immunoprecipitation [IP] buffer [PBS containing 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 10 mM Na pyrophosphate, and 50 mM NaF]) as described above. Equal amounts of protein per sample were separated by SDS-PAGE and analyzed by Western blotting with a rabbit polyclonal Ab specific for SV (28) or the anti-E2 MAb, G5 (54), and the Supersignal picogram or femtogram quantity detection system (Pierce).
Metabolic labeling and analysis of intracellular proteins. At various times after infection, SV-infected and mock-infected cells were washed twice with PBS, incubated in Met- and Cys-free medium for 1 h, and labeled with Tran35S-label (100 μCi/ml; ICN Biomedicals, Irvine, Calif.) for 1 h. Labeled cells were washed three times with ice-cold PBS and lysed with RIPA buffer (10 mM Tris [pH 7.0] with 1% Igepal-630, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA; protease inhibitors were added immediately before use). Cell lysates were placed on ice for 30 min to allow complete lysis and then centrifuged at 9,000 x g for 5 min at 4°C to remove insoluble debris. The 35S incorporated into protein was quantified by precipitation with 20% trichloroacetic acid, and equal counts per minute per lysate sample were used for protein analysis by SDS-PAGE and autoradiography.
To analyze the change in protein expression in CSM14.1 cells over time, cells were labeled and washed as described above and lysed with ice-cold 1% (wt/vol) CHAPS in IP buffer (protease inhibitors were added immediately before use). After complete lysis and removal of insoluble cellular debris, we immunoprecipitated SV proteins from samples to which equal counts per minute per lysate were added using a rabbit polyclonal Ab specific for SV at 4°C overnight. Immunocomplexes were isolated using protein G immobilized to agarose beads (Pierce), washed twice with 1% CHAPS-IP buffer and once with IP buffer, and analyzed by SDS-PAGE and autoradiography.
Statistics. Differences between geometric means of virus replication levels of undifferentiated and differentiated CSM14.1 cells or between the levels of viability of infected and mock-infected cells were determined by Student's t test using the RAqua 1.8.1 software (Free Software Foundation, Boston, Mass.).
RESULTS
The immortalized cell line CSM14.1 can differentiate into neurons. CSM14.1 cells in medium with 10% FBS actively proliferated at 31°C and then stopped proliferating and differentiated when they were shifted to medium with reduced serum (1%) at the nonpermissive temperature (37 to 39°C) (Fig. 1). During the process of differentiation, a substantial number of cells died. Differentiated CSM14.1 cells displayed a neuronal morphology, with large cell bodies and extensive process formation. To determine whether this change in morphology correlated with the expression of neuronal markers, lysates of cells at various times after differentiation were assessed for the expression of TH, MAP-2, and GFAP by Western blotting (Fig. 2). Compared to undifferentiated cells, differentiated CSM14.1 cells expressed increased levels of two neuron-specific cell markers, TH and MAP-2, and reduced levels of an astrocyte marker, GFAP (Fig. 2A and B). Immunofluorescent staining demonstrated that the majority of the differentiated cells expressed TH, NeuN, NF-68, and NSE (Fig. 2C). For subsequent experiments, cells were differentiated for 3 to 4 weeks.
SV replication and CPE of undifferentiated and differentiated CSM14.1 cells. After establishing the neuronal characteristics of differentiated CSM14.1 cells, susceptibility to SV infection and virus-induced cell death were tested. The production of new infectious virus was detected in cultures of undifferentiated cells at 7 h and reached peaks of approximately 1010.5 PFU/ml and 104.5 PFU/cell by 24 h after infection (Fig. 3A). Undifferentiated cells developed cytopathic effects (CPE), including cell rounding, detachment, and apoptotic blebs, and essentially all were dead by 4 days after infection (Fig. 4A). In contrast, the production of new virus was first detected in cultures of differentiated cells at 11 h after infection, and virus production continued to increase through 72 h (Fig. 3A). Amounts of virus produced were substantially lower in differentiated cells than in undifferentiated cells. Peak virus production in undifferentiated cells was 107.2 PFU/ml and 103.1 PFU/cell. Differentiated cells displayed little to no CPE and survived for more than 6 days after infection (Fig. 4B).
To determine whether these differences in levels of virus replication were due to the culture conditions or to the differentiation state of the cells, undifferentiated CSM14.1 cells were infected and cultured at 31°C with 10% FBS or at 39°C with 1% FBS and virus production was assessed over 24 h (Fig. 3B). The levels of virus replication were the same, indicating that higher temperature and lower FBS concentration were not the reasons for restricted virus replication in differentiated cells.
Viral and cellular protein synthesis in undifferentiated and differentiated CSM14.1 cells. Immunofluorescent staining was used to identify virus-infected cells. At 24 h after infection, all undifferentiated cells expressed detectable levels of the E2 glycoprotein, while approximately 50% of differentiated cells could be identified to be infected by this criterion (Fig. 5A). Costaining with Ab to E2 and TH at this time indicated that more than 95% of the infected cells also expressed TH (data not shown). E2 antigen was detected in all differentiated cells by day 3 after infection. Similar results were obtained when cells were assessed using a polyclonal Ab to SV (data not shown).
Examination by Western blotting of SV structural proteins in infected cells showed detectable levels of capsid, E1/E2, and the precursor E2 protein, pE2, in undifferentiated cells by 24 h after infection (Fig. 5B) and in differentiated cells by day 2 after infection. There was a lower level of expression of E2 relative to that of pE2 in differentiated cells (E2/pE2 ratio, 0.0426), compared to that in undifferentiated cells (E2/pE2 ratio, 2.14) (Fig. 5C).
SV infection is characteristically associated with a shutdown of host protein synthesis (47). To examine overall protein synthesis, BHK, undifferentiated CSM14.1, and differentiated CSM14.1 cells were infected with SV, and newly synthesized proteins were labeled with 35S for 1 h at various times after infection and analyzed by SDS-PAGE and autoradiography (Fig. 6). Mock-infected cells synthesized many cellular proteins. By 24 h after infection, the synthesis of SV proteins was predominant in BHK cells and in undifferentiated CSM14.1 cells (Fig. 6A). Viral structural proteins, E1/E2, pE2, and capsid, were readily identified in infected BHK cells and in undifferentiated CSM14.1 cells within 24 h after infection. On the other hand, viral proteins were not clearly detected in the differentiated CSM14.1 cells until day 2 and were maximal on day 3 after infection. Differentiated CSM14.1 cells survived SV infection and gradually restored cellular protein synthesis (Fig. 6A). No newly synthesized viral proteins were detected in differentiated cells by immunoprecipitation on day 6 after infection (Fig. 6B).
DISCUSSION
An important host factor determining the outcome of alphavirus infection of neurons is age (21, 29), but there has not been a convenient in vitro model for characterizing this aspect of infection. These studies have shown that both undifferentiated and differentiated CSM14.1 neuronal cells could be infected with SV and that the levels of virus replication and CPE were dependent on the maturity of the cells. Undifferentiated CSM14.1 cells were efficiently infected, produced large amounts of virus, and died within 3 to 4 days. Differentiated CSM14.1 cells were less efficiently infected, produced virus more slowly, had 10- to 100-fold-lower peak virus production, and survived for many days. E2 glycoprotein processing was more efficient in undifferentiated than in differentiated cells. Virus infection led to a shutdown of cellular protein synthesis in both undifferentiated and differentiated CSM14.1 cells, but differentiated cells spontaneously restored host protein synthesis. Restoration of cellular protein synthesis was accompanied by a reduction in viral protein synthesis and a decreased production of infectious virus. These characteristics of virus replication and neuronal survival are typical of the responses to SV infection in immature and mature mice (18, 29).
Several experimental approaches have been used to study the survival of neurons after SV infection and the response of these cells to immune mediators. The AT3 rat prostatic adenocarcinoma cell line has been transfected with the cellular oncogene Bcl-2, an antiapoptotic protein. AT3-Bcl-2 cells are more resistant than AT3 cells to virus-induced cell death (35), exhibit Ab-mediated down-regulation of production of infectious virus, but die 2 to 3 days after infection (9, 10). Thus, AT3-Bcl-2 cells have the advantage of a continuous cell line but do not reflect the survival of mature neurons in vivo. Primary neuronal cells, such as rodent cortical and DRG neurons, have also been studied. Cortical neurons and undifferentiated DRG neurons collected from embryonic mice or rats are highly susceptible to virus-induced cell death (34, 41). DRG neurons differentiated for 2 to 3 weeks in culture remain alive for several days after infection and have been used successfully to study Ab-mediated clearance of virus (34, 54). However, the preparation of DRG neurons is labor-intensive and the number of cells that can be obtained is limited.
CSM14.1 cells, which can be propagated without losing their ability to differentiate, have been used previously to study neuronal differentiation (23, 30) and the effects of various factors on neurons (12, 13, 38, 57, 58) but have not been used to study responses to virus infection. Consistent with previous studies (11, 23), differentiating CSM14.1 cells sequentially expressed general and dopaminergic neuron-specific markers in a manner similar to that observed in developing rodent brain (23). In contrast to what was found in one previous study using an MAb which detected no GFAP in CSM14.1 cells (23), we found that GFAP was detectable in undifferentiated cells by using a polyclonal Ab and was down-regulated with differentiation. GFAP, an intermediate filament protein, is commonly used as an astrocyte marker but has been found in many nonglial populations of cells, such as hepatic stellate cells (45), chondrocytes of epiglottic cartilage (4), and fibroblasts (24). The inversely proportional relationship between GFAP expression and the time of CSM14.1 cell differentiation is consistent with the conclusion that CSM14.1 cells assume a more neuronal phenotype as they differentiate.
Undifferentiated CSM14.1 cells were very susceptible to SV infection and produced new virus within 8 h. With differentiation, CSM14.1 cells became less susceptible to SV infection and produced new infectious virus more slowly than undifferentiated cells. These differences were not due to the culture conditions because undifferentiated cells infected under the culture conditions for differentiated cells (39°C and 1% FBS) replicated virus efficiently.
Immunocytochemical staining suggested that all undifferentiated cells, but not all differentiated cells, were infected initially. In cultures of differentiated cells, the infection spread gradually, with all cells being infected within 3 days. SV structural proteins were present in neuronal processes that contacted neighboring cells, possibly facilitating the spread of virus from cell to cell rather than the spread by release of infectious virions into the media. The delay in the appearance of viral structural proteins detectable by Western blotting in differentiated CSM14.1 cells may reflect the initial infection of fewer cells. However, peak virus production on a per-cell basis was also lower in differentiated than in undifferentiated cells. The level of fully processed E2 relative to that of pE2 was lower in differentiated than in undifferentiated cells. Less-efficient posttranslational processing of pE2 into E2 may contribute to the reduced and slower production of SV in differentiated cells. Furthermore, if pE2 is incorporated into virions, production of noninfectious particles may also delay the spread of infection in differentiated CSM14.1 cell cultures.
Shutdown of host protein synthesis was evident by 24 h after infection in undifferentiated CSM14.1 cells, and these cells eventually died. In differentiated cells, the ratio of viral to host proteins synthesized gradually increased, peaked by day 3, and decreased thereafter, suggesting that differentiated neurons controlled virus replication and restored cellular protein synthesis. Cell survival and the reappearance of cellular protein synthesis in differentiated CSM14.1 cells after infection indicated an ability of these cells to control virus replication. Control of virus replication has also been described for interferon-producing cells infected with a strain of SV with mutated nonstructural P2 (16), suggesting a potential role for interferon in this process.
This pattern of virus growth and the association between neuronal maturation and reduced susceptibility to virus infection resembles the time course of viral replication and outcome of SV infection in mice (18, 21, 29). Increasing age correlates with reduced virus production and reduced virus-induced mortality and is not attributable to maturation of the immune response (18, 29). In newborn mice, peak virus titers are reached within 1 to 2 days after infection and neuronal apoptosis is observed (36). In weanling mice, peak virus titers are reached 3 to 4 days after infection, cell-to-cell spread is observed, and there is minimal apoptosis (17, 50). Furthermore, in the absence of an antiviral immune response, SV-infected neurons of weanling mice survive, partially control virus replication, and continue to produce virus for many weeks after infection (3, 34).
Age-dependent resistance to virus replication is also a characteristic of a number of other neurotropic viruses, including Semliki Forest virus (SFV) (14, 15, 43), Japanese encephalitis virus (JEV) (32, 42, 56), mouse hepatitis virus (48), measles virus (19, 22), reovirus (39), blue-tongue virus (5), parvoviruses (6, 7), influenza virus (26), and herpesviruses (1, 2, 40, 55). As with the AR339 strain of SV, infection of neonatal mice with the A7(74) strain of SFV leads to fatal encephalitis, but mice that are at least 2 weeks of age recover uneventfully (44). A7(74) replicates productively, spreads efficiently, and leads to the death of neurons during axonogenesis and synaptogenesis, whereas replication is restricted in mature neurons (14, 15, 43). Type 3 reovirus, induces an acute necrotizing and lethal encephalitis in newborn mice but a nonfatal infection in older animals (39). Age-dependent resistance of mice to infection with SFV and reovirus is not explained by developmental changes in the adaptive immune response but rather by an intrinsic ability of mature neurons to restrict viral replication (15, 18, 49). Similarly, JEV preferentially infects immature neurons, and animals older than 2 weeks survive infection (42, 56).
The susceptibility of immature neurons to JEV may be related to a decreased expression of neuronal surface molecules acting as viral receptors as the neurons mature (32), and changes in the abundance of neuronal receptors may also play a role in age-dependent susceptibility to SV (53), but even after infection, less virus is produced by mature than by immature neurons, indicating an additional postentry restriction of replication. In addition to receptor expression, potentially important age-dependent determinants of resistance that have been identified include decreased expression of proapoptotic molecules and inflammatory response genes and increased expression of the neuroprotective chemokine fractalkine and of interferon-inducible genes (25, 33, 36).
CSM14.1 cells demonstrate development of resistance as a function of neuronal maturation and provide a system in which the cellular basis for the neuronal restriction of SV can be studied. This in vitro model system should allow further studies of the molecular mechanisms that determine the age-dependent interactions of neurons with SV and the mechanisms of host control of virus replication in neurons.
ACKNOWLEDGMENTS
These studies were supported by research grants RO1 NS18596 and NS38932 (D.E.G.) and medical scientist training program grant T32 GMO7309 (P.S.V.) from the National Institutes of Health.
We appreciate the helpful suggestions of George Oyler, Irina Shatz, and Jeffrey Rothstein; assistance from Marcia Lyons; and support from the Cellular and Molecular Medicine Graduate Training Program at the Johns Hopkins University School of Medicine.
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ABSTRACT
Terminally differentiated, mature neurons are essential cells that are not easily regenerated. Neurotropic viruses, such as Sindbis virus (SV), cause encephalomyelitis through their ability to replicate in neurons. SV causes the death of immature neurons, while mature neurons can often survive infection. The lack of a reproducible and convenient neuronal cell culture system has hindered a detailed study of the differences in levels of virus replication between immature and mature neurons and the molecular events involved in virus clearance from mature neurons. We have characterized SV replication in immortalized CSM14.1 rat neuronal cells that can be differentiated into neurons. During differentiation, CSM14.1 cells ceased dividing, developed neuronal morphology, and expressed neuron-specific cell markers. SV infection of undifferentiated CSM14.1 cells was efficient and resulted in high levels of virus replication and cell death. SV infection of differentiated CSM14.1 cells was less efficient and resulted in the production of 10- to 100-fold less virus and cell survival. In undifferentiated cells, SV induced a rapid shutdown of cellular protein synthesis and pE2 was efficiently processed to E2 (ratio of E2 to pE2, 2.14). In differentiated cells, the SV-induced shutdown of cellular protein synthesis was transient and pE2 was the primary form of E2 in cells (ratio of E2 to pE2, 0.0426). We conclude that age-dependent restriction of virus replication is an intrinsic property of maturing neurons and that the CSM14.1 cell line is a convenient model system for investigating the interactions of alphaviruses with neurons at various stages of differentiation.
INTRODUCTION
Viral encephalitis due to infection with arthropod-borne viruses is an important cause of mortality and often results in significant long-term neurological deficits in those that survive (8, 31, 46). The enveloped, message-sense RNA virus Sindbis virus (SV) is the prototype alphavirus in the family Togaviridae and is related to viruses known to cause encephalitis and arthritis in humans (47). Neurons are the primary target cell for SV in mice (28), and the outcome of infection is determined by both host and viral factors (20, 21, 37, 50, 52). An important host determinant is the maturity of the neuronal population infected. Immature animals replicate virus in the central nervous system (CNS) to higher titers than mature animals and are highly susceptible to fatal disease, dying within 3 to 4 days after infection (20, 29, 37). Adult animals infected with the same strain of SV survive and, in the absence of a virus-specific immune response, develop persistent infection (20, 34). Immunocompetent mature animals can recover from infection and clear virus from the CNS through noncytolytic mechanisms involving anti-viral glycoprotein antibody (34) and gamma interferon (3). However, an understanding of the mechanisms underlying these virus-host interactions has been hampered by the lack of a convenient in vitro system for study.
The availability of a reproducible and convenient in vitro system is essential to understand the molecular mechanism(s) of age-dependent virus-neuron interactions and noncytolytic clearance. Several cell culture systems have been used to study aspects of SV-induced cell death and immunity-mediated virus clearance, such as neuroblastoma cells, primary cortical neuron cultures, dorsal root ganglion (DRG) neurons, and cells overexpressing the antiapoptotic protein Bcl-2 (10, 34, 41, 51, 54). However, none of these systems offers the possibility of studying large numbers of well-characterized cells while truly mimicking the responses of immature and mature neurons to infection.
To develop a cell culture system that models the interaction of SV with neurons in vivo, we have used CSM14.1 cells (11, 57), a temperature-sensitive, immortalized rat nigral neuron cell line that can be differentiated in vitro. CSM14.1 cells were derived by immortalization of primary rat embryonic day 14 mesencephalic neural cells with a retroviral vector containing tsA58, a temperature-sensitive mutant of the simian virus 40 large tumor antigen (11). We have studied undifferentiated and differentiated CSM14.1 cells for expression of neuron-specific cell markers and susceptibility to SV infection and have shown that they mimic the in vivo properties of SV infection of immature and mature neurons.
MATERIALS AND METHODS
Cell culture. The CSM14.1 cell line was obtained from Dale Bredesen (Buck Institute for Age Research, Novato, Calif.) (11, 57, 58) and was maintained and passaged in Dulbecco's modified Eagle medium (DMEM) (Gibco, Grand Island, N.Y.) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco), 2 mM L-glutamine (Gibco), 50 μg of gentamicin (Quality Biological, Inc, Gaithersburg, Md.) per ml, 100 U of penicillin/ml, and 100 μg of streptomycin (Gibco) per ml in a humidified 5% CO2 incubator at 31°C. For cell differentiation, CSM14.1 cells were plated in six-well plates under the permissive conditions described above. Once the cells reached confluence, they were washed once with phosphate-buffered saline, pH 7.4 (PBS), and switched to the nonpermissive culture conditions of DMEM-1% FBS and an incubation temperature of 37 to 39°C. Two different stocks of CSM14.1 cells were used in this study. One was previously infected with SV, cured of the infection, and tested negative for viral RNA by reverse transcriptase PCR (CSM14.1c), while the other had not been previously exposed to SV (CSM14.1). Essentially all experiments were performed with both stocks of cells and exhibited no differences. The types of cells used to generate the data in the figures are indicated in the figure legend for each experiment.
Baby hamster kidney 21 (BHK-21) cells grown in DMEM-10% FBS were used for production of virus stocks and for virus quantification by plaque formation.
Analysis of expression of neural proteins. For Western blot analysis of neuronal cell markers, cells were washed three times with ice-cold Tris-buffered saline (TBS; 10 mM Tris HCl [pH 7.5], 150 mM NaCl) and lysed with ice-cold lysis buffer (TBS containing 1% Igepal CA-630, 0.1% SDS, and 0.5% sodium deoxycholate; protease inhibitors were added immediately before use). Brain and liver tissues were homogenized in lysis buffer using a Fast Prep machine (Bio 101, Carlsbad, Calif.). After cellular debris was removed by centrifugation, equal amounts of lysate proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane (Hybond-C Extra; Amersham, Piscataway, N.J.). Antibodies (Ab) specific for tyrosine hydroxylase (TH; polyclonal; Chemicon, Temecula, Calif.), microtubule-associated protein 2 (MAP-2; monoclonal; Sigma-Aldrich, St. Louis, Mo.), glial fibrillary acidic protein (GFAP; polyclonal; Chemicon), and actin (monoclonal; Chemicon) were used according to the manufacturers' recommendations. Levels of expression of cell markers were quantified relative to the amount of actin by using Image Gauge software (Fuji Photo Film Co., Ltd.). The intensity of each band over a specified surface area was quantified in arbitrary units determined by the software. Background intensity was subtracted from the total intensity, and the resulting difference was divided by the surface area to obtain specific intensity over area (in square millimeters). The quantified intensity per surface area of each cell marker was then divided by that of actin to get the relative level of protein expression.
For immunofluorescence staining for TH, neuron-specific nuclear protein (NeuN; monoclonal; Chemicon)-, neurofilament 68 (NF-68; polyclonal; Chemicon)-, neuron-specific enolase (NSE; polyclonal; Chemicon)-, and GFAP-stained CSM14.1 cells were plated on glass coverslips in six-well plates. At various times after the initiation of differentiation, cells were washed with PBS, fixed with 4% paraformaldehyde, permeabilized, and blocked with permeablization-blocking buffer (PBS plus 5% bovine serum albumin [BSA], 0.4% Triton X-100, and 1% normal goat serum [NGS] for TH and NeuN staining; or PBS plus 1% BSA, 0.4% Triton X-100, and 4% NGS for NF-68, NSE, and GFAP staining) and stained with Ab in dilution buffer (PBS containing 5% BSA and 1% NGS for TH and NeuN staining; PBS containing 1% BSA, 0.4% Triton X-100, and 1% NGS for NF-68 and GFAP staining; or PBS containing 1% BSA and 1% NGS for NSE staining). Ab specific for TH, NeuN, NF-68, NSE, or GFAP were used according to the manufacturers' recommendations. Cells were stained with a secondary Ab conjugated with Alexa-fluor 488 (green; Molecular Probes, Eugene, Oreg.) for TH and NeuN or with fluorescein (Pierce, Rockford, Ill.) for NF-68, NSE, and GFAP. For TH and NeuN, cellular nuclei were also stained with Hoechst dye (Molecular Probes). The fluorescent signals were visualized with a fluorescence microscope (Nikon, Melville, N.Y.).
Viruses. The 633 strain of SV, which contains glutamine at E2-55, was used (52). Recombinant SV expressing enhanced green fluorescent protein (eGFP) as a reporter gene was constructed using 633 cDNA engineered to contain a second subgenomic promoter (27). The eGFP gene was inserted into the BstEII cloning site to create 633-eGFP. Full-length viral RNA was transcribed in vitro with an SP6 promoter from an XhoI-linearized plasmid and transfected into BHK cells with Lipofectin (Invitrogen, Carlsbad, Calif.). At 24 to 48 h after transfection, supernatant fluid containing progeny virus was collected, assayed by plaque formation on BHK cells, and stored in aliquots at –80°C.
In vitro SV infection, detection of virus, and determination of cell viability. Cells plated in six-well plates were infected with SV 633-eGFP (multiplicity of infection [MOI] of 5, determined on BHK cells) diluted in DMEM-1% FBS. After 1 h, the cells were washed once with PBS and fresh medium was added. Supernatant fluids were collected at various times after infection and analyzed for virus production by plaque assay. For virus growth curves, the amount of virus was normalized to the viable cell number at the time of infection and expressed as the number of log10 PFU per cell. Cell viability was determined by trypan blue exclusion.
Immunofluorescence and Western blot analyses of SV structural proteins. For immunofluorescent staining of the SV E2 protein, infected and mock-infected CSM14.1 cells grown on glass coverslips in six-well plates were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with ice-cold acetone. After several washes with PBS, cells were stained with anti-E2 monoclonal Ab (MAb) G5 (54) for 30 min on ice, washed six times with PBS, and stained with a secondary Ab conjugated with Alexa-fluor 594 (red; Molecular Probes) for 30 min on ice. Nuclei were then stained with Hoechst dye and visualized as before.
For Western blot analysis of SV structural proteins, cells were washed three times with ice-cold PBS and lysed with ice-cold 1% (wt/vol) CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} lysis buffer (CHAPS in immunoprecipitation [IP] buffer [PBS containing 5 mM EDTA, 5 mM EGTA, 1 mM Na3VO4, 10 mM Na pyrophosphate, and 50 mM NaF]) as described above. Equal amounts of protein per sample were separated by SDS-PAGE and analyzed by Western blotting with a rabbit polyclonal Ab specific for SV (28) or the anti-E2 MAb, G5 (54), and the Supersignal picogram or femtogram quantity detection system (Pierce).
Metabolic labeling and analysis of intracellular proteins. At various times after infection, SV-infected and mock-infected cells were washed twice with PBS, incubated in Met- and Cys-free medium for 1 h, and labeled with Tran35S-label (100 μCi/ml; ICN Biomedicals, Irvine, Calif.) for 1 h. Labeled cells were washed three times with ice-cold PBS and lysed with RIPA buffer (10 mM Tris [pH 7.0] with 1% Igepal-630, 0.1% SDS, 0.1% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA; protease inhibitors were added immediately before use). Cell lysates were placed on ice for 30 min to allow complete lysis and then centrifuged at 9,000 x g for 5 min at 4°C to remove insoluble debris. The 35S incorporated into protein was quantified by precipitation with 20% trichloroacetic acid, and equal counts per minute per lysate sample were used for protein analysis by SDS-PAGE and autoradiography.
To analyze the change in protein expression in CSM14.1 cells over time, cells were labeled and washed as described above and lysed with ice-cold 1% (wt/vol) CHAPS in IP buffer (protease inhibitors were added immediately before use). After complete lysis and removal of insoluble cellular debris, we immunoprecipitated SV proteins from samples to which equal counts per minute per lysate were added using a rabbit polyclonal Ab specific for SV at 4°C overnight. Immunocomplexes were isolated using protein G immobilized to agarose beads (Pierce), washed twice with 1% CHAPS-IP buffer and once with IP buffer, and analyzed by SDS-PAGE and autoradiography.
Statistics. Differences between geometric means of virus replication levels of undifferentiated and differentiated CSM14.1 cells or between the levels of viability of infected and mock-infected cells were determined by Student's t test using the RAqua 1.8.1 software (Free Software Foundation, Boston, Mass.).
RESULTS
The immortalized cell line CSM14.1 can differentiate into neurons. CSM14.1 cells in medium with 10% FBS actively proliferated at 31°C and then stopped proliferating and differentiated when they were shifted to medium with reduced serum (1%) at the nonpermissive temperature (37 to 39°C) (Fig. 1). During the process of differentiation, a substantial number of cells died. Differentiated CSM14.1 cells displayed a neuronal morphology, with large cell bodies and extensive process formation. To determine whether this change in morphology correlated with the expression of neuronal markers, lysates of cells at various times after differentiation were assessed for the expression of TH, MAP-2, and GFAP by Western blotting (Fig. 2). Compared to undifferentiated cells, differentiated CSM14.1 cells expressed increased levels of two neuron-specific cell markers, TH and MAP-2, and reduced levels of an astrocyte marker, GFAP (Fig. 2A and B). Immunofluorescent staining demonstrated that the majority of the differentiated cells expressed TH, NeuN, NF-68, and NSE (Fig. 2C). For subsequent experiments, cells were differentiated for 3 to 4 weeks.
SV replication and CPE of undifferentiated and differentiated CSM14.1 cells. After establishing the neuronal characteristics of differentiated CSM14.1 cells, susceptibility to SV infection and virus-induced cell death were tested. The production of new infectious virus was detected in cultures of undifferentiated cells at 7 h and reached peaks of approximately 1010.5 PFU/ml and 104.5 PFU/cell by 24 h after infection (Fig. 3A). Undifferentiated cells developed cytopathic effects (CPE), including cell rounding, detachment, and apoptotic blebs, and essentially all were dead by 4 days after infection (Fig. 4A). In contrast, the production of new virus was first detected in cultures of differentiated cells at 11 h after infection, and virus production continued to increase through 72 h (Fig. 3A). Amounts of virus produced were substantially lower in differentiated cells than in undifferentiated cells. Peak virus production in undifferentiated cells was 107.2 PFU/ml and 103.1 PFU/cell. Differentiated cells displayed little to no CPE and survived for more than 6 days after infection (Fig. 4B).
To determine whether these differences in levels of virus replication were due to the culture conditions or to the differentiation state of the cells, undifferentiated CSM14.1 cells were infected and cultured at 31°C with 10% FBS or at 39°C with 1% FBS and virus production was assessed over 24 h (Fig. 3B). The levels of virus replication were the same, indicating that higher temperature and lower FBS concentration were not the reasons for restricted virus replication in differentiated cells.
Viral and cellular protein synthesis in undifferentiated and differentiated CSM14.1 cells. Immunofluorescent staining was used to identify virus-infected cells. At 24 h after infection, all undifferentiated cells expressed detectable levels of the E2 glycoprotein, while approximately 50% of differentiated cells could be identified to be infected by this criterion (Fig. 5A). Costaining with Ab to E2 and TH at this time indicated that more than 95% of the infected cells also expressed TH (data not shown). E2 antigen was detected in all differentiated cells by day 3 after infection. Similar results were obtained when cells were assessed using a polyclonal Ab to SV (data not shown).
Examination by Western blotting of SV structural proteins in infected cells showed detectable levels of capsid, E1/E2, and the precursor E2 protein, pE2, in undifferentiated cells by 24 h after infection (Fig. 5B) and in differentiated cells by day 2 after infection. There was a lower level of expression of E2 relative to that of pE2 in differentiated cells (E2/pE2 ratio, 0.0426), compared to that in undifferentiated cells (E2/pE2 ratio, 2.14) (Fig. 5C).
SV infection is characteristically associated with a shutdown of host protein synthesis (47). To examine overall protein synthesis, BHK, undifferentiated CSM14.1, and differentiated CSM14.1 cells were infected with SV, and newly synthesized proteins were labeled with 35S for 1 h at various times after infection and analyzed by SDS-PAGE and autoradiography (Fig. 6). Mock-infected cells synthesized many cellular proteins. By 24 h after infection, the synthesis of SV proteins was predominant in BHK cells and in undifferentiated CSM14.1 cells (Fig. 6A). Viral structural proteins, E1/E2, pE2, and capsid, were readily identified in infected BHK cells and in undifferentiated CSM14.1 cells within 24 h after infection. On the other hand, viral proteins were not clearly detected in the differentiated CSM14.1 cells until day 2 and were maximal on day 3 after infection. Differentiated CSM14.1 cells survived SV infection and gradually restored cellular protein synthesis (Fig. 6A). No newly synthesized viral proteins were detected in differentiated cells by immunoprecipitation on day 6 after infection (Fig. 6B).
DISCUSSION
An important host factor determining the outcome of alphavirus infection of neurons is age (21, 29), but there has not been a convenient in vitro model for characterizing this aspect of infection. These studies have shown that both undifferentiated and differentiated CSM14.1 neuronal cells could be infected with SV and that the levels of virus replication and CPE were dependent on the maturity of the cells. Undifferentiated CSM14.1 cells were efficiently infected, produced large amounts of virus, and died within 3 to 4 days. Differentiated CSM14.1 cells were less efficiently infected, produced virus more slowly, had 10- to 100-fold-lower peak virus production, and survived for many days. E2 glycoprotein processing was more efficient in undifferentiated than in differentiated cells. Virus infection led to a shutdown of cellular protein synthesis in both undifferentiated and differentiated CSM14.1 cells, but differentiated cells spontaneously restored host protein synthesis. Restoration of cellular protein synthesis was accompanied by a reduction in viral protein synthesis and a decreased production of infectious virus. These characteristics of virus replication and neuronal survival are typical of the responses to SV infection in immature and mature mice (18, 29).
Several experimental approaches have been used to study the survival of neurons after SV infection and the response of these cells to immune mediators. The AT3 rat prostatic adenocarcinoma cell line has been transfected with the cellular oncogene Bcl-2, an antiapoptotic protein. AT3-Bcl-2 cells are more resistant than AT3 cells to virus-induced cell death (35), exhibit Ab-mediated down-regulation of production of infectious virus, but die 2 to 3 days after infection (9, 10). Thus, AT3-Bcl-2 cells have the advantage of a continuous cell line but do not reflect the survival of mature neurons in vivo. Primary neuronal cells, such as rodent cortical and DRG neurons, have also been studied. Cortical neurons and undifferentiated DRG neurons collected from embryonic mice or rats are highly susceptible to virus-induced cell death (34, 41). DRG neurons differentiated for 2 to 3 weeks in culture remain alive for several days after infection and have been used successfully to study Ab-mediated clearance of virus (34, 54). However, the preparation of DRG neurons is labor-intensive and the number of cells that can be obtained is limited.
CSM14.1 cells, which can be propagated without losing their ability to differentiate, have been used previously to study neuronal differentiation (23, 30) and the effects of various factors on neurons (12, 13, 38, 57, 58) but have not been used to study responses to virus infection. Consistent with previous studies (11, 23), differentiating CSM14.1 cells sequentially expressed general and dopaminergic neuron-specific markers in a manner similar to that observed in developing rodent brain (23). In contrast to what was found in one previous study using an MAb which detected no GFAP in CSM14.1 cells (23), we found that GFAP was detectable in undifferentiated cells by using a polyclonal Ab and was down-regulated with differentiation. GFAP, an intermediate filament protein, is commonly used as an astrocyte marker but has been found in many nonglial populations of cells, such as hepatic stellate cells (45), chondrocytes of epiglottic cartilage (4), and fibroblasts (24). The inversely proportional relationship between GFAP expression and the time of CSM14.1 cell differentiation is consistent with the conclusion that CSM14.1 cells assume a more neuronal phenotype as they differentiate.
Undifferentiated CSM14.1 cells were very susceptible to SV infection and produced new virus within 8 h. With differentiation, CSM14.1 cells became less susceptible to SV infection and produced new infectious virus more slowly than undifferentiated cells. These differences were not due to the culture conditions because undifferentiated cells infected under the culture conditions for differentiated cells (39°C and 1% FBS) replicated virus efficiently.
Immunocytochemical staining suggested that all undifferentiated cells, but not all differentiated cells, were infected initially. In cultures of differentiated cells, the infection spread gradually, with all cells being infected within 3 days. SV structural proteins were present in neuronal processes that contacted neighboring cells, possibly facilitating the spread of virus from cell to cell rather than the spread by release of infectious virions into the media. The delay in the appearance of viral structural proteins detectable by Western blotting in differentiated CSM14.1 cells may reflect the initial infection of fewer cells. However, peak virus production on a per-cell basis was also lower in differentiated than in undifferentiated cells. The level of fully processed E2 relative to that of pE2 was lower in differentiated than in undifferentiated cells. Less-efficient posttranslational processing of pE2 into E2 may contribute to the reduced and slower production of SV in differentiated cells. Furthermore, if pE2 is incorporated into virions, production of noninfectious particles may also delay the spread of infection in differentiated CSM14.1 cell cultures.
Shutdown of host protein synthesis was evident by 24 h after infection in undifferentiated CSM14.1 cells, and these cells eventually died. In differentiated cells, the ratio of viral to host proteins synthesized gradually increased, peaked by day 3, and decreased thereafter, suggesting that differentiated neurons controlled virus replication and restored cellular protein synthesis. Cell survival and the reappearance of cellular protein synthesis in differentiated CSM14.1 cells after infection indicated an ability of these cells to control virus replication. Control of virus replication has also been described for interferon-producing cells infected with a strain of SV with mutated nonstructural P2 (16), suggesting a potential role for interferon in this process.
This pattern of virus growth and the association between neuronal maturation and reduced susceptibility to virus infection resembles the time course of viral replication and outcome of SV infection in mice (18, 21, 29). Increasing age correlates with reduced virus production and reduced virus-induced mortality and is not attributable to maturation of the immune response (18, 29). In newborn mice, peak virus titers are reached within 1 to 2 days after infection and neuronal apoptosis is observed (36). In weanling mice, peak virus titers are reached 3 to 4 days after infection, cell-to-cell spread is observed, and there is minimal apoptosis (17, 50). Furthermore, in the absence of an antiviral immune response, SV-infected neurons of weanling mice survive, partially control virus replication, and continue to produce virus for many weeks after infection (3, 34).
Age-dependent resistance to virus replication is also a characteristic of a number of other neurotropic viruses, including Semliki Forest virus (SFV) (14, 15, 43), Japanese encephalitis virus (JEV) (32, 42, 56), mouse hepatitis virus (48), measles virus (19, 22), reovirus (39), blue-tongue virus (5), parvoviruses (6, 7), influenza virus (26), and herpesviruses (1, 2, 40, 55). As with the AR339 strain of SV, infection of neonatal mice with the A7(74) strain of SFV leads to fatal encephalitis, but mice that are at least 2 weeks of age recover uneventfully (44). A7(74) replicates productively, spreads efficiently, and leads to the death of neurons during axonogenesis and synaptogenesis, whereas replication is restricted in mature neurons (14, 15, 43). Type 3 reovirus, induces an acute necrotizing and lethal encephalitis in newborn mice but a nonfatal infection in older animals (39). Age-dependent resistance of mice to infection with SFV and reovirus is not explained by developmental changes in the adaptive immune response but rather by an intrinsic ability of mature neurons to restrict viral replication (15, 18, 49). Similarly, JEV preferentially infects immature neurons, and animals older than 2 weeks survive infection (42, 56).
The susceptibility of immature neurons to JEV may be related to a decreased expression of neuronal surface molecules acting as viral receptors as the neurons mature (32), and changes in the abundance of neuronal receptors may also play a role in age-dependent susceptibility to SV (53), but even after infection, less virus is produced by mature than by immature neurons, indicating an additional postentry restriction of replication. In addition to receptor expression, potentially important age-dependent determinants of resistance that have been identified include decreased expression of proapoptotic molecules and inflammatory response genes and increased expression of the neuroprotective chemokine fractalkine and of interferon-inducible genes (25, 33, 36).
CSM14.1 cells demonstrate development of resistance as a function of neuronal maturation and provide a system in which the cellular basis for the neuronal restriction of SV can be studied. This in vitro model system should allow further studies of the molecular mechanisms that determine the age-dependent interactions of neurons with SV and the mechanisms of host control of virus replication in neurons.
ACKNOWLEDGMENTS
These studies were supported by research grants RO1 NS18596 and NS38932 (D.E.G.) and medical scientist training program grant T32 GMO7309 (P.S.V.) from the National Institutes of Health.
We appreciate the helpful suggestions of George Oyler, Irina Shatz, and Jeffrey Rothstein; assistance from Marcia Lyons; and support from the Cellular and Molecular Medicine Graduate Training Program at the Johns Hopkins University School of Medicine.
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