Murine coronavirus receptors are differentially expressed in the central nervous system and play virus strain-dependent roles in neuronal spread

Abstract

Coronavirus infection of the murine central nervous system (CNS) provides a model for studies of viral encephalitis and demyelinating disease. Mouse hepatitis virus (MHV) neurotropism varies by strain: MHV-A59 causes mild encephalomyelitis and demyelination, while the highly neurovirulent strain JHM.SD (MHV-4) causes fatal encephalitis with extensive neuronal spread of virus. In addition, while neurons are the predominant CNS cell type infected in vivo, the canonical receptor for MHV, the carcinoembryonic antigen family member CEACAM1a, has been demonstrated only on endothelial cells and microglia. In order to investigate whether CEACAM1a is also expressed in other cell types, ceacam1a mRNA expression was quantified in murine tissues and primary cells. As expected, among CNS cell types, microglia expressed the highest levels of ceacam1a, but lower levels were also detected in oligodendrocytes, astrocytes, and neurons. Given the low levels of neuronal expression of ceacam1a, primary neurons from wild-type and ceacam1a knockout mice were inoculated with MHV to determine the extent to which CEACAM1a-independent infection might contribute to CNS infection. While both A59 and JHM.SD infected small numbers of ceacam1a knockout neurons, only JHM.SD spread efficiently to adjacent cells in the absence of CEACAM1a. Quantification of mRNA for the ceacam1a-related genes ceacam2 and psg16 (bCEA), which encode proposed alternative MHV receptors, revealed low ceacam2 expression in microglia and oligodendrocytes and psg16 expression exclusively in neurons; however, only CEACAM2 mediated infection in human 293T cells. Therefore, neither CEACAM2 nor PSG16 is likely to be an MHV receptor on neurons, and the mechanism for CEACAM1a-independent neuronal spread of JHM.SD remains unknown.

FIG. 1.

MHV infection of CNS cell types. C57BL/6 mice inoculated i.c. with 50 PFU of rA59 or rJHM.SD were sacrificed at days 3 (A) and 5 (B) postinfection. Formalin-fixed, paraffin-embedded tissues were sectioned and dual immunolabeled for GFAP, Iba1, or MAP2 (red) and MHV nucleocapsid protein (green). Arrowheads indicate double-positive cells. Magnification, ×400. Data are representative of two independent experiments.

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.

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.

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.

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.

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.

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.

FIG. 8.

MHV infection of 293T cells transfected with receptor. Human 293T cells were transfected with expression plasmids encoding CEACAM1a, CEACAM2, or PSG16 or with empty vector by use of FuGENE 6 transfection reagent. (A) Cells were infected at 36 h posttransfection with approximately 1 PFU/cell of rA59 or rJHM.SD or mock infected, fixed at 8 h postinfection, and immunolabeled for MHV nucleocapsid protein. (B) Quantification of infected cells. Error bars represent standard errors of the means (n = 3). (C) GFP transfection control showing transfection efficiency. Magnification, ×200. Data are representative of two or more independent experiments.

FIG. 9.

MHV infection and surface expression of TVA-CEA chimeras. Human 293T cells were transfected with expression plasmids encoding CEACAM1a, CEACAM2, and PSG16 extracellular domains with the signal sequence and membrane anchor domains of the avian retrovirus receptor TVA. Plasmids encoding wild-type CEACAM1a and empty vector were transfected as controls. TM, TVA transmembrane anchor; GPI, TVA glycosylphosphatidylinositol anchor. (A) Cells were infected at 48 h posttransfection with approximately 5 PFU/cell of rA59-EGFP or rA59/S_JHM.SD-EGFP and were fixed at 16 h postinfection for EGFP detection. (B) TVA-CEA-transfected cells were immunolabeled with mouse M2 anti-FLAG antibody and Alexa 488-conjugated goat anti-mouse IgG and were analyzed by flow cytometry. “Mock” represents untransfected 293T cells immunolabeled in parallel. (C) Summary of percent positive cells and median fluorescence intensity for TVA-CEA-transfected cells. Positive cells were defined as those with a fluorescence intensity of >98% that of the mock-transfected cells. Median fluorescence intensity was calculated for the positive population.