Targeted disruption of the Ceacam1 (MHVR) gene leads to reduced susceptibility of mice to mouse hepatitis virus infection

Abstract

The CEACAM1 glycoproteins (formerly called biliary glycoproteins; BGP, C-CAM, CD66a, or MHVR) are members of the carcinoembryonic antigen family of cell adhesion molecules. In the mouse, splice variants of CEACAM1 have either two or four immunoglobulin (Ig) domains linked through a transmembrane domain to either a short or a long cytoplasmic tail. CEACAM1 has cell adhesion activity and acts as a signaling molecule, and long-tail isoforms inhibit the growth of colon and prostate tumor cells in rodents. CEACAM1 isoforms serve as receptors for several viral and bacterial pathogens, including the murine coronavirus mouse hepatitis virus (MHV) and Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis in humans. To elucidate the mechanisms responsible for the many biological activities of CEACAM1, we modified the expression of the mouse Ceacam1 gene in vivo. Manipulation of the Ceacam1 gene in mouse embryonic stem cells that contained the Ceacam1a allele yielded a partial knockout. We obtained one line of mice in which the insert in the Ceacam1a gene had sustained a recombination event. This resulted in the markedly reduced expression of the two CEACAM1a isoforms with four Ig domains, whereas the expression of the two isoforms with two Ig domains was doubled relative to that in wild-type BALB/c (+/+) mice. Homozygous (p/p) Ceacam1a-targeted mice (Ceacam1aDelta4D) had no gross tissue abnormalities and were viable and fertile; however, they were more resistant to MHV A59 infection and death than normal (+/+) mice. Following intranasal inoculation with MHV A59, p/p mice developed markedly fewer and smaller lesions in the liver than +/+ or heterozygous (+/p) mice. The titers of virus produced in the livers were 50- to 100-fold lower in p/p mice than in +/p or +/+ mice. p/p mice survived a dose 100-fold higher than the lethal dose of virus for +/+ mice. +/p mice were intermediate between +/+ and p/p mice in susceptibility to liver damage, virus growth in liver, and susceptibility to killing by MHV. Ceacam1a-targeted mice provide a new model to study the effects of modulation of receptor expression on susceptibility to MHV infection in vivo.

FIG. 1

Targeting of the Ceacam1 gene. (A) The mouse Ceacam1 gene encodes nine exons. The ATG initiation codon is located in the first exon, whereas two stop codons can be alternatively used, one in exon 8 [TGA(S), used in the translation of isoforms encoding a short cytoplasmic domain], or another in exon 9 [TGA(L), used in the translation of isoforms encoding a long cytoplasmic domain]. The broken lines over and under the gene structure represent the alternative splicing events that produce CEACAM1 isoforms with either two or four Ig domains and either a short or a long cytoplasmic domain. (B) The Ceacam1 gene produces four major alternatively splice variants. The CEACAM1/D1-4 variants contain D1 to D4 Ig domains (identified in the boxes) that are linked through a transmembrane domain (TM) to either a short (S) or a long (L) tail. This alternative splicing event is due to the inclusion of 53-bp exon 7 (hatched box with exon number over the box), resulting in a shift of the ORF and the translation of a 73-aa tail. (C) Targeting construct. The targeting construct that led to a recombination event in ES cells had the TK-neo^r selection cassette (stippled) inserted into a unique SalI site in the second intron. The construct contained two engineered TGA stop codons at the beginning of exon 2 that were inserted by overlapping mutagenesis with the oligonucleotides indicated beneath the construct. These TGA codons were eliminated in the recombination event. A herpes simplex virus thymidine kinase-negative selection cassette was included in the unique HindIII site within intron 5. Three probes, identified by the black boxes, were used in Southern analyses. The recombinant allele producing a novel EcoRI-cleaved fragment of 4.2 kb, due to the insertion of TK-neo^r, was detected with the NcoI promoter probe. prom., promoter; N-term., N terminal. (D) Southern analyses of genomic DNAs from different inbred strains of mice. Mice express three Ceacam-related genes: Ceacam1 (1), Ceacam2 (2), and Ceacam10 (10). The numbers next to the various restriction fragments in the gels indicate the position of each gene in the particular digest of genomic DNA. Samples of 5 μg of genomic DNA extracted from each mouse strain were digested with either EcoRI, BamHI or HindIII restriction enzyme and run on 0.75% agarose gels. The DNA fragments were transferred to GeneScreen Plus and probed with a ³²P-labeled 395-bp NcoI fragment located in the proximal promoter region. Note that the 129Sv/J Ceacam2-specific EcoRI restriction fragment is shorter than that of other mouse strains. The 1.7-kb Ceacam1-specific HindIII fragment present in BALB/c and C57BL/6 mice is not found in strain 129Sv/J. Instead, this fragment migrates as an 8.0-kb fragment due to the mutation of the HindIII site present in intron 1 of other inbred strains. (E) Genotyping of ES cells and mice. Recombination events in ES cells and genotypes of mice obtained from heterozygous matings were detected by cleaving genomic DNA with EcoRI. The samples were run on 0.75% agarose gels and hybridized with ³²P-labeled probes described in Materials and Methods and shown in panel C. (Panel a) The NcoI probe is located in the upstream promoter region and hybridizes to all three mouse Ceacam-related genes (1, 2, and 10). The targeted allele in the F3 ES cells and mice is shortened from the original 12 kb to 4.2 kb. (Panel b) The neo probe detects one band of approximately 8.0 kb. Only one integration site was detected in the F3 ES cell line. The genomic DNA from +/+ mice did not hybridize with this probe, whereas the +/p and p/p mice were positive. (Panel c) The Ceacam1-specific probe, located in the N-terminal domain of the gene (shown in panel C), detected a 12-kb EcoRI fragment in genomic DNA prepared from all mice except p/p mice. In addition, this probe hybridized to the targeted 4.2-kb fragment in the F3 ES cell line and in +/p and p/p mice. (Panel d) The Ceacam2-specific probe, also from the N-terminal domain of its respective gene, hybridized to either a 9.6-kb fragment or a 7.7-kb fragment, depending on the contribution from the BALB/c or 129Sv/J mice, respectively.

FIG. 1

Targeting of the Ceacam1 gene. (A) The mouse Ceacam1 gene encodes nine exons. The ATG initiation codon is located in the first exon, whereas two stop codons can be alternatively used, one in exon 8 [TGA(S), used in the translation of isoforms encoding a short cytoplasmic domain], or another in exon 9 [TGA(L), used in the translation of isoforms encoding a long cytoplasmic domain]. The broken lines over and under the gene structure represent the alternative splicing events that produce CEACAM1 isoforms with either two or four Ig domains and either a short or a long cytoplasmic domain. (B) The Ceacam1 gene produces four major alternatively splice variants. The CEACAM1/D1-4 variants contain D1 to D4 Ig domains (identified in the boxes) that are linked through a transmembrane domain (TM) to either a short (S) or a long (L) tail. This alternative splicing event is due to the inclusion of 53-bp exon 7 (hatched box with exon number over the box), resulting in a shift of the ORF and the translation of a 73-aa tail. (C) Targeting construct. The targeting construct that led to a recombination event in ES cells had the TK-neo^r selection cassette (stippled) inserted into a unique SalI site in the second intron. The construct contained two engineered TGA stop codons at the beginning of exon 2 that were inserted by overlapping mutagenesis with the oligonucleotides indicated beneath the construct. These TGA codons were eliminated in the recombination event. A herpes simplex virus thymidine kinase-negative selection cassette was included in the unique HindIII site within intron 5. Three probes, identified by the black boxes, were used in Southern analyses. The recombinant allele producing a novel EcoRI-cleaved fragment of 4.2 kb, due to the insertion of TK-neo^r, was detected with the NcoI promoter probe. prom., promoter; N-term., N terminal. (D) Southern analyses of genomic DNAs from different inbred strains of mice. Mice express three Ceacam-related genes: Ceacam1 (1), Ceacam2 (2), and Ceacam10 (10). The numbers next to the various restriction fragments in the gels indicate the position of each gene in the particular digest of genomic DNA. Samples of 5 μg of genomic DNA extracted from each mouse strain were digested with either EcoRI, BamHI or HindIII restriction enzyme and run on 0.75% agarose gels. The DNA fragments were transferred to GeneScreen Plus and probed with a ³²P-labeled 395-bp NcoI fragment located in the proximal promoter region. Note that the 129Sv/J Ceacam2-specific EcoRI restriction fragment is shorter than that of other mouse strains. The 1.7-kb Ceacam1-specific HindIII fragment present in BALB/c and C57BL/6 mice is not found in strain 129Sv/J. Instead, this fragment migrates as an 8.0-kb fragment due to the mutation of the HindIII site present in intron 1 of other inbred strains. (E) Genotyping of ES cells and mice. Recombination events in ES cells and genotypes of mice obtained from heterozygous matings were detected by cleaving genomic DNA with EcoRI. The samples were run on 0.75% agarose gels and hybridized with ³²P-labeled probes described in Materials and Methods and shown in panel C. (Panel a) The NcoI probe is located in the upstream promoter region and hybridizes to all three mouse Ceacam-related genes (1, 2, and 10). The targeted allele in the F3 ES cells and mice is shortened from the original 12 kb to 4.2 kb. (Panel b) The neo probe detects one band of approximately 8.0 kb. Only one integration site was detected in the F3 ES cell line. The genomic DNA from +/+ mice did not hybridize with this probe, whereas the +/p and p/p mice were positive. (Panel c) The Ceacam1-specific probe, located in the N-terminal domain of the gene (shown in panel C), detected a 12-kb EcoRI fragment in genomic DNA prepared from all mice except p/p mice. In addition, this probe hybridized to the targeted 4.2-kb fragment in the F3 ES cell line and in +/p and p/p mice. (Panel d) The Ceacam2-specific probe, also from the N-terminal domain of its respective gene, hybridized to either a 9.6-kb fragment or a 7.7-kb fragment, depending on the contribution from the BALB/c or 129Sv/J mice, respectively.

FIG. 2

Expression of CEACAM1 in various tissues. (A) Expression of CEACAM1 proteins in various mouse tissues. The colon, liver, and kidneys were excised from +/+, +/p, and p/p mice and snap-frozen. The frozen tissues were powdered and extracted with lysis buffer. Samples of 200 μg were analyzed by SDS–8% PAGE and transferred to Immobilon membranes. The CEACAM1 isoforms were detected by immunoblotting with rabbit anti-mouse 231 or 2456 polyclonal antibody and ¹²⁵I-labeled protein A. (B) Expression of CEACAM1-L isoforms. Total lysate protein samples of 1 mg prepared from the livers of +/+, +/p, and p/p mice were immunoprecipitated with 5 μg of antibody 836 IgG, raised to CEACAM1-L. Immune complexes were collected and separated by SDS-PAGE, and the CEACAM1-L protein was detected with the same antibody. (C) Quantitation of CEACAM1 isoforms. The proteins on blots from several representative experiments were quantified using a Fuji BioImaging system, and the results were computed relative to the expression in +/+ mice. Error bars indicate standard deviations. D, domains.

FIG. 3

CEACAM1 immunostaining of tissue sections from wild-type and p/p mice. Tissues were immunostained with CEACAM1-specific polyclonal antibody 231 and lightly counterstained with hematoxylin. (a, c, and e) Sections from +/+ mice. (b, d, and f) Sections from p/p mice. (a and b) Colon. Cross-sections of colonic crypts in p/p mice (b) have weaker CEACAM1 immunostaining of the luminal membrane (arrowheads) than those in +/+ mice (a). (c and d) Liver. Hepatocyte bile canaliculi contacts (arrowheads) are CEACAM1 positive in +/+ mice (c), whereas they express CEACAM1 only weakly or not at all in p/p mice (d). (e and f) Kidneys. Collecting tubules of the kidneys were strongly positive for CEACAM1 in +/+ mice (arrowheads) (e), whereas those of p/p mice were faint and generally negative (f). Magnification, approximately ×37.

FIG. 4

Representative liver lesions of MHV A59-infected mice at 3 days after inoculation. +/+, +/p, and p/p mice were inoculated intranasally with 10⁶ PFU of hepatotropic MHV A59 and sacrificed at 3 or 5 days postinoculation (dpi). (A and B) Very large areas of necrosis in the livers of +/+ mice. (C and D) Intermediate-sized lesions in the livers of +/p mice. (F and G) Scattered small aggregates of inflammatory cells (arrows) with no areas of hepatocyte necrosis in p/p mice. (E and H) +/p mice (E) and p/p mice (H) were inoculated intranasally with 10⁶ PFU of MHV A59 and sacrificed at 5 dpi. At 5 dpi, areas of hepatocyte necrosis in +/p mice (F) had increased in size relative to the lesions at day 3 (C), while the lesions in p/p livers remained very small. Magnifications: A, C, E, F, and H, ×91; B, D, and G, approximately ×164.

FIG. 5

Quantitation of lesions per section of liver from MHV A59-inoculated mice. +/+, +/p, and p/p mice were inoculated intranasally with 10⁴, 10⁶, or 10⁸ PFU of virus per mouse, and livers were harvested at 3, 5, or 7 days postinoculation. Several experiments are illustrated. The number of lesions per section of mouse liver is shown on the y axis. Each bar indicates the number of lesions per liver section harvested from one mouse at the time indicated on the x axis. The following were not done: +/+ mice inoculated with 10⁴ PFU at day 7 and 10⁸ PFU; and +/p mice inoculated with 10⁴ and 10⁸ PFU at day 7. +/+ animals inoculated with 10⁶ PFU died or were euthanatized by day 3 or 4, whereas +/p and p/p mice survived. p/p mice were highly resistant to disease, even at doses of 10⁸ PFU/mouse. Plus signs over the bars indicate livers that had more than 50 lesions. Asterisks on the x axis indicate no liver lesions.