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.     

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.     

The murine coronavirus mouse hepatitis virus strain A59 from persistently infected murine cells exhibits an extended host range.

In murine 17 Cl 1 cells persistently infected with murine coronavirus mouse hepatitis virus strain A59 (MHV-A59), expression of the virus receptor glycoprotein MHVR was markedly reduced (S. G. Sawicki, J. H. Lu, and K. V. Holmes, J. Virol. 69:5535-5543, 1995). Virus isolated from passage 600 of the persistently infected cells made smaller plaques on 17 Cl 1 cells than did MHV-A59. Unlike the parental MHV-A59, this variant virus also infected the BHK-21 (BHK) line of hamster cells. Virus plaque purified on BHK cells (MHV/BHK) grew more slowly in murine cells than did MHV-A59, and the rate of viral RNA synthesis was lower and the development of the viral nucleocapsid (N) protein was slower than those of MHV-A59. MHV/BHK was 100-fold more resistant to neutralization with the purified soluble recombinant MHV receptor glycoprotein (sMHVR) than was MHV-A59. Pretreatment of 17 Cl 1 cells with anti-MHVR monoclonal antibody CC1 protected the cells from infection with MHV-A59 but only partially protected them from infection with MHV/BHK. Thus, although MHV/BHK could still utilize MHVR as a receptor, its interactions with the receptor were significantly different from those of MHV-A59. To determine whether a hemagglutinin esterase (HE) glycoprotein that could bind the virions to 9-O-acetylated neuraminic acid moieties on the cell surface was expressed by MHV/BHK, an in situ esterase assay was used. No expression of HE activity was detected in 17 Cl 1 cells infected with MHV/BHK, suggesting that this virus, like MHV-A59, bound to cell membranes via its S glycoprotein. MHV/BHK was able to infect cell lines from many mammalian species, including murine (17 Cl 1), hamster (BHK), feline (Fcwf), bovine (MDBK), rat (RIE), monkey (Vero), and human (L132 and HeLa) cell lines. MHV/BHK could not infect dog kidney (MDCK I) or swine testis (ST) cell lines. Thus, in persistently infected murine cell lines that express very low levels of virus receptor MHVR and which also have and may express alternative virus receptors of lesser efficiency, there is a strong selective advantage for virus with altered interactions with receptor (D. S. Chen, M. Asanaka, F. S. Chen, J. E. Shively, and M. M. C. Lai, J. Virol. 71:1688-1691, 1997; D. S. Chen, M. Asanaka, K. Yokomori, F.-I. Wang, S. B. Hwang, H.-P. Li, and M. M. C. Lai, Proc. Natl. Acad. Sci. USA 92:12095-12099, 1995; P. Nedellec, G. S. Dveksler, E. Daniels, C. Turbide, B. Chow, A. A. Basile, K. V. Holmes, and N. Beauchemin, J. Virol. 68:4525-4537, 1994). Possibly, in coronavirus-infected animals, replication of the virus in tissues that express low levels of receptor might also select viruses with altered receptor recognition and extended host range.     

Pseudotype virions formed between mouse hepatitis virus and lactate dehydrogenase-elevating virus (LDV) mediate LDV replication in cells resistant to infection by LDV virions.

Infection of cultures of peritoneal macrophages with both lactate dehydrogenase-elevating virus (LDV) and mouse hepatitis virus (MHV) resulted in the formation of pseudotype virions containing LDV RNA which productively infected cells that are resistant to infection by intact LDV virions but not to infection by MHV. These cells were mouse L-2 and 3T3-17Cl-1 cells as well as residual peritoneal macrophages from persistently LDV-infected mice. Productive LDV infection of these cells via pseudotype virions was inhibited by antibodies to the MHV spike protein or to the MHV receptor, indicating that LDV RNA entered the cells via particles containing the MHV envelope. Simultaneous exposure of L-2 cells to both LDV and MHV resulted in infection by MHV but not by LDV. The results indicate that an internal block to LDV replication is not the cause of the LDV nonpermissiveness of many cell types, including the majority of the macrophages in an adult mouse. Instead, LDV permissiveness is restricted to a subpopulation of mouse macrophages because only these cells possess a surface component that acts as an LDV receptor.     

Mouse susceptibility to mouse hepatitis virus infection is linked to viral receptor genotype.

We have reported that the receptor for mouse hepatitis virus (MHV) expressed in MHV-susceptible BALB/c mice (MHVR1) has 10 to 30 times the virus-binding activity of the MHV receptor expressed in MHV-resistant SJL mice (MHVR2) (N. Ohtsuka, Y. K. Yamada, and F. Taguchi, J. Gen. Virol. 77:1683-1992, 1996). This fact indicates the possibility that the difference in MHV susceptibility between BALB/c and SJL mice is determined by the virus-binding activity of the receptor. To test this possibility, we have examined MHV susceptibility in mice with the homozygous MHVR1 gene (R1/R1 genotype), mice with the MHVR1 and MHVR2 genes (R1/R2 genotype), and mice with the homozygous MHVR2 gene (R2/R2 genotype) produced by cross and backcross mating between BALB/c and SJL mice. All 63 F2 and backcrossed mice with the MHVR1 gene (R1/R1 and R1/R2) were susceptible to MHV infection, and all 57 with the homozygous MHVR2 gene (R2/R2) were resistant. We have also examined the MHV receptor genotypes of several mouse strains that were reported to be susceptible to MHV infection. All of those mice had the MHVR1 gene. These results suggest the possibility that the viral receptor determines the susceptibility of the whole animal to MHV infection.     

Intracellular Complexes of Viral Spike and Cellular Receptor Accumulate during Cytopathic Murine Coronavirus Infections

Murine hepatitis virus (MHV) infections exhibit remarkable variability in cytopathology, ranging from acutely cytolytic to essentially asymptomatic levels. In this report, we assess the role of the MHV receptor (MHVR) in controlling this variable virus-induced cytopathology. We developed human (HeLa) cell lines in which the MHVR was produced in a regulated fashion by placing MHVR cDNA under the control of an inducible promoter. Depending on the extent of induction, MHVR levels ranged from less than ~1,500 molecules per cell (designated R^lo) to ~300,000 molecules per cell (designated R^hi). Throughout this range, the otherwise MHV-resistant HeLa cells were rendered susceptible to infection. However, infection in the R^lo cells occurred without any overt evidence of cytopathology, while the corresponding R^hi cells died within 14 h after infection. When the HeLa-MHVR cells were infected with vaccinia virus recombinants encoding MHV spike (S) proteins, the R^hi cells succumbed within 12 h postinfection; R^lo cells infected in parallel were intact, as judged by trypan blue exclusion. This acute cytopathology was not due solely to syncytium formation between the cells producing S and MHVR, because fusion-blocking antiviral antibodies did not prevent it. These findings raised the possibility of an intracellular interaction between S and MHVR in the acute cell death. Indeed, we identified intracellular complexes of S and MHVR via coimmunoprecipitation of endoglycosidase H-sensitive forms of the two proteins. We suggest that MHV infections can become acutely cytopathic once these intracellular complexes rise above a critical threshold level.     

Maintenance of pluripotency in mouse embryonic stem cells persistently infected with murine coronavirus.

A persistently coronavirus-infected embryonic stem (ES) cell line A3/MHV was established by infecting an ES cell line, A3-1, with mouse hepatitis virus type-2. Although almost all A3/MHV cells were found infected, both A3/MHV and A3-1 cells expressed comparable levels of cell surface differentiation markers. In addition, A3/MHV cells retained the ability to form embryoid bodies. These results suggest that persistent coronavirus infection does not affect the differentiation of ES cells.     

Receptor-independent spread of a highly neurotropic murine coronavirus JHMV strain from initially infected microglial cells in mixed neural cultures

Although neurovirulent mouse hepatitis virus (MHV) strain JHMV multiplies in a variety of brain cells, expression of its receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM 1) (MHVR) is restricted only in microglia. The present study was undertaken to clarify the mechanism of an extensive JHMV infection in the brain by using neural cells isolated from mouse brain. In contrast to wild-type (wt) JHMV, a soluble-receptor-resistant mutant (srr7) infects and spreads solely in an MHVR-dependent fashion (F. Taguchi and S. Matsuyama, J. Virol. 76:950-958, 2002). In mixed neural cell cultures, srr7 infected a limited number of cells and infection did not spread, although wt JHMV induced syncytia in most of the cells. srr7-infected cells were positive for GS-lectin, a microglia marker. Fluorescence-activated cell sorter analysis showed that about 80% of the brain cells stained with anti-MHVR antibody (CC1) were also positive for GS-lectin. Pretreatment of those cells with CC1 prevented virus attachment to the cell surface and also blocked virus infection. These results show that microglia express functional MHVR that mediates JHMV infection. As expected, in microglial cell-enriched cultures, both srr7and wt JHMV produced syncytia in a majority of cells. Treatment with CC1 of mixed neural cell cultures and microglia cultures previously infected with wt virus failed to block the spread of infection, indicating that wt infection spreads in an MHVR-independent fashion. Thus, the present study indicates that microglial cells are the major population of the initial target for MHV infection and that the wt spreads from initially infected microglia to a variety of cells in an MHVR-independent fashion.     

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.     

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.     

Persistent reovirus infections of L cells select mutations in viral attachment protein sigma1 that alter oligomer stability.

During maintenance of L-cell cultures persistently infected with reovirus, mutations are selected in viruses and cells. Cells cured of persistent infection support growth of viruses isolated from persistently infected cultures (PI viruses) significantly better than that of wild-type (wt) viruses. In a previous study, the capacity of PI virus strain L/C to grow better than wt strain type 1 Lang (T1L) in cured cells was mapped genetically to the S1 gene (R. S. Kauffman, R. Ahmed, and B. N. Fields, Virology 131:79-87, 1983), which encodes viral attachment protein sigma1. To investigate mechanisms by which mutations in S1 confer growth of PI viruses in cured cells, we determined the S1 gene nucleotide sequences of L/C virus and six additional PI viruses isolated from independent persistently infected L-cell cultures. The S1 sequences of these viruses contained from one to three mutations, and with the exception of PI 2A1 mutations in each S1 gene resulted in changes in the deduced amino acid sequence of sigma1 protein. Using electrophoresis conditions that favor migration of sigma1 oligomers, we found that sigma1 proteins of L/C, PI 1A1, PI 3-1, and PI 5-1 migrated as monomers, whereas sigma1 proteins of wt reovirus and PI 2A1 migrated as oligomers. These findings suggest that mutations in sigma1 protein affecting stability of sigma1 oligomers are important for the capacity of PI viruses to infect mutant cells selected during persistent infection. Since no mutation was found in the deduced amino acid sequence of PI 2A1 sigma1 protein, we used T1L X PI 2A1 reassortant viruses to identify viral genes associated with the capacity of this PI virus to grow better than wt in cured cells. The capacity of PI 2A1 to grow better than T1L in cured cells was mapped to the S4 gene, which encodes outer-capsid protein sigma3. This finding suggests that in some cases, mutations in sigma3 protein in the absence of sigma1 mutations confer growth of PI viruses in mutant cells. To confirm the importance of the S1 gene in PI virus growth in cured cells, we used T1L X PI 3-1 reassortant viruses to genetically map the capacity of this PI virus to grow better than wt in cured cells. In contrast to our results using PI 2A1, we found that growth of PI 3-1 in cured cells was determined by the sigma1-encoding S1 gene. Given that the sigma1 and sigma3 proteins play important roles in reovirus disassembly, findings made in this study suggest that stability of the viral outer capsid is an important determinant of the capacity of reoviruses to adapt to host cells during persistent infection.     

Cells and viruses with mutations affecting viral entry are selected during persistent infections of L cells with mammalian reoviruses.

Previous studies demonstrated that both cellular and viral mutants are selected during maintenance of persistent infections established in murine L cells with high-passage stocks of mammalian reoviruses. In particular, when one culture was cured of persistent infection, the resulting cells were found to support the growth of viruses isolated from persistently infected cultures (termed PI viruses here) better than that of wild-type (wt) viruses (R. Ahmed, W. M. Canning, R. S. Kauffman, A. H. Sharpe, J. V. Hallum, and B. N. Fields, Cell 25:325-332, 1981). To address the nature of cellular and viral mutations selected during maintenance of persistent reovirus infections, we established independent, persistently infected cultures with L cells and high-passage stocks of wt reovirus. These cultures served as sources of new PI viruses and cured cells for study. We found that although wt viruses grew poorly in cured cells when infection was initiated with intact virions, they grew well in cured cells when infection was initiated with infectious subvirion particles generated from virions by in vitro treatment with chymotrypsin. This finding indicates that the block to growth of wt viruses in cured cells involves an early step that is unique to infection by virions, such as proteolytic processing in an endocytic compartment. We also found that PI viruses grew better than wt viruses in L cells treated with ammonium chloride, a weak base that inhibits the pH decrease in endosomes and lysosomes. Because ammonium chloride blocks an early step in infection by intact virions, probably the proteolytic processing of viral outer capsid proteins by acid-dependent cellular proteases in late endosomes or lysosomes, this finding indicates that PI viruses differ from wt viruses with respect to viral entry into cells. Therefore, these results indicate that both cells and viruses evolve mutations that affect one or more early steps in the viral growth cycle during maintenance of L-cell cultures persistently infected with reoviruses.     

Molecular anatomy of mouse hepatitis virus persistence: coevolution of increased host cell resistance and virus virulence.

Persistent infection of murine astrocytoma (DBT) cells with mouse hepatitis virus (MHV) has been established. From this in vitro virus-host system, persistence is mediated at the level of cellular MHV receptor (MHVR) expression and increased virus virulence. MHV persistence selects for resistant host cell populations which abate virus replication. Reductions in MHVR expression were significantly associated with increased host resistance, and transfection of MHVR into resistant host cells completely restored the capacity of cells to support efficient replication of MHV strain A59. The emergence of resistant host cells coselected for variant viruses that had increased avidity for MHVR and also recognized different receptors for entry into resistant cells. These data illustrate that MHV persistence in vitro provides a model to identify critical sites of virus-host interaction at the cellular level which are altered during the evolution of host cell resistance to viral infection and the coevolution of virus virulence.     

Persistent infection with herpes simplex virus type 1 in an Ia antigen-positive murine macrophage cell line

The interaction of herpes simplex virus type 1 (HSV-1) with murine macrophage cell lines was examined. The cell lines appeared to be moderately permissive for HSV-1 replication, though the yield of the virus was limited compared with that in Vero cells. Furthermore, the murine macrophage cell line SL-1, bearing Ia antigen, was persistently infected with HSV-1 for over one year, and was designated SL-1/KOS. Persistent infection could not be established in an Ia antigen-negative macrophage cell line, SL-4. In the SL-1/KOS culture, there was a small number of infected cells as revealed by infectious center assay. Treatment with monoclonal antibody against HSV-1 cured the persistent infection. Therefore maintenance of the persistent infection is considered to be due to a carrier culture consisting of a minority of infected cells and a majority of uninfected cells. In the SL-1/KOS cultures a low level of interferon (IFN) was found. When a large amount of exogenous recombinant murine IFN-beta (10(5)-10(6) international units/ml) was added to the culture, virus production diminished to undetectable levels. These results suggest that IFN plays an important role in the maintenance of persistent infection. In long-term persistently infected cultures, syncytium formation appeared and the virus from such cultures had a different DNA structure from that of the virus originally used for infection as revealed by restriction endonuclease analysis.     

Role of the host cell in persistent viral infection: Coevolution of L cells and reovirus during persistent infection

Mutant L cells, designated LR cells, were isolated after “curing” a persistently infected cell line (L/C) with antireovirus serum. The LR cells were shown to be virus-free; no reovirus was detectable by infectious center assays, plaque assays, presence of viral proteins, presence of viral dsRNA and immunofluorescence studies. Persistent infections were readily established in LR cells following infection with either cloned, low passage wild-type reovirus or cloned, low passage reovirus isolated from carrier cultures. Reovirus isolated from carrier cultures, however, grew much better than wild-type reovirus in LR cells and showed complete dominance over wild-type reovirus in coinfection experiments. Infection of LR cells with wild-type reovirus resulted in a low-level persistent infection with inefficient viral replication; these mutant L cells were partially resistant to infection with wild-type reovirus. In contrast, infection of the mutant L cells with virus isolated from the persistently infected cells resulted in a persistent infection accompanied with efficient viral replication. Infection of the original L cells with either wild-type reovirus or reovirus isolated from the persistently infected cells resulted in a lytic infection with no surviving cells. Thus the host cell plays a crucial role in the maintenance of persistent reovirus infection. Our results show that there is a coevolution of both mutant L cells and mutant reovirus during persistent infection.     

Soluble Receptor Potentiates Receptor-Independent Infection by Murine Coronavirus

Mouse hepatitis virus (MHV) infection spreads from MHV-infected DBT cells, which express the MHV receptor CEACAM1 (MHVR), to BHK cells, which are devoid of the receptor, by intercellular membrane fusion (MHVR-independent fusion). This mode of infection is a property of wild-type (wt) JHMV cl-2 virus but is not seen in cultures infected with the mutant virus JHMV srr7. In this study, we show that soluble MHVR (soMHVR) potentiates MHVR-independent fusion in JHMV srr7-infected cultures. Thus, in the presence of soMHVR, JHMV srr7-infected DBT cells overlaid onto BHK cells induce BHK cell syncytia and the spread of JHMV srr7 infection. This does not occur in the absence of soMHVR. soMHVR also enhanced wt virus MHVR-independent fusion. These effects were dependent on the concentration of soMHVR in the culture and were specifically blocked by the anti-MHVR monoclonal antibody CC1. Together with these observations, direct binding of soMHVR to the virus spike (S) glycoprotein as revealed by coimmunoprecipitation demonstrated that the effect is mediated by the binding of soMHVR to the S protein. Furthermore, fusion of BHK cells expressing the JHMV srr7 S protein was also induced by soMHVR. These results indicated that the binding of soMHVR to the S protein expressed on the DBT cell surface potentiates the fusion of MHV-infected DBT cells with nonpermissive BHK cells. We conclude that the binding of soMHVR to the S protein converts the S protein to a fusion-active form competent to mediate cell-cell fusion, in a fashion similar to the fusion of virus and cell membranes.     

Efficiency of viral entry determines the capacity of murine erythroleukemia cells to support persistent infections by mammalian reoviruses.

To determine mechanisms by which persistent viral infections are established and maintained, we initiated persistent infections of murine erythroleukemia (MEL) cells by using reovirus strains type 3 Abney and type 3 Dearing. Establishment of persistent reovirus infections of MEL cells was not associated with a significant cytopathic effect despite the presence of high titers of infectious virus in the cultures (>10(5) PFU/ml of culture lysate). Maintenance of persistently infected MEL-cell cultures was associated with coevolution of mutant viruses and cells. Mutant viruses produced greater yields than the parental wild-type (wt) strains in MEL cells cured of persistent infection and in cells treated with ammonium chloride, a weak base that blocks viral disassembly. Mutant cells supported growth of wt infectious subvirion particles, which are disassembly intermediates generated in vitro by treatment of virions with chymotrypsin, substantially better than growth of wt virions. These findings indicate that viral and cellular mutations selected during maintenance of persistently infected MEL-cell cultures affect acid-dependent proteolysis of virions during entry into cells. We also found that wt infectious subvirion particles produce greater yields than wt virions in wt MEL cells, which suggests that inefficient viral disassembly in MEL cells favors establishment of persistent infection. Therefore, steps in reovirus replication leading to viral disassembly appear to be critical determinants of the capacity of MEL cells to support both establishment and maintenance of persistent reovirus infections.     

Establishment of Vero E6 cell clones persistently infected with severe acute respiratory syndrome coronavirus

Little information is available on persistent infection of severe acute respiratory syndrome (SARS) coronavirus (CoV). In this study, we established persistent infection of SARS-CoV in the Vero E6 cell line. Acute infection of Vero E6 with SARS-CoV produced a lytic infection with characteristic rounding cytopathic effects (CPE) and the production of a large number of infectious particles in the culture fluid within 3 days post-infection. Upon subsequent culturing of the remaining adherent cells, the cells gradually proliferated and recovered normal morphology similar to that of the parental cells, and continued to produce large numbers of infectious viral particles during the observation period of 5 months. Among a total of 87 cell clones obtained from the persistently infected Vero E6, only four cell clones (named #13, #18, #21, and #34) were positive for viral RNA. Clones #13, #18, and #34 shifted to viral RNA-negative during subsequent cultures, while #21 continuously produced infectious particles at a high rate. The SARS-CoV receptor, angiotensin-converting enzyme 2, was almost completely down regulated from the cell surface of persistently infected cells. Western blot analysis as well as electron microscopy indicated that the ratios of spike to nucleocapsid protein in clone #21 as well as its parental persistently infected cells were lower than that in the cells in the acute phase of infection. These Vero E6 cells persistently infected with SARS-CoV may be useful for clarifying the mechanism of the persistent infection and also for elucidating the possible pathophysiologic significance of such long-term maintenance of this virus.     

Persistent infection with bovine herpesvirus-1 (infectious bovine rhinotracheitis virus) in cultured hamster cells

Summary Bovine herpesvirus-1 infection in hamster embryo cells was found to be dependent upon input multiplicity; productive infection was achieved at input multiplicities greater than one, while persistent infection was established when input multiplicities were about 0.5. This persistence was characterized by a noncyclic, minimal degree of cytopathic effect with a low level of released virus. Maintenance of the persistently infected cultures did not require external supportive measures. Subcultivation of the persistently infected cultures led to virus replication followed by CPE and then cell regrowth. Within 3 to 4 weeks after subcultivation a persistent infection was re-established. The possible mechanism for the bovine herpesvirus persistence in hamster cells is discussed. This work was supported in part by Public Health Service Research Contract FDA 233-74-1035 and by Research Grant AI-08648 from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.     

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.