Genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues.

The molecular mechanism of genetic resistance of inbred mouse strains to mouse hepatitis virus, a murine coronavirus, was studied by comparing virus binding to plasma membranes of intestinal epithelium or liver from susceptible BALB/c and resistant SJL/J mice with a new solid-phase assay for virus-binding activity. Virus bound to isolated membranes from susceptible mice, but not to membranes from resistant mice. F1 progeny of SJL/J X BALB/c mice had an intermediate level of virus-binding activity on their enterocyte and hepatocyte membranes. This correlated well with previous studies showing that susceptibility to mouse hepatitis virus strain A59 is controlled by a single autosomal dominant gene (M. S. Smith, R. E. Click, and P. G. W. Plagemann, J. Immunol. 133:428-432). Because virus binding was not prevented by treating membranes with sodium dodecyl sulfate, the virus-binding molecule could be identified by a virus overlay protein blot assay. Virus bound to a single broad band of Mr 100,000 to 110,000 in membranes from hepatocytes or enterocytes of susceptible BALB/c and semisusceptible C3H mice, but no virus-binding band was detected in comparable preparations of resistant SJL/J mouse membranes. Therefore, SJL/J mice may be resistant to mouse hepatitis virus A59 infection because they lack a specific virus receptor which is present on the plasma membranes of target cells from genetically susceptible BALB/c and semisusceptible C3H mice.     

Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein and not homologous CEA-related glycoproteins of other species. These results suggest that MHV-A59 binds to a mouse-specific epitope of MHVR, and they support the hypothesis that the species specificity of MHV-A59 infection may be due to the specificity of the virus-receptor interaction.     

Bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis viruses.

Murine coronaviruses such as mouse hepatitis virus (MHV) infect mouse cells via cellular receptors that are isoforms of biliary glycoprotein (Bgp) of the carcinoembryonic antigen gene family (G. S. Dveksler, C. W. Dieffenbach, C. B. Cardellichio, K. McCuaig, M. N. Pensiero, G.-S. Jiang, N. Beauchemin, and K. V. Holmes, J. Virol. 67:1-8, 1993). The Bgp isoforms are generated through alternative splicing of the mouse Bgp1 gene that has two allelic forms called MHVR (or mmCGM1), expressed in MHV-susceptible mouse strains, and mmCGM2, expressed in SJL/J mice, which are resistant to MHV. We here report the cloning and characterization of a new Bgp-related gene designated Bgp2. The Bgp2 cDNA allowed the prediction of a 271-amino-acid glycoprotein with two immunoglobulin domains, a transmembrane, and a putative cytoplasmic tail. There is considerable divergence in the amino acid sequences of the N-terminal domains of the proteins coded by the Bgp1 gene from that of the Bgp2-encoded protein. RNase protection assays and RNA PCR showed that Bgp2 was expressed in BALB/c kidney, colon, and brain tissue, in SJL/J colon and liver tissue, in BALB/c and CD1 spleen tissue, in C3H macrophages, and in mouse rectal carcinoma CMT-93 cells. When Bgp2-transfected hamster cells were challenged with MHV-A59, MHV-JHM, or MHV-3, the Bgp2-encoded protein served as a functional MHV receptor, although with a lower efficiency than that of the MHVR glycoprotein. The Bgp2-mediated virus infection could not be inhibited by monoclonal antibody CC1 that is specific for the N-terminal domain of MHVR. Although CMT-93 cells express both MHVR and Bgp2, infection with the three strains of MHV was blocked by pretreatment with monoclonal antibody CC1, suggesting that MHVR was the only functional receptor in these cells. Thus, a novel murine Bgp gene has been identified that can be coexpressed in inbred mice with the Bgp1 glycoproteins and that can serve as a receptor for MHV strains when expressed in transfected hamster cells.     

Purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis virus (MHV)-A59 from mouse liver and identification of a nonfunctional, homologous protein in MHV-resistant SJL/J mice.

The receptor for mouse hepatitis virus strain A59 (MHV-A59) is a 110- to 120-kilodalton (kDa) glycoprotein which is expressed in MHV-susceptible mouse strains on the membranes of hepatocytes, intestinal epithelial cells, and macrophages. SJL/J mice, which are highly resistant to MHV-A59, were previously shown to lack detectable levels of receptor by using either solid-phase virus receptor assays or binding of a monoclonal anti-receptor antibody (MAb) which blocks infection of MHV-susceptible mouse cells. This MAb was used for affinity purification of the receptor glycoprotein from livers of MHV-susceptible Swiss Webster mice. The MHV receptor and an antigenically related protein of 48 to 58 kDa were copurified and then separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 15 amino acids of the receptor were sequenced, and a synthetic peptide of this amino acid sequence was prepared. Rabbit antiserum made against this peptide bound to the MHV receptor glycoprotein and the 48- to 58-kDa protein from livers of MHV-susceptible BALB/c mice and Swiss Webster mice and from the intestinal brush border of BALB/c mice. In immunoblots of intestinal brush border and hepatocyte membranes of MHV-resistant SJL/J mice, the antibody against the amino terminus of the receptor identified proteins that are 5 to 10 kDa smaller than the MHV receptor and the 48- to 58-kDa related protein from Swiss Webster or BALB/c mice. Thus, SJL/J mice express a protein which shares some sequence homology with the MHV receptor but which lacks virus-binding activity and is not recognized by the blocking anti-receptor MAb. These results suggest that resistance of SJL/J mice to MHV-A59 may be due to absence or mutation of the virus-binding domain in the nonfunctional receptor homolog in SJL/J mice.     

Role of Mouse Hepatitis Virus (MHV) Receptor Murine CEACAM1 in the Resistance of Mice to MHV Infection: Studies of Mice with Chimeric mCEACAM1a and mCEACAM1b

Although most inbred mouse strains are highly susceptible to mouse hepatitis virus (MHV) infection, the inbred SJL line of mice is highly resistant to its infection. The principal receptor for MHV is murine CEACAM1 (mCEACAM1). Susceptible strains of mice are homozygous for the 1a allele of mCeacam1, while SJL mice are homozygous for the 1b allele. mCEACAM1a (1a) has a 10- to 100-fold-higher receptor activity than does mCEACAM1b (1b). To explore the hypothesis that MHV susceptibility is due to the different MHV receptor activities of 1a and 1b, we established a chimeric C57BL/6 mouse (cB61ba) in which a part of the N-terminal immunoglobulin (Ig)-like domain of the mCeacam1a (1a) gene, which is responsible for MHV receptor function, is replaced by the corresponding region of mCeacam1b (1b). We compared the MHV susceptibility of these chimeric mice to that of SJL and B6 mice. B6 mice that are homozygous for 1a are highly susceptible to MHV-A59 infection, with a 50% lethal dose (LD₅₀) of 10^2.5 PFU, while chimeric cB61ba mice and SJL mice homozygous for 1ba and 1b, respectively, survived following inoculation with 10⁵ PFU. Unexpectedly, cB61ba mice were more resistant to MHV-A59 infection than SJL mice as measured by virus replication in target organs, including liver and brain. No infectious virus or viral RNA was detected in the organs of cB61ba mice, while viral RNA and infectious virus were detected in target organs of SJL mice. Furthermore, SJL mice produced antiviral antibodies after MHV-A59 inoculation with 10⁵ PFU, but cB61ba mice did not. Thus, cB61ba mice are apparently completely resistant to MHV-A59 infection, while SJL mice permit low levels of MHV-A59 virus replication during self-limited, asymptomatic infection. When expressed on cultured BHK cells, the mCEACAM1b and mCEACAM1ba proteins had similar levels of MHV-A59 receptor activity. These results strongly support the hypothesis that although alleles of mCEACAM1 are the principal determinants of mouse susceptibility to MHV-A59, other as-yet-unidentified murine genes may also play a role in susceptibility to MHV.Differences in susceptibility to a number of viral infections have been documented among inbred mouse strains (20). These differences have been studied as models for the various degrees of susceptibility of individual humans to some viral infections. Numerous host factors have been found to be involved in such differences (2, 15). For example, allelic variations in the virus receptor and coreceptor for HIV-1 are important host factors influencing susceptibility to HIV-1 infection (36).A virus receptor is a molecule with which the virus interacts at an initial step of infection. Therefore, receptors are crucial host determinants of virus susceptibility (15, 16). A variety of receptor proteins has been identified for many different viruses, including the murine coronavirus mouse hepatitis virus (MHV) (12, 50). The principal receptor for MHV is murine carcinoembryonic antigen-related cell adhesion molecule 1 (mCEACAM1; previously called Bgp or MHVR [3]), which is in the immunoglobulin (Ig) superfamily (12, 50). Four isoforms of mCEACAM1a (1a) are expressed on the plasma membranes of a variety of murine cells and tissues (14). The two mCEACAM1 isoforms with a molecular mass of 100 to 120 kDa are composed of four Ig-like ectodomains, a transmembrane (TM) domain, and either a long or a short cytoplasmic tail (Cy) (3, 22). Two other isoforms consist of two Ig-like domains, with either long or short Cy (3, 22). The N-terminal (N) domain is responsible for virus binding (10, 24), the induction of conformational changes in the viral spike protein (S), and membrane fusion during virus entry and syncytium formation (13, 24). The replacement of the N-terminal domain of mCEACAM1a with that of the murine homolog of the poliovirus receptor (PVR) yields a functional receptor for MHV (10), and Ceacam1a-knockout mice are completely resistant to infection with the hepatotropic A59 strain of MHV (17, 25).Wild mice have two alleles of the mCeacam1 gene, called mCeacam1a and mCeacam1b. Inbred mouse strains that are homozygous for mCeacam1a, including BALB/c, C57BL/6 (B6), C3H, and A/J mice, etc., are highly susceptible to infection with strains of MHV. In contrast, the SJL line of inbred mice, which is resistant to death from MHV infection, is homozygous for the mCeacam1b allele (5, 11, 50). The most extensive differences in amino acid sequence between mCEACAM1a and mCEACAM1b are found in the N-terminal domain, where the virus-binding region is located (21, 22, 32). It was initially reported by Boyle et al. that mCEACAM1a proteins had MHV-A59 virus-binding activity in a virus overlay protein blot, while mCEACAM1b did not (5). Those authors speculated that the different viral affinities of these mCEACAM1 proteins may account for the various MHV-A59 susceptibilities of BALB/c mice compared to those of SJL mice (49). However, Yokomori and Lai (53) and Dveksler et al. (11) previously showed that when recombinant CEACAM1a and CEACAM1b proteins are expressed at high levels on cultured cells, both proteins have MHV-A59 receptor activity. Yokomori and Lai suggested that the difference in MHV susceptibility between BALB/c and SJL mice does not depend solely upon the interaction of the virus with mCEACAM1 proteins (52, 53). Dveksler et al. suggested that small differences in MHV-A59 receptor activity between mCEACAM1a and mCEACAM1b could result in very large biological differences during multiple cycles of infection in in vivo infection (11). We then quantitatively showed that recombinant mCEACAM1a expressed in BHK cells has 10- to 30-times-higher MHV-binding activity than mCEACAM1b (31). Similar results were observed in other laboratories (7, 32). Because the mCeacam1 gene is located on chromosome 7 (34) and the gene controlling MHV-A59 susceptibility and the resistance of BALB/c mice versus SJL mice is also located on chromosome 7 close to the mCeacam1 gene (40), we speculated that the mCeacam1 gene is identical to the gene that determines the susceptibility and/or resistance of mice to MHV-A59 and MHV-JHM infection.To examine the above-described hypothesis, we used progeny mice produced by crossing BALB/c and SJL mice. F₂ mice and F₁ mice backcrossed to SJL mice were examined for the mCeacam1 genotype and for MHV-JHM susceptibility (30). Mice homozygous for mCeacam1a (1a/1a) and heterozygous mice (1a/1b) were susceptible to lethal MHV-JHM infection, while mice homozygous for mCeacam1b (1b/1b) were not killed by inoculation with MHV-JHM. These data are consistent with the hypothesis that the susceptibility of mice to MHV is determined by the mCeacam1a allele (30). However, this classical genetic analysis could not prove that mCeacam1 alone determines the susceptibility or resistance of mice to MHV-JHM infection, because this methodology cannot rule out the possibility that a different unknown host gene located close to mCeacam1 on chromosome 7 could also affect MHV-JHM susceptibility. Therefore, we used gene replacement in B6 embryonic stem (ES) cells to create a mouse strain in which the exon encoding the N-terminal part of the N-terminal Ig domain of mCeacam1a was replaced with the corresponding region of mCeacam1b from SLJ mice. We bred the chimeric mCeacam1 gene on the B6 background (called B6 chimeric mCeacam1ba, or cB61ba). We compared these mice, wild-type B6 mice, and SJL mice for their susceptibilities to MHV-A59 infection. We confirmed that the expression of mCEACAM1a makes mice susceptible to lethal infection with MHV-A59. However, surprisingly, we found that cB61ba mice were profoundly resistant to MHV-A59 infection, while the virus could replicate at low levels in SJL mice in a self-limited, unapparent infection. Our results suggest that one or more as-yet-unidentified murine genes may also contribute to murine susceptibility and/or resistance to MHV-A59 infection.     

Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV.

The cellular receptor for murine coronavirus mouse hepatitis virus (MHV)-A59 is a member of the carcinoembryonic antigen (CEA) family of glycoproteins in the immunoglobulin superfamily. We isolated a cDNA clone (MHVR1) encoding the MHV receptor. The sequence of this clone predicts a 424-amino-acid glycoprotein with four immunoglobulinlike domains, a transmembrane domain, and a short intracytoplasmic tail, MHVR1 is closely related to the murine CEA-related clone mmCGM1 (Mus musculus carcinoembryonic antigen gene family member). Western blot (immunoblot) analysis performed with antireceptor antibodies detected a glycoprotein of 120 kDa in BHK cells stably transfected with MHVR1. This corresponds to the size of the MHV receptor expressed in mouse intestine and liver. Human and hamster fibroblasts transfected with MHVR1 became susceptible to infection with MHV-A59. Like MHV-susceptible mouse fibroblasts, the MHVR1-transfected human and hamster cells were protected from MHV infection by pretreatment with monoclonal antireceptor antibody CC1. Thus, the 110- to 120-kDa CEA-related glycoprotein encoded by MHVR1 is a functional receptor for murine coronavirus MHV-A59.     

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

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.     

The receptor for mouse hepatitis virus in the resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection.

The SJL mouse strain is resistant to infection by some strains of the murine coronavirus mouse hepatitis virus (MHV), such as JHM and A59. The block to virus infection has been variously attributed to defects in virus receptors or virus spread. Since the cellular receptors for MHV, mmCGM1 and mmCGM2, have recently been identified as members of the carcinoembryonic antigen family, we reexamined the possible defectiveness of the MHV receptors in SJL mouse strain. Cloning and sequencing of the cDNAs of both mmCGMs RNAs from SJL mice revealed that they were identical in size to those of the susceptible C57BL/6 (B6) mouse. There was some sequence divergence in the N terminus of the mmCGM molecules between the two mouse strains, resulting in a different number of potential glycosylation sites. This was confirmed by in vitro translation of the mmCGM RNAs, which showed that the glycosylated mmCGM2 of SJL was smaller than that of B6 mice. However, transfection of either mmCGM1 or mmCGM2 from SJL mice into MHV-resistant Cos 7 cells rendered the cells susceptible to MHV infection. The ability of the SJL mmCGM molecules to serve as MHV receptors was comparable to that of those from B6. These molecules are expressed in SJL mouse brain and liver in a similar ratio and in amounts equivalent to those in the B6 mouse. Furthermore, we demonstrated that an SJL-derived cell line was susceptible to A59 but resistant to JHM infection. We concluded that the MHV receptor molecules in the SJL mouse are functional and that the resistance of SJL mice to infection by some MHV strains most likely results from some other factor(s) required for virus entry or some other step(s) in virus replication.     

Duration of mouse hepatitis virus infection: studies in immunocompetent and chemically immunosuppressed mice

The duration of mouse hepatitis virus (MHV) infection was examined in mice inoculated intranasally with selected strains of MHV. Following inoculation with virulent MHV-JHM, genetically susceptible BALB/c mice and resistant CD1 mice had detectable virus in the brain at 1 month, but not later intervals up to 12 months. BALB/c mice infected with avirulent MHV-S or MHV-1 had no detectable virus in brains at 1 month or thereafter. Immunosuppression of BALB/c mice with treatment regimens of hydrocortisone acetate or cyclophosphamide at 1 and 2 months after infection with MHV-JHM did not activate detectable virus in liver or increase the prevalence or degree of brain infection. Immunosuppression with these drugs during the acute phase of MHV-JHM infection influenced MHV infection, based on virus quantification in livers, but timing of drug treatment relative to MHV infection was critical. Mice infected with MHV developed IgG serum antibody titers that persisted without decline for up to 1 year after infection. Antibody titers varied with mouse genotype and infecting virus. These studies, using intranasal inoculation, support the conclusions of others, using other routes of inoculation, that MHV infection is not persistent in adult, immunocompetent mice.     

Variations in genetic control of susceptibility to Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease. I. Differences between susceptible SJL/J and resistant BALB/c strains map near the T cell beta-chain constant gene on chromosome 6

In some susceptible mouse strains, intracerebral (IC) inoculation of Theiler's murine encephalomyelitis virus (TMEV) results in a persistent infection leading to chronic demyelinating disease. Previous genetic analyses between susceptible SJL/J and resistant C57BL/6 mice indicated a role for multiple unlinked genes in the development of clinical and histopathological disease, including a major influence of the D region of the H-2 complex. In this study, genetic analysis of a different strain combination (susceptible SJL/J and resistant BALB/c) also demonstrates the involvement of multiple genes, but the H-2 genotype (H-2s and H-2d, respectively) does not appear to contribute significantly to susceptibility differences. In both segregation studies and recombinant-inbred (R-I) analysis, clinical and histopathological disease occurs in both H-2s homozygotes and H-2d homozygotes (as well as H-2s/H-2d heterozygotes), with the actual frequency related to the proportion of non-H-2 genome from the susceptible strain. There appear to be at least two non-H-2 genes involved in differential susceptibility of SJL/J and BALB/c to TMEV-induced disease. Analysis of R-I strains generated from BALB/c and SJL/J progenitors indicates linkage of at least one of these non-H-2 genes to those encoding the constant portion of the beta-chain of the T cell receptor on chromosome 6. Many genes may actually be involved, but each strain comparison defines a different subset of these loci--only those at which the two strains in question carry "functionally" different alleles. Thus, different strain comparisons may accent the roles of different genes in resistance to the same infectious organism or disease process. In addition to the genes identified thus far, there may be yet other genes contributing to development of TMEV-induced disease, but their recognition may require analysis of still other strain combinations.     

Ceacam1a-/- mice are completely resistant to infection by murine coronavirus mouse hepatitis virus A59

CEACAM1a glycoproteins are members of the immunoglobulin (Ig) superfamily and the carcinoembryonic antigen family. Isoforms expressing either two or four alternatively spliced Ig-like domains in mice have been found in a number of epithelial, endothelial, or hematopoietic tissues. CEACAM1a functions as an intercellular adhesion molecule, an angiogenic factor, and a tumor cell growth inhibitor. Moreover, the mouse and human CEACAM1a proteins are targets of viral or bacterial pathogens, respectively, including the murine coronavirus mouse hepatitis virus (MHV), Haemophilus influenzae, Neisseria gonorrhoeae, and Neisseria meningitidis, as well as Moraxella catarrhalis in humans. We have shown that targeted disruption of the Ceacam1a (MHVR) gene resulting in a partial ablation of the protein in mice (p/p mice) led to reduced susceptibility to MHV-A59 infection of the modified mice in the BALB/c background. We have now engineered and produced a Ceacam1a-/- mouse that exhibits complete ablation of the CEACAM1a protein in every tissue where it is normally expressed. We report that 3-week-old Ceacam1a-/- mice in the C57BL/6 genetic background are fully resistant to MHV-A59 infection by both intranasal and intracerebral routes. Whereas virus-inoculated wild-type +/+ C57BL/6 mice showed profound liver damage and spinal cord demyelination under these conditions, Ceacam1a-/- mice displayed normal livers and spinal cords. Virus was recovered from liver and spinal cord tissues of +/+ mice but not of -/- mice. These results indicate that CEACAM1a is the sole receptor for MHV-A59 in both liver and brain and that its deletion from the mouse renders the mouse completely resistant to infection by this virus.     

Identification of quantitative trait loci for susceptibility to mouse adenovirus type 1

Adult SJL/J mice are highly susceptible to mouse adenovirus type 1 (MAV-1) infections, whereas other inbred strains, including BALB/cJ, are resistant (K. R. Spindler, L. Fang, M. L. Moore, C. C. Brown, G. N. Hirsch, and A. K. Kajon, J. Virol. 75:12039-12046, 2001). Using congenic mouse strains, we showed that the H-2(s) haplotype of SJL/J mice is not associated with susceptibility to MAV-1. Susceptibility of MAV-1-infected (BALB/cJ x SJL/J)F(1) mice was intermediate between that of SJL/J mice and that of BALB/cJ mice, indicating that susceptibility is a genetically controlled quantitative trait. We mapped genetic loci involved in mouse susceptibility to MAV-1 by analysis of 192 backcross progeny in a genome scan with 65 simple sequence length polymorphic markers. A major quantitative trait locus (QTL) was detected on chromosome 15 (Chr 15) with a highly significant logarithm of odds score of 21. The locus on Chr 15 alone accounts for 40% of the total trait variance between susceptible and resistant strains. QTL modeling of the data indicated that there are a number of other QTLs with small effects that together with the major QTL on Chr 15 account for 54% of the trait variance. Identification of the major QTL is the first step in characterizing host genes involved in susceptibility to MAV-1.     

Interaction of mouse hepatitis virus (MHV) spike glycoprotein with receptor glycoprotein MHVR is required for infection with an MHV strain that expresses the hemagglutinin-esterase glycoprotein.

In addition to the spike (S) glycoprotein that binds to carcinoembryonic antigen-related receptors on the host cell membrane, some strains of mouse coronavirus (mouse hepatitis virus [MHV]) express a hemagglutinin esterase (HE) glycoprotein with hemagglutinating and acetylesterase activity. Virions of strains that do not express HE, such as MHV-A59, can infect mouse fibroblasts in vitro, showing that the HE glycoprotein is not required for infection of these cells. The present work was done to study whether interaction of the HE glycoprotein with carbohydrate moieties could lead to virus entry and infection in the absence of interaction of the S glycoprotein with its receptor glycoprotein, MHVR. The DVIM strain of MHV expresses large amounts of HE glycoprotein, as shown by hemadsorption, acetylesterase activity, and immunoreactivity with antibodies directed against the HE glycoprotein of bovine coronavirus. A monoclonal anti-MHVR antibody, MAb-CC1, blocks binding of virus S glycoprotein to MHVR and blocks infection of MHV strains that do not express HE. MAb-CC1 also prevented MHV-DVIM infection of mouse DBT cells and primary mouse glial cell cultures. Although MDCK-I cells express O-acetylated sialic acid residues on their plasma membranes, these canine cells were resistant to infection with MHV-A59 and MHV-DVIM. Transfection of MDCK-I cells with MHVR cDNA made them susceptible to infection with MHV-A59 and MHV-DVIM. Thus, the HE glycoprotein of an MHV strain did not lead to infection of cultured murine neural cells or of nonmurine cells that express the carbohydrate ligand of the HE glycoprotein. Therefore, interaction of the spike glycoprotein of MHV with its carcinoembryonic antigen-related receptor glycoprotein is required for infectivity of MHV strains whether or not they express the HE glycoprotein.     

A role for naturally occurring variation of the murine coronavirus spike protein in stabilizing association with the cellular receptor.

Murine hepatitis virus (MHV), a coronavirus, initiates infection by binding to its cellular receptor (MHVR) via spike (S) proteins projecting from the virion membrane. The structures of these S proteins vary considerably among MHV strains, and this variation is generally considered to be important in determining the strain-specific pathologies of MHV infection, perhaps by affecting the interaction between MHV and the MHVR. To address the relationships between S variation and receptor binding, assays capable of measuring interactions between MHV and MHVR were developed. The assays made use of a novel soluble form of the MHVR, sMHVR-Ig, which comprised the virus-binding immunoglobulin-like domain of MHVR fused to the Fc portion of human immunoglobulin G1. sMHVR-Ig was stably expressed as a disulfide-linked dimer in human 293 EBNA cells and was immobilized to Sepharose-protein G via the Fc domain. The resulting Sepharose beads were used to adsorb radiolabelled MHV particles. At 4 degrees C, the beads specifically adsorbed two prototype MHV strains, MHV JHM (strain 4) and a tissue culture-adapted mutant of MHV JHM, the JHMX strain. A shift to 37 degrees C resulted in elution of JHM but not JHMX. This in vitro observation of JHM (but not JHMX) elution from its receptor at 37 degrees C was paralleled by a corresponding 37 degrees C elution of receptor-associated JHM (but not JHMX) from tissue culture cells. The basis for this difference in maintenance of receptor association was correlated with a large deletion mutation present within the JHMX S protein, as sMHVR-Ig exhibited relatively thermostable binding to vaccinia virus-expressed S proteins containing the deletion. These results indicate that naturally occurring mutations in the coronavirus S protein affect the stability of the initial interaction with the host cell and thus contribute to the likelihood of successful infection by incoming virions. These changes in virus entry features may result in coronaviruses with novel pathogenic properties.     

Differences between BALB/c and C57BL/6 mice in mouse hepatitis virus replication in primary hepatocyte culture.

We previously showed that an intraperitoneal infection with mouse hepatitis virus (MHV) resulted in acute hepatic failure accompanying extremely elevated viral growth in the liver in interferon-gamma-deficient BALB/c (BALB-GKO), but not C57BL/6 (B6-GKO) mice. To examine the basis of the strain difference against MHV infection in interferon-gamma-deficient mice, viral replication in primary hepatocyte cultures from BALB/c and B6 mice with or without the IFN-gamma gene was compared in vitro. The MHV replication in BALB/c hepatocytes with or without the IFN-gamma gene was significantly higher than that in B6 hepatocytes with or without the IFN-gamma gene, suggesting that there is a strain difference in MHV replication in hepatocytes. Since a significant difference in MHV replication in hepatocytes was not observed between wild type and IFN-gamma-deficient mice of the same genetic background, the phenomenon is thought to be independent of IFN-gamma. However, pretreatment of hepatocytes with recombinant mouse interferon-gamma inhibited MHV replication in a dose-dependent fashion. The results are discussed with respect to the pathology of MHV infection in mice with or without the IFN-gamma gene.     

Nematospiroides dubius: genetic control of immunity to infections of mice

Inbred strains of mice differ in their susceptibility and resistance to challenge infections with Nematospiroides dubius. In our studies, F1 hybrid mice from resistant SJL and susceptible CBA parents were resistant to N. dubius challenge infections. Only 22% of backcrosses to SJL were susceptible while backcrosses to CBA had a wide range of susceptibility. Male mice were more susceptible than female mice. In another experiment, inbred strains of mice were compared in their ability to resist N. dubius challenge infection: SJL and A.SW (H-2s) mice became resistant after one immunizing infection, A, A/He (both H-2a), as well as BALB/c and DBA/2 (both H-2d) mice became resistant after two immunizing infections, while C57BL/6 (H-2b), C3H/He, CBA, and AKR (H-2k) mice remained susceptible. The resistance to reinfections was characterized by reduction of worm burdens between Days 6 and 14 postinfection. It was concluded that (1) resistance to N. dubius challenge infections is inherited in a dominant fashion and that multiple genes may influence such response, which in turn might be modulated by the Y chromosome; (2) both MHC and non-MHC genes may influence, in conjunction with the number of exposures to parasite antigens, the resistance to challenge infections.     

Mutational Analysis of the Virus and Monoclonal Antibody Binding Sites in MHVR,the Cellular Receptor of the Murine Coronavirus Mouse Hepatitis Virus Strain A59

The primary cellular receptor for mouse hepatitis virus (MHV), a murine coronavirus, is MHVR (also referred to as Bgp1^a or C-CAM), a transmembrane glycoprotein with four immunoglobulin-like domains in the murine biliary glycoprotein (Bgp) subfamily of the carcinoembryonic antigen (CEA) family. Other murine glycoproteins in the Bgp subfamily, including Bgp1^b and Bgp2, also can serve as MHV receptors when transfected into MHV-resistant cells. Previous studies have shown that the 108-amino-acid N-terminal domain of MHVR is essential for virus receptor activity and is the binding site for monoclonal antibody (MAb) CC1, an antireceptor MAb that blocks MHV infection in vivo and in vitro. To further elucidate the regions of MHVR required for virus receptor activity and MAb CC1 binding, we constructed chimeras between MHVR and other members of the CEA family and tested them for MHV strain A59 (MHV-A59) receptor activity and MAb CC1 binding activity. In addition, we used site-directed mutagenesis to introduce selected amino acid changes into the N-terminal domains of MHVR and these chimeras and tested the abilities of these mutant glycoproteins to bind MAb CC1 and to function as MHV receptors. Several recombinant glycoproteins exhibited virus receptor activity but did not bind MAb CC1, indicating that the virus and MAb binding sites on the N-terminal domain of MHVR are not identical. Analysis of the recombinant glycoproteins showed that a short region of MHVR, between amino acids 34 and 52, is critical for MHV-A59 receptor activity. Additional regions of the N-terminal variable domain and the constant domains, however, greatly affected receptor activity. Thus, the molecular context in which the amino acids critical for MHV-A59 receptor activity are found profoundly influences the virus receptor activity of the glycoprotein.Initial events in virus infection of a cell include attachment of the virus to the cell, entry, and disassembly of the virion. For most viruses, attachment is mediated through a specific interaction between the virus attachment protein and a cell surface receptor. Previous studies identified the murine biliary glycoprotein MHVR (also referred to as Bgp1^a or C-CAM) as the primary cellular receptor for murine coronavirus mouse hepatitis virus strain A59 (MHV-A59) (20, 53). This glycoprotein, isolated from liver and intestinal brush border membranes of MHV-sensitive BALB/c mice, binds to MHV-A59 virions in a solid-phase viral overlay protein blot assay (9) and is recognized by an antireceptor monoclonal antibody (MAb CC1) that protects cells expressing MHVR from infection by MHV-A59 in vivo and in vitro (20, 52, 53). A cDNA encoding an allelic variant of MHVR, Bgp1^b (also referred to as mmCGM2) (38), was isolated from cells of MHV-resistant SJL/J mice (18, 53), and a second murine biliary glycoprotein, Bgp2, which is expressed in the colons of both BALB/c and SJL/J mice, also has been characterized (38). MHVR and Bgp1^b consist of an N-terminal immunoglobulin (Ig)-like variable domain, three Ig-like constant domains, a transmembrane domain, and a cytoplasmic tail. The Bgp2 glycoprotein exhibits a similar structure except that it contains only one constant domain. The Bgp1^b and Bgp2 glycoproteins can serve as functional receptors for MHV-A59 when overexpressed in MHV-A59-resistant hamster cells in transient transfection assays, but these glycoproteins do not bind virus in solid-phase binding assays and are not recognized by MAb CC1 (18, 38). Natural splice variants of MHVR and Bgp1^b yield glycoproteins containing the N-terminal and fourth Ig-like domains, the transmembrane domain, and the cytoplasmic tail (18, 21, 53).A secreted three Ig domain murine glycoprotein called bCEA, a pregnancy-specific glycoprotein in the murine carcinoembryonic antigen (CEA) family, is expressed in C57BL/6 mouse brain and placenta and exhibits a low level of MHV-A59 receptor activity when expressed in COS-7 cells (11). To date, the only murine CEA-related glycoprotein shown to have no MHV receptor activity in transient transfection assays in MHV-A59-resistant hamster cells is Cea10 (formerly referred to as mmCGM3), a secreted glycoprotein consisting of two variable Ig-like domains that does not bind MHV-A59 or MAb CC1 (26, 32).Deletion mutagenesis studies showed that MHV-A59 and MAb CC1 bind to the N-terminal Ig-like variable domain of MHVR (21). A recombinant chimeric glycoprotein containing the N-terminal domain of MHVR and the second, third, transmembrane, and cytoplasmic domains of the mouse poliovirus receptor (Pvr) homolog serves as a functional receptor for MHV-A59 when expressed in hamster cells (17). Furthermore, a soluble recombinant glycoprotein consisting of only the N-terminal domain of MHVR can inhibit MHV-A59 infectivity in a concentration-dependent manner (19). MAb CC1 recognizes both the MHVR/mph chimera and the soluble N-terminal domain of MHVR in immunoblot assays. A chimeric glycoprotein consisting of the N-terminal domain of Cea10, the three constant domains, transmembrane region, and cytoplasmic tail of MHVR, however, does not bind MHV-A59 or MAb CC1 (32).Sequence analysis of the various receptor-like glycoproteins in the murine CEA family shows that the 108-amino-acid N-terminal domains of MHVR, Bgp1^b, and Cea10 are significantly different, with 29 amino acid differences between MHVR and Bgp1^b and 43 amino acid differences between MHVR and Cea10 (18, 26, 32). These glycoproteins also differ significantly in their receptor activities. A detailed analysis of the virus and MAb binding sites in the N-terminal domain of MHVR was done to elucidate the molecular basis for these observed differences in the receptor activities of the murine CEA-related glycoproteins. We have constructed a series of recombinant chimeric glycoproteins and tested their abilities to serve as functional receptors for MHV-A59 in transient transfection assays. The abilities of MAb CC1 to protect transfected cells from infection by MHV-A59 and to bind the recombinant glycoproteins in an immunoblot assay also were examined. Results of these assays indicate that amino acids 34 to 52 of the glycoprotein are critical for receptor activity and that binding of the MAb is very sensitive to any changes in the tertiary structure of MHVR. Site-directed mutagenesis studies confirmed the importance of these residues. Thus, this small region of the N-terminal domain of MHVR is a critical determinant of MHV receptor activity. These residues alone, however, are not sufficient for optimal receptor activity. Additional amino acids within the N-terminal domain of MHVR and the three Ig-like constant domains of MHVR also profoundly affect receptor activity. The data suggest that these domains either influence the conformation of the virus-binding site or affect events subsequent to virus binding that are required for infection.     

Hierarchy of effects of the MHC and T cell receptor beta-chain genes in susceptibility to Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible mice with Theiler's murine encephalomyelitis virus induces a demyelinating disease that is similar to human multiple sclerosis. This murine model for human multiple sclerosis is apparently immune-mediated and the genes involved in the immune response influence the outcome of disease susceptibility as observed with human multiple sclerosis. These genes include the MHC and TCR genes. However, the functional relationships among these genes on the disease susceptibility has not yet been studied. In this study, we demonstrate that the effect of the H-2s genotype from susceptible SJL/J mice overrides the resistant effect of the BALB/c TCR beta-chain gene in CXJ recombinant-inbred and BALB.S congenic mice. These results strongly suggest the presence of a hierarchy of genes involved in the immune response in Theiler's murine encephalomyelitis virus-induced demyelinating disease.