Viruses and inoculations.

Recombinant A59 (rA59) (previously referred to as RA59, S_A59R13, or wtR13), recombinant JHM.SD (rJHM.SD) (previously referred to as RJHM and derived from the MHV-4 isolate of JHM.SD), and a chimeric recombinant expressing the JHM.SD spike in the A59 background (rA59/S_JHM.SD) (previously referred to as SJHM/RA59 or S₄R22) were described previously (37, 41, 42). Recombinant viruses of the same genotypes, but expressing enhanced green fluorescent protein (EGFP) in place of gene 4 (8, 27), were used to monitor infection in primary cells. Viruses were propagated in murine 17Cl1 fibroblasts and titrated by standard plaque assay on murine L2 fibroblasts. To remove cell fragments from the virus preparations, filtered virus stocks were prepared by passage through a 0.22-μm filter apparatus and were retitrated after filtration. For i.c. inoculations, 4-week-old mice were anesthetized with isoflurane, and 50 PFU of virus diluted in 30 μl phosphate-buffered saline (PBS) containing 0.75% bovine serum albumin (BSA) was injected into the left cerebral hemisphere. Mock infections were performed with lysate from 17Cl1 fibroblasts.

Immunofluorescence.

For identification of infected cell types, brains were perfused with PBS, fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sagittally sectioned. Sections were deparaffinized and rehydrated, treated with antigen-unmasking solution (Vector Labs), blocked with 1.5% normal goat serum, and dual immunolabeled with a mouse monoclonal antibody (1.16.1) directed against the MHV nucleocapsid protein (a gift from Julian Leibowitz, Texas A&M University) and a rabbit polyclonal antibody directed against either glial fibrillary acidic protein (GFAP), for astrocytes (Dako USA); Iba1, for microglia (Wako Pure Chemical Industries); OLIG2, for oligodendrocytes (Millipore); or microtubule-associated protein 2 (MAP2), for neurons (a gift from Virginia Lee, University of Pennsylvania). Primary antibodies were detected with Alexa Fluor 488- and 594-conjugated goat anti-mouse and goat anti-rabbit (Invitrogen). Cultured cells were fixed in PBS containing 4% paraformaldehyde (Electron Microscopy Sciences), blocked with 1.5% normal goat serum, and immunolabeled as described above, with the exception of microglia, which were labeled with rat monoclonal anti-CD11b (Abcam) and goat anti-rat-Alexa Fluor 594 (Invitrogen). Fluorescence was visualized with a Nikon Eclipse TE2000-U microscope, and images were acquired using Spot imaging software (Diagnostic Instruments).

Primary hepatocyte cultures.

Primary hepatocytes were prepared from 8-week-old mice by in situ perfusion and digestion with liver perfusion medium (Invitrogen) and liver digestion medium (Invitrogen), respectively, followed by mechanical disruption and Percoll separation as previously described (48). Hepatocytes were seeded on BioCoat collagen I-coated plates (BD Biosciences) and were cultured overnight in RPMI 1640 medium containing 10% fetal bovine serum (FBS).

Primary glial cultures.

Mixed glial cells were prepared from the cortices of P1-3 neonates and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin for 5 to 14 days. Mixed glial cultures were enriched for either astrocytes or microglia based on differential adhesion to tissue culture plastic as previously described (1). Astrocyte cultures were routinely 90 to 95% pure, as determined by positive immunostaining for GFAP and negative staining for MAP2, CD11b, and OLIG2. Microglia cultures were routinely ≥98% pure, as determined by positive immunostaining for CD11b and negative staining for MAP2, GFAP, and OLIG2. Oligodendrocytes were prepared from forebrains of P1-3 neonates by trypsin digestion and were cultured in DMEM containing 10% FBS, 1× nonessential amino acid solution (Invitrogen), 2 mM l-glutamine, 100 U/ml penicillin, and 100 ng/ml streptomycin as previously described (13). After 24 h, culture medium was replaced with neurobasal medium containing B-27 supplement, 10 ng/ml bovine basic fibroblast growth factor (R&D Systems), 2 ng/ml recombinant human platelet-derived growth factor (R&D Systems), and 1 ng/ml recombinant human NT-3 (PeproTech Inc.). After 7 days, oligodendrocytes were removed from underlying astrocytes by gentle rinsing and were seeded onto poly-d-lysine-coated tissue culture plates. Oligodendrocyte cultures were routinely 90 to 95% pure, as determined by positive immunostaining for OLIG2 and negative immunostaining for MAP2, CD11b, and GFAP.

Primary neuronal cultures.

Hippocampal neurons were prepared from E15-16 mouse embryos, seeded onto poly-l-lysine-coated coverslips or tissue culture plates, and cultured in neurobasal medium containing B-27 supplement (Invitrogen), 100 U/ml penicillin, 100 ng/ml streptomycin, 2 mM l-glutamine, and 4 μg/ml glutamate for 4 days in the absence of an astrocyte feeder layer, as previously described (2, 40, 43). Cortical neurons prepared from E18 mouse embryos were provided by Marc Dichter (University of Pennsylvania) (9, 50). Neuron cultures of both types were routinely 95 to 98% pure, as determined by positive immunostaining for MAP2 and negative immunostaining for CD11b, GFAP, and OLIG2. After 4 days in vitro, neuron cultures were inoculated with virus diluted in neurobasal medium for 1 h at 37°C, washed, and cultured for an additional 24 to 72 h in neurobasal medium. To quantify extracellular virus, neuron supernatants were collected at various times postinfection and stored at −80°C until titration. Fresh neurobasal medium was added to the remaining cells, and intracellular virus was released by repeated freeze-thaw cycles, with freezing to −80°C.

Cell lines.

Murine DBT astrocytoma cells, 17Cl1 fibroblasts, and L2 fibroblasts were cultured in DMEM supplemented with 10% FBS, 10 mM HEPES, 2 mM l-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, and 2.5 μg/ml amphotericin B. Human 293T cells were cultured in high-glucose DMEM containing 10% FBS, 100 U/ml penicillin, and 100 ng/ml streptomycin.

Quantitative reverse transcription-PCR (qRT-PCR).

RNAs were isolated from liver, brain, and spinal cord tissues by homogenization in TRIzol reagent (Invitrogen), followed by phenol-chloroform extraction and purification with an RNeasy Mini kit (Qiagen) as previously described (49). RNAs were isolated from cultured cells by use of an RNeasy Mini kit according to the manufacturer's instructions. Tissue and cellular RNAs were DNase treated using a Turbo DNA-free kit (Ambion), and quantitative PCRs without reverse transcriptase were performed to ensure adequate removal of genomic DNA. For cDNA synthesis, 350 ng RNA was combined with 0.5 mM deoxynucleoside triphosphate (dNTP) mix (0.5 mM, total concentration; Invitrogen) and 50 ng random hexamers (Invitrogen) in a total volume of 13 μl, heated to 65°C in a PCR thermocycler for 3 min, and cooled to room temperature. Next, 1× first-strand buffer (Invitrogen), 5 mM dithiothreitol (DTT) (Invitrogen), 200 U SuperScript III reverse transcriptase (Invitrogen), and nuclease-free water were added for a final reaction volume of 20 μl, and the mixture was heated to 50°C for 60 min followed by 70°C for 15 min. cDNA was stored at −20°C until use. Quantitative PCR was performed in duplicate, using 2 μl cDNA, 12.5 μl iQ SYBR Green Supermix (Bio-Rad), and 0.4 μM (each) forward and reverse primers (listed in Table ​Table1)1) in a total volume of 25 μl in an iQ5 iCycler (Bio-Rad). For copy number determination, cycle threshold (C_T) values were compared to a plasmid standard curve run in parallel for each target. Copy numbers are expressed as numbers of target cDNA copies per million cDNA copies of the mouse beta-actin gene (actb). For relative expression compared to mock-infected samples, C_T values were normalized to actb levels, resulting in a ΔC_T value (ΔC_T = C_{T target} − C_{T actb}). ΔΔC_T values were then calculated (ΔΔC_T = ΔC_{T infected} − ΔC_{T mock}), and results are expressed as fold changes compared to mock-infected samples (2^−ΔΔCT).

TABLE 1.

Primer sequences for qRT-PCR

Target	Forward primer (5′-3′)	Reverse primer (5′-3′)	Product size (bp)	GenBank accession no.
Mouse targets
Beta-actin gene	CAGATGTGGATCAGCAAGCAGGA	CGCAGCTCAGTAACAGTCCGCCTA	90	NM_007393
ceacam1a
Total	GCCTCAGCACATCTCCACAAAG	CCTCAATGGTGACTTCAGCAGTG	109	NM_001039185
2D	CCTGGCGCTTGGAGCCTTTG	GCTGAGTCACTGGCTGGTGT	263	NM_001039187
4D	GGCCTCAGTAGGACCACAGTCAAGA	CGACAAGCAGGTCAGGGTCACAG	120	NM_001039185
S	GCATCGTGATTGGAGTTGTG	GTTGTCAGAAGGAGCCAGATCC	99	NM_001039187
L	GGGAAGTGACCAGCGAGAT	GTGGTTGGAGGCTGAGGGTTTGT	52	NM_001039185
ceacam2	CATCTCCACAAAGGGCAGGTTCC	TGTGCCAGCTAAAGGCCGAGAC	187	NM_007543
psg16	GCTTCGAGGGCTTTCCTGGTACA	TGTCGGGCAATCTCAAGGTTCCTA	63	NM_007676, U34272
MHV target
mRNA7 (nucleocapsid)	GGCGTCCGTACGTACCC	GGTCAGCCCAAGTGGTC	167	X00990

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Plasmids.

pLXSN-BgpC (CEACAM1a-2S) and pLXSN-BgpD (CEACAM1a-4L), used for ceacam1a splice variant standard curves, were provided by Nicole Beauchemin (McGill University). pCMV-SPORT6-CEACAM1, pCMV-SPORT6-CEACAM2, pCMV-SPORT6-PSG16, and pCMV-SPORT6-ACTB (mouse beta-actin partial clone) were purchased from Open Biosystems (Thermo Scientific). pCB-TVA-CEACAM1-TM, pCB-TVA-CEACAM2-TM, and pCB-TVA-PSG16-TM were made by replacing the AgeI-SacII fragment of pCB-TVA 950 (46) (obtained from Paul Bates) with PCR products containing the extracellular domains for the pCMV-SPORT6 plasmids listed above. pCB-TVA-CEACAM1-GPI, pCB-TVA-CEACAM2-GPI, and pCB-TVA-PSG16-GPI were constructed by replacing the AgeI-SacII fragment of pCB-TVA 800 (obtained from Paul Bates) as described above.

ceacam1a mRNA is expressed in murine tissues, primary cells, and cell lines.

Both rA59 and rJHM.SD infect multiple cell types, as described above, but microglia are the only CNS cell type previously demonstrated to express CEACAM1a protein (44). While we were able to detect CEACAM1a on microglia by flow cytometry, we were unable to demonstrate CEACAM1a protein on neurons or any glial cell types by immunoblotting, immunohistochemistry, or immunohistochemical amplification techniques such as tyramide signal amplification (PerkinElmer) (data not shown). Therefore, a qRT-PCR approach was taken to assess the expression of ceacam1a mRNA in murine tissues, primary CNS cells, and cell lines to determine whether susceptible CNS cells might express low levels of CEACAM1a protein not detectable by routine methods. Primers were designed to amplify a region of the N-terminal Ig-like domain. This domain contains the site of MHV spike binding and is present in all four known ceacam1a splice variants, thus allowing amplification of total ceacam1a mRNA. The results are expressed as numbers of cDNA copies of ceacam1a per million cDNA copies of actb. To determine if mRNA expression data parallel known CEACAM1a protein expression data, RNAs isolated from livers, brains, and spinal cords of 4-week-old C57BL/6 mice were subjected to qRT-PCR analysis for total ceacam1a mRNA, using the primer set listed in Table ​Table1.1. Consistent with published data showing that the liver expresses more CEACAM1a protein than the brain (17, 59), ceacam1a mRNA levels in the liver were approximately 10- to 100-fold higher than those in both the brain and spinal cord in WT mice (Fig. ​(Fig.22 A). To ensure the specificity of these primers, qRT-PCR analysis was performed on tissue RNA from ceacam1a^−/− (KO) mice. As expected, no product was amplified from ceacam1a^−/− RNA (Fig. ​(Fig.2A),2A), confirming the specificity of the assay.

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FIG. 2.

ceacam1a mRNA expression in murine tissues, primary cells, and cell lines. RNAs isolated from 4-week-old C57BL/6 mouse tissues (A), primary cell cultures (B), cell lines (C), 2- to 14-week-old C57BL/6 mouse tissues (D), and C57BL/6 mouse brains (E and F) from mice inoculated i.c. with 50 PFU of rA59 or rJHM.SD were analyzed by qRT-PCR for expression of total ceacam1a mRNA (A to E) or mRNA7 (F). Results are expressed as numbers of cDNA copies of ceacam1a per million cDNA copies of actb (A to D) or as fold changes compared to mock-infected controls (E and F). Error bars represent standard errors of the means (n = 3). Data are representative of two or more independent experiments. LIV, liver; BR, brain; SC, spinal cord; WT, wild type; KO, ceacam1a^−/−; HEP, hepatocyte; MIC, microglia; AST, astrocyte; OLI, oligodendrocyte; CN, cortical neuron; HN, hippocampal neuron.

To evaluate which CNS cell types express ceacam1a, enriched primary cell cultures were generated from WT mice as described in Materials and Methods. RNAs isolated from these cell cultures were then subjected to qRT-PCR analysis of total ceacam1a expression. Primary hepatocytes and microglia, two cell types known to express CEACAM1a protein (17, 44), were used as positive controls. As expected, both hepatocytes and microglia were positive for ceacam1a expression, with hepatocytes expressing approximately 10-fold higher levels than microglia (Fig. ​(Fig.2B).2B). Interestingly, ceacam1a expression was readily detected in both astrocyte and oligodendrocyte cultures, albeit at lower levels than in microglia (Fig. ​(Fig.2B).2B). While ceacam1a expression was also detected in cortical and hippocampal neuron cultures, these expression levels were extremely low (Fig. ​(Fig.2B).2B). Importantly, since primary cell cultures are enriched but not entirely pure, immunofluorescence labeling was performed as described in Materials and Methods to identify contaminating cell types in the individual cultures. Microglia cultures were routinely ≥98% pure, with rare astrocyte and oligodendrocyte contamination (data not shown). Astrocyte cultures were routinely 90 to 95% pure, with contaminating microglia and rare oligodendrocytes (data not shown). Oligodendrocyte cultures were routinely 90 to 95% pure, with contaminating astrocytes and rare microglia (data not shown). Neuron cultures were routinely 95 to 98% pure, with contaminating astrocytes, oligodendrocytes, and rare microglia (data not shown). It is important to consider these low levels of contaminating cells in interpreting the qRT-PCR results obtained for primary cell cultures.

Murine DBT, 17Cl1, and L2 cell lines are routinely used for propagation and/or titration of MHV. DBT cells are derived from astrocytoma tissues in CDF1 mice (22), whereas 17Cl1 and L2 fibroblasts are derived from the BALB/c 3T3 cell line and the C3H/An L929 cell line, respectively (52). Given the different mouse strains of origin as well as ongoing propagation in tissue culture, it was important to characterize the levels of ceacam1a expression in these cell lines. To this end, RNAs isolated from DBT, 17Cl1, and L2 cells were subjected to qRT-PCR analysis of total ceacam1a expression. As expected, ceacam1a mRNA was readily detected in all three cell lines (Fig. ​(Fig.2C),2C), consistent with their permissiveness to MHV infection in vitro. Furthermore, all cell lines examined showed similar levels of ceacam1a expression (Fig. ​(Fig.2C2C).

WT mice exhibit age-dependent differences in susceptibility to CNS infection with some neurotropic strains of MHV, including rA59. Since ceacam1a expression is developmentally regulated in mouse embryos (20), it was of interest to determine if ceacam1a mRNA levels in the CNS changed with age in postnatal mice. To this end, RNAs were isolated from liver and brain tissues of WT mice aged 2 to 14 weeks. This age range encompasses the different subsets of weanling and adult mice typically used for MHV studies. Interestingly, ceacam1a expression levels were relatively stable in both the liver and brain at all ages observed (Fig. ​(Fig.2D),2D), suggesting that age-related differences in MHV susceptibility are not due to CEACAM1a expression.

Previous flow cytometric studies demonstrated that CEACAM1a expression is downregulated on microglia during acute infection of BALB/c mice with the J.2.2v-1 variant of JHM.SD; this downregulation, largely mediated by CD4 T cells, is restored following viral control (44). To determine if MHV infection affected ceacam1a expression in the whole brain (including brain-resident cells not easily observed by flow cytometry), C57BL/6 mice were inoculated i.c. with 50 PFU of rA59 or rJHM.SD, and RNAs were isolated on days 1 to 5 postinfection. This period was chosen to precede the CD4 T-cell peak in the brain (23) in order to examine whether acute infection and/or early host responses also affected ceacam1a expression. Within this period of acute infection, ceacam1a expression levels were unchanged compared to those in mock-infected brains (Fig. ​(Fig.2E).2E). Importantly, these ceacam1a expression levels remained constant despite robust levels of virus replication, as demonstrated by qRT-PCR analysis of nucleocapsid mRNA (mRNA7) (Fig. ​(Fig.2F).2F). Therefore, early host responses do not affect ceacam1a expression.

ceacam1a splice variant mRNAs are differentially expressed in murine tissues and primary cells.

The CEACAM1a proteins containing two Ig-like domains (2D) have been suggested to be less efficient receptors than those expressing all four domains (4D) (3), which has the potential to affect MHV tropism; for example, if neurons were to express predominantly 2D isoforms, one might hypothesize that neurotropic strains are better able to use 2D isoforms than are nonneurotropic strains. In addition, the CEACAM1a isoforms with short cytoplasmic tails (S) lack some of the signaling functions of the long-tailed (L) isoforms (18, 25), which could affect infection outcomes. To more fully characterize the expression patterns of ceacam1a in CNS tissues and cell types, primer sets were designed to detect 2D versus 4D ceacam1a splice variants and short versus long cytoplasmic tails (S versus L). Primer sets are listed in Table ​Table1,1, and a schematic depicting primer amplification sites is shown in Fig. ​Fig.33 A. While traditional RT-PCR can be used to distinguish all four splice variants based on product size alone, such assays are complicated by differences in amplification efficiency for shorter versus longer splice variants. While the qRT-PCR assay described here does not allow for distinction of each individual splice variant (2S, 2L, 4S, and 4L), it does allow for more careful quantification of 2D versus 4D species and S versus L species. As shown in Fig. ​Fig.3A,3A, ​,2D2D primers amplify a region at the splice junction between D1 and D4, whereas 4D primers amplify a region at the junction of D3 and D4. Similarly, S primers amplify the region where exon 7 has been removed by splicing, whereas L primers encompass a portion of exon 7 (Fig. ​(Fig.3A).3A). Plasmids carrying ceacam1a-2S (BgpC) and ceacam1a-4L (BgpD) were used to construct standard curves for copy number determination by qRT-PCR.

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FIG. 3.

ceacam1a splice variant expression in murine tissues, primary cells, and cell lines. (A) Schematic representing ceacam1a splice variants. (B to G) RNAs isolated from 4-week-old C57BL/6 mouse tissues (B and E), primary cell cultures (C and F), and cell lines (D and G) were analyzed by qRT-PCR for ceacam1a expression of 2 versus 4 Ig-like domains (B to D) or short versus long cytoplasmic tails (E to G). Results are expressed as numbers of cDNA copies of ceacam1a per million cDNA copies of actb. Error bars represent standard errors of the means (n = 3). *, P ≤ 0.05; **, P ≤ 0.005 (determined by paired t tests). Data are representative of two independent experiments. 2D, two extracellular Ig-like domains; 4D, four extracellular Ig-like domains; S, short cytoplasmic tail due to exclusion of exon 7; L, long cytoplasmic tail due to inclusion of exon 7.

Using these primer sets, the tissue and cellular RNAs described for Fig. ​Fig.22 were analyzed by qRT-PCR to compare splice variant expression. As shown in Fig. ​Fig.3B,3B, ​,4D4D variants were expressed at significantly higher levels than those of 2D variants in the liver, whereas 2D variants were expressed more highly in the brain (2D variants also predominated in the spinal cord, but the difference was not statistically significant). Interestingly, while primary hepatocytes mimicked the expression (4D > 2D) observed in the liver, most primary CNS cell types also expressed 4D variants at higher levels than those of the 2D variants (Fig. ​(Fig.3C).3C). This difference in brain expression versus primary cells may reflect the contribution of an additional cell type in vivo, such as endothelial cells, that was not analyzed in the primary cell panel. A notable exception in the primary cell panel was the oligodendrocyte cultures, which expressed slightly more 2D than 4D species, though the difference was not statistically significant (Fig. ​(Fig.3C).3C). The absence of the 4D > 2D pattern seen in other primary cell types suggests that oligodendrocytes themselves express ceacam1a and that the total ceacam1a expression level shown in Fig. ​Fig.2B2B is not due entirely to contaminating microglia or astrocytes. Cell line expression of ceacam1a isoforms also differed slightly, with DBT cells expressing relatively equal levels of 2D and 4D variants, while 17Cl1 fibroblasts expressed higher levels of 4D species than 2D species; L2 fibroblasts also expressed predominantly 2D species, but the difference was not statistically significant (Fig. ​(Fig.3D).3D). The pattern of 2D versus 4D splice variants did not suggest any significance for MHV tropism.

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FIG. 4.

MHV infection and replication in wild-type neuron cultures. Hippocampal neurons generated from C57BL/6 mice were inoculated with 1 PFU/cell of rA59, rA59/S_JHM.SD, or rJHM.SD. (A) Cells were fixed at 72 h postinfection and dual immunolabeled for MHV nucleocapsid protein (green) and MAP2 (red). Magnification, ×200. Cell lysates (B) and culture supernatants (C) were collected at 24, 48, and 72 h postinfection, and infectious virus was titrated by standard plaque assay on L2 cell monolayers. Error bars represent standard errors of the means (n = 3); the dotted line indicates the limit of detection. Data are representative of two or more independent experiments.

Since the length of the cytoplasmic tail may affect infection outcome, tissue and cellular RNAs were analyzed for expression of ceacam1a mRNA species encoding S versus L cytoplasmic tails. Interestingly, the liver exhibited relatively even expression of S and L variants, while L variants predominated over S variants in the brain (L variants also predominated in the spinal cord, but the difference was not statistically significant) (Fig. ​(Fig.3E).3E). In terms of S versus L tails, the primary cells analyzed mimicked the expression seen in the tissues from which they were derived (Fig. ​(Fig.3F).3F). Expression in all three cell lines showed a similar pattern as well, with L species predominating slightly over S species in DBT cells and L2 fibroblasts (L species appeared more highly expressed in 17Cl1 fibroblasts as well, but the difference was not statistically significant) (Fig. ​(Fig.3G).3G). Again, we did not detect any expression pattern which would explain differences in MHV tropism.

rJHM.SD spreads in wild-type neuron cultures despite low levels of infectious virus.

To determine whether the low levels of ceacam1a message detected in enriched primary neuron cultures were significant for MHV infection, we infected neurons from wild-type and ceacam1a^−/− mice with rA59 and rJHM.SD and assayed for virus entry and spread. Hippocampal neuron cultures were generated from embryonic C57BL/6 mice and inoculated with 1 PFU/cell of filtered rA59, rJHM.SD, or chimeric recombinant A59 virus expressing the JHM.SD spike (rA59/S_JHM.SD); virus stocks were filtered prior to neuronal inoculations to remove residual cell debris. Recombinant versions of these viruses expressing EGFP were used in parallel to monitor infection over time and revealed widespread infection with all virus strains by 72 h postinfection (data not shown). Therefore, cells were fixed at 72 h postinfection and immunolabeled for MAP2 and the MHV nucleocapsid protein as described in Materials and Methods. As expected, all three viruses were able to infect and spread in wild-type cultured neurons, as evidenced by colocalization of MAP2 and MHV antigen (Fig. ​(Fig.44 A); however, foci of rJHM.SD- and rA59/S_JHM.SD-infected neurons were typically larger than rA59-infected foci, consistent with previous results (42). To investigate whether spread in neuron cultures was occurring between adjacent cells or via virus release, cell lysates and culture supernatants were collected at 24, 48, and 72 h postinfection, and infectious virus was titrated by standard plaque assay on L2 cell monolayers. Interestingly, while rA59 replicated to relatively high titers within cells, rJHM.SD and rA59/S_JHM.SD titers in cell lysates were approximately 10- to 100-fold lower than rA59 titers at all three time points (Fig. ​(Fig.4B).4B). Virus release from infected neuron cultures paralleled the intracellular differences, with extracellular rA59 titers exceeding rJHM.SD titers by up to 1,000-fold by 72 h postinfection (Fig. ​(Fig.4C).4C). Thus, both rJHM.SD and rA59/S_JHM.SD spread well in wild-type neurons, despite relatively low levels of virus replication and release, suggesting that spread between adjacent cells is likely to be the major route of rJHM.SD dissemination in neurons.

rJHM.SD spreads extensively in ceacam1a^−/− neurons.

To determine if MHV entry and spread in neurons are dependent on the CEACAM1a receptor, hippocampal neurons were generated from embryonic ceacam1a^−/− mice and inoculated with 1 PFU/cell of filtered rA59, rJHM.SD, or chimeric rA59/S_JHM.SD. Recombinant versions of these viruses expressing EGFP were again used in parallel to monitor the spread of infection (data not shown). Cells were fixed at 72 h postinfection and immunolabeled for MAP2 and MHV nucleocapsid protein as described for Fig. ​Fig.4.4. Surprisingly, all three viruses were able to infect ceacam1a^−/− neurons, as evidenced by colocalization of MAP2 and MHV antigen (Fig. ​(Fig.5).5). However, foci of infection were much more rare (typically 1 to 3 infected foci per coverslip) for all virus strains in ceacam1a^−/− neuron cultures than the numerous coalescing foci of infection observed in wild-type neuron cultures (data not shown). Thus, virus infection of neuron cultures was much less efficient in the absence of CEACAM1a. Strikingly, though all viruses were able to enter individual cells in the ceacam1a^−/− neuron cultures, only rJHM.SD and rA59/S_JHM.SD spread efficiently to adjacent cells (Fig. ​(Fig.5).5). By 72 h postinfection, rA59 remained localized to one or two ceacam1a^−/− neurons, while viruses expressing the JHM.SD spike spread similarly in ceacam1a^−/− and wild-type cultures (Fig. ​(Fig.4A4A and ​and5).5). Similar differences in spread were observed in blocking experiments in which wild-type neurons were infected with rA59 or rJHM.SD and then treated with CEACAM1a-blocking antibody to evaluate neuronal spread in the absence of CEACAM1a (data not shown). Thus, while MHV entry into neural cells appears largely dependent on ceacam1a expression, only rA59 appears to require ceacam1a expression for efficient spread among adjacent neurons. This finding is consistent with previous studies showing that ceacam1a^−/− mice are susceptible to infection with viruses expressing the JHM.SD spike but resistant to rA59 (33). Taken together, these results imply that the low levels of ceacam1a detected in enriched primary neuron cultures are significant for infection by A59 but not for infection by JHM.SD.

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FIG. 5.

MHV infection in ceacam1a^−/− neuron cultures. Hippocampal neurons generated from ceacam1a^−/− mice were inoculated with 1 PFU/cell of rA59, rA59/S_JHM.SD, or rJHM.SD. Cells were fixed at 72 h postinfection and dual immunolabeled for MHV nucleocapsid protein (green) and MAP2 (red). Magnification, ×200. Data are representative of two or more independent experiments.

ceacam2 mRNA is expressed at low levels in the murine liver and CNS.

To determine if an alternative CEACAM1a-related receptor might be positioned to mediate viral infection/spread in ceacam1a^−/− mice and neuron cultures, primers were designed to amplify the N-terminal Ig-like or equivalent domain of ceacam2 and psg16 (bCEA), two ceacam1a-related genes that encode proteins that serve inefficiently as MHV receptors when overexpressed in vitro (5, 38). Primer sequences are shown in Table ​Table1.1. Using the tissue and cellular RNAs described for Fig. ​Fig.2,2, qRT-PCR was performed to detect expression of ceacam2, the most closely related gene to ceacam1a in the mouse. Results are expressed as numbers of cDNA copies of ceacam2 per million cDNA copies of actb. As shown in Fig. ​Fig.66 A, ceacam2 mRNA was expressed at similarly low levels in both liver and CNS tissues of WT mice. Notably, CNS expression of ceacam2 was relatively unchanged in the absence of ceacam1a (KO), while liver expression was slightly increased compared to that in WT mice (Fig. ​(Fig.6A);6A); however, only one set of KO tissues (n = 3) was available for qRT-PCR analysis. Interestingly, despite detectable levels of ceacam2 mRNA in both liver and CNS tissues, low to no expression of ceacam2 was observed in the primary cell types studied, with only microglia and oligodendrocyte cultures showing consistent low-level expression (Fig. ​(Fig.6B).6B). Thus, other resident cell types, such as endothelial cells, likely contribute to the ceacam2 expression observed in the liver and CNS. Interestingly, when cell lines were observed, ceacam2 expression was detectable in L2 and, to a lesser extent, DBT cells but not in 17Cl1 cells (Fig. ​(Fig.6C).6C). As with ceacam1a, ceacam2 expression stayed relatively constant in mice from 2 to 14 weeks of age (Fig. ​(Fig.6D)6D) and was not markedly affected by MHV infection (Fig. ​(Fig.6E6E).

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FIG. 6.

ceacam2 mRNA expression in murine tissues, primary cells, and cell lines. RNAs isolated from 4-week-old C57BL/6 mouse tissues (A), primary cell cultures (B), cell lines (C), 2- to 14-week-old C57BL/6 mouse tissues (D), and C57BL/6 mouse brains from mice inoculated i.c. with 50 PFU of rA59 or rJHM.SD (E) were analyzed by qRT-PCR for expression of ceacam2 mRNA. Results are expressed as numbers of cDNA copies of ceacam2 per million cDNA copies of actb (A to D) or as fold changes compared to mock-infected controls (E). Error bars represent standard errors of the means (n = 3). Data are representative of two or more independent experiments.

PSG16 (bCEA) mRNA is expressed in the murine CNS and primary neurons.

Using the tissue and cellular RNAs described for Fig. ​Fig.2,2, qRT-PCR was performed to detect expression of psg16 (bCEA), a gene more distantly related to ceacam1a and described solely for the murine CNS (5). The results are expressed as numbers of cDNA copies of psg16 per million cDNA copies of actb. Consistent with the previously published data (5), psg16 expression was readily detected in the brains and spinal cords of both WT and KO mice, while expression was negligible in the liver (Fig. ​(Fig.77 A). Interestingly, when RNAs from primary cells were examined, psg16 expression was detected exclusively in neurons (Fig. ​(Fig.7B).7B). As expected given the data in Fig. ​Fig.7B,7B, DBT, 17Cl1, and L2 cells were all negative for psg16 expression (Fig. ​(Fig.7C).7C). As with ceacam1a and ceacam2, psg16 expression remained constant in the murine CNS in mice from 2 to 14 weeks of age (Fig. ​(Fig.7D)7D) and was not significantly affected by MHV infection (Fig. ​(Fig.7E).7E). The neuronal localization of psg16 expression suggested that PSG16 might substitute for CEACAM1a to facilitate MHV entry or spread in neurons. Therefore, the functionality of this receptor was investigated.

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FIG. 7.

psg16 (bCEA) mRNA expression in murine tissues, primary cells, and cell lines. RNAs isolated from 4-week-old C57BL/6 mouse tissues (A), primary cell cultures (B), cell lines (C), 2- to 14-week-old C57BL/6 mouse tissues (D), and C57BL/6 mouse brains from mice inoculated i.c. with 50 PFU rA59 or rJHM.SD (E) were analyzed by qRT-PCR for expression of psg16 mRNA. Results are expressed as numbers of cDNA copies of psg16 per million cDNA copies of actb (A to D) or as fold changes compared to mock-infected controls (E). Error bars represent standard errors of the means (n = 3). Data are representative of two or more independent experiments.

We thank Jayasri Das Sarma, Marc Dichter, Margaret Maronski, Judith Grinspan, and Mary Reid for assistance with primary cell cultures, Nicole Beauchemin and Kathryn Holmes for ceacam1a^−/− mice and reagents, and Jessica Roth-Cross and Kristine Rose for helpful advice and discussions.

This work was supported by NIH grant AI-60021 to S.R.W. S.J.B. and E.P.S. were partially supported by NIH training grant NS-07180. J.M.P. was partially supported by NIH training grant AI-07634.