Acute infection of mice with lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of their macrophages

The infection of mice with lactic dehydrogenase virus (LDV) is characterized by elevated levels of various plasma enzymes such as lactic dehydrogenase, malic dehydrogenase, and others. This elevation is probably the consequence of a defect in the clearance capacity of the virus-affected reticuloendothelial cells, which were found to serve as the targets for LDV infection. Since macrophages play a pivotal role in the induction and regulation of cellular immune responses, we tested the antigen-presenting capacity of macrophages from LDV-infected mice, using a system in which in vitro reactivation of memory T cells depends on specific antigen presentation by macrophages. Our experiments revealed that the antigen-presenting capacity of spleen, lymph node, and peritoneal antigen-presenting macrophages from LDV-infected mice was impaired. This impairment, however, was not due to a defective cellular concentration capacity of antigen, since no difference in the uptake of radiolabeled antigen by uninfected and acutely LDV-infected macrophages was observed. Similarly one cannot attribute the impaired presentation capacity to suppressor cells, since we found that LDV-infected macrophages are not differentially immunosuppressive in the specific in vitro assays used. The analysis of peritoneal macrophages for their expression of Ia antigens revealed that the proportion of Ia-positive macrophages among the LDV-infected peritoneal cells is reduced in comparison to their proportion in noninfected mice. Our results suggest, therefore, that infection of macrophages by LDV is followed by an impairment of their antigen-presenting capacity, probably due to a reduction in the relative proportion of Ia-positive macrophages. These results indicate that the virus either impairs the expression of membrane-associated antigen-presenting components (such as the Ia determinants), thus damaging antigen presentation, or is responsible for the elimination of Ia-positive cells from the peritoneum.     

Comparison of the ability of lactate dehydrogenase-elevating virus and its virion RNA to infect murine leukemia virus-infected or -uninfected cell lines.

Lactate dehydrogenase-elevating virus (LDV) has a strict species specificity. Cells or cell lines other than a particular subset of mouse primary macrophages which can support LDV replication in vitro have not been identified. LDV induces neurological disorders in old C58 or AKR strains, in which the involvement of multiple copies of the endogenous N-tropic murine leukemia virus (MuLV) genome and the Fv-1 locus of the mouse has been implicated. Our previous studies have demonstrated that LDV could infect and replicate in cell lines of the mouse or other species in vitro when they were infected with MuLV. The significance of and the precise mechanism underlying this phenomenon, however, remain unclear. We demonstrated in this study the efficient infection and replication of the virus in vitro by inoculation of its RNA mixed with liposome. No significant difference either in the efficiency of RNA transfection or in the ability to support its replication was observed among the various species' cell lines examined. In addition, by RNA transfection the virus replicated with equal efficiency in MuLV-infected and -uninfected cells or in macrophages derived from mice irrespective of their age. In contrast, the pattern of the infection by virus particles was quite different; LDV replication was observed only in macrophages (particularly from newborn mice) and MuLV-infected cells. By using various LDV isolates, it was demonstrated that the capability of replication between neurovirulent, LDV type C, and the other avirulent strains was almost the same in mouse cell lines when their RNA was introduced into the cells. Higher infectivity of LDV-C to MuLV-infected cells may be due to its efficient incorporation of the particles into MuLV-infected cells.     

The leader polypeptide of Theiler's murine encephalomyelitis virus is required for the assembly of virions in mouse L cells

Deletion of the entire leader polypeptide of the GDVII strain of Theiler's murine encephalomyelitis virus (TMEV) results in the production of an attenuated virus that grows in baby hamster kidney (BHK) cells but cannot grow at all in mouse L-929 cells. This study examined the reasons for the failure of dl-L, the GDVII variant that lacks the leader polypeptide, to grow in mouse cells. At low multiplicities of infection, it was difficult to detect any viral proteins in mouse cells. However, levels of positive- and negative-strand RNA molecules were only moderately reduced in these infections. Viral RNA showed no major defect in translatability, as the mutant viral RNA was nearly as efficient as that of the wild-type (WT) virus in directing protein synthesis in vitro in assays using extracts prepared from mouse L cells. Viral protein synthesis was detected in dl-L-infected mouse cells as multiplicities of infection were increased and approached the levels observed in WT infections. Despite this, there was a total lack of virus production in high-multiplicity infections, and this was found to correlate with the failure of viral proteins and early virion precursors to assemble into virions in mouse cells. Thus, the inability of dl-L to grow in mouse cells reflects complex effects on various stages of the virus infection but is primarily a defect in virus assembly.     

Redirecting coronavirus to a nonnative receptor through a virus-encoded targeting adapter

Murine hepatitis coronavirus (MHV)-A59 infection depends on the interaction of its spike (S) protein with the cellular receptor mCEACAM1a present on murine cells. Human cells lack this receptor and are therefore not susceptible to MHV. Specific alleviation of the tropism barrier by redirecting MHV to a tumor-specific receptor could lead to a virus with appealing properties for tumor therapy. To demonstrate that MHV can be retargeted to a nonnative receptor on human cells, we produced bispecific adapter proteins composed of the N-terminal D1 domain of mCEACAM1a linked to a short targeting peptide, the six-amino-acid His tag. Preincubation of MHV with the adapter proteins and subsequent inoculation of human cells expressing an artificial His receptor resulted in infection of these otherwise nonsusceptible cells and led to subsequent production of progeny virus. To generate a self-targeted virus able to establish multiround infection of the target cells, we subsequently incorporated the gene encoding the bispecific adapter protein as an additional expression cassette into the MHV genome through targeted RNA recombination. When inoculated onto murine LR7 cells, the resulting recombinant virus indeed expressed the adapter protein. Furthermore, inoculation of human target cells with the virus resulted in a His receptor-specific infection that was multiround. Extensive cell-cell fusion and rapid cell killing of infected target cells was observed. Our results show that MHV can be genetically redirected via adapters composed of the S protein binding part of mCEACAM1a and a targeting peptide recognizing a nonnative receptor expressed on human cells, consequently leading to rapid cell death. The results provide interesting leads for further investigations of the use of coronaviruses as antitumor agents.     

Natural cytotoxicity against mouse hepatitis virus-infected target cells. I. Correlation of cytotoxicity with virus binding to leukocytes

Spleen cells from uninfected control mice selectively lysed BALB/c 3T3 fibroblasts infected with mouse hepatitis virus (MHV), a murine coronavirus. Lysis of infected cells occurred within 3 hr, and histocompatibility between effector and target cells was not required. This natural, cell-mediated, virus-associated cytotoxicity differed from NK cell- and T cell-mediated lysis. Spleen cells from animals infected with MHV were enriched in NK activity and were more cytotoxic to YAC-1 target cells, but did not show enhanced cytotoxicity for MHV-infected target cells. Spleen cells from beige mice, which are deficient in NK cell activity, were able to lyse MHV-infected target cells, as were spleen cells from nude mice, which are deficient in T cell activity. Lysis of MHV-infected target cells could be mediated by cells from the spleen and, to a lesser extent, by cells from the bone marrow, but not by resident peritoneal cells or thymocytes. We suggest the term "virus killer (VK) activity" for this phenomenon. VK activity of splenocytes from different mouse strains correlated with the ability of the splenocytes to bind purified radiolabeled MHV virions. MHV virions caused agglutination of spleen leukocytes from susceptible mouse strains, indicating that leukocyte agglutination or adsorption may provide a useful assay for coronaviruses such as MHV which lack hemagglutinating activity. SJL mouse splenocytes did not bind MHV and did not lyse infected targets. MHV bound relatively well to splenocytes of other mouse strains, but poorly to thymocytes and erythrocytes. Binding of MHV to leukocytes was not influenced by 6 mM EDTA or EGTA, indicating a lack of requirement for Mg++ or Ca++. VK activity was also resistant to EDTA and EGTA, in contrast to NK activity, which was sensitive to those chelating agents. VK activity was also unaffected by actinomycin D, cycloheximide, or puromycin, indicating that new protein synthesis was not required for lysis. Antibody to interferon-alpha/beta did not block lysis, nor was there substantially enhanced lysis mediated by leukocytes from mice infected with virus and thus exposed to high levels of interferon. VK activity was blocked by antibody directed against the peplomeric glycoprotein E2 of MHV. VK activity required infected target cells, because cells with adsorbed MHV virions were not lysed by splenocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

In vitro replication of mouse hepatitis virus strain A59.

An in vitro replication system for mouse hepatitis virus (MHV) strain A59 was developed using lysolecithin to produce cell extracts. In extracts of MHV-infected cells, radiolabeled UMP was incorporated at a linear rate for up to 1 h into RNA, which hybridized to MHV-specific cDNA probes and migrated in denaturing formaldehyde-agarose gels to the same position as MHV genomic RNA. The incorporation of [32P]UMP into genome-sized RNA in vitro correlated with the observed increase of [3H]uridine incorporation in MHV-infected cells labeled in vivo. Incorporation of [32P]UMP into genome-sized RNA was inhibited when extracts were incubated with puromycin. The addition to the assay of antiserum to the MHV-A59 nucleocapsid protein N inhibited synthesis of genome-sized RNA by 90% compared with the addition of preimmune serum. In contrast, antiserum to the E1 or E2 glycoproteins did not significantly inhibit RNA replication. In vitro-synthesized RNA banded in cesium chloride gradients as a ribonucleoprotein complex with the characteristic density of MHV nucleocapsids isolated from virions. These experiments suggest that ongoing protein synthesis is necessary for replication of MHV genomic RNA and indicate that the N protein plays an important role in MHV replication.     

Lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral RNA in germinal centers, concomitant with polyclonal activation of B cells.

Lactate dehydrogenase-elevating virus (LDV) replicates primarily and most likely solely in a subpopulation of macrophages in extraneuronal tissues. Infection of mice, regardless of age, with LDV leads to the rapid cytocidal replication of the virus in these cells, resulting in the release of large amounts of LDV into the circulation. The infection then progresses into life-long, asymptomatic, low-level viremic persistence, which is maintained by LDV replication in newly generated LDV-permissive cells which escapes all antiviral immune responses. In situ hybridization studies of tissue sections of adult FVB mice revealed that by 1 day postinfection (p.i.), LDV-infected cells were present in practically all tissues but were present in the highest numbers in the lymph nodes, spleen, and skin. In the central nervous system, LDV-infected cells were restricted to the leptomeninges. Most of the infected cells had disappeared at 3 days p.i., consistent with the cytocidal nature of the LDV infection, except for small numbers in lymph node, spleen, liver, and testis tissues. These tissues harbored infected cells until at least 90 days p.i. The results suggest that the generation of LDV-permissive cells during the persistent phase is restricted to these tissues. The continued presence of LDV-infected cells in testis tissue suggests the possibility of LDV release in semen and sexual transmission. Most striking was the accumulation of large amounts of LDV RNA in newly generated germinal centers of lymph nodes and the spleen. The LDV RNA was not associated with infected cells but was probably associated with virions or debris of infected, lysed cells. The appearance of LDV RNA in germinal centers in these mice coincided in time with the polyclonal activation of B cells, which leads to the accumulation of polyclonal immunoglobulin G2a and low-molecular-weight immune complexes in the circulation.     

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR^−/−) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-β in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-β production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-β production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-β mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-β by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.     

Glycosaminoglycan profile of peritoneal and bone marrow-derived macrophages. Changes associated with macrophage activation.

1. We have analysed the glycosaminoglycan patterns of peritoneal and bone marrow-derived macrophages obtained from four different mouse strains which are resistant (A/J) or susceptible (BALB/c, DBA and C-57) to murine hepatitis virus type 3 (MHV3) infection. The glycosaminoglycans were biosynthetically labelled by exposing the macrophages to 35S-sulphate. The medium and cell fractions were collected and the 35S-glycosaminoglycans formed were identified by a combination of agarose gel electrophoresis and enzymatic degradation with bacterial mucopolysaccharidases. 2. Both peritoneal and bone marrow-derived macrophages synthesize and secrete a mixture of dermatan sulphate, heparan sulphate and chondroitin sulphate. Dermatan sulphate is the main glycosaminoglycan and most of the synthesized glycosaminoglycans are released to the culture medium. 3. The glycosaminoglycan patterns vary depending on the macrophage source. Bone marrow-derived cells synthesize glycosaminoglycans at lower rates, release a lower glycosaminoglycan percentage to the culture medium and express higher amounts of heparan sulphate in comparison with their peritoneal counterparts. Furthermore, LPS-induced activation leads to an increased glycosaminoglycan expression in bone marrow-derived macrophages and to a decrease in 35S-glycosaminoglycans of peritoneal macrophages from BALB/c, A/J and C-57 mice. 4. We have not established any correlation between macrophage glycosaminoglycans and resistance to MHV3 infection, since the glycosaminoglycan patterns of resistant (A/J) and susceptible (BALB/c, DBA and C-57) mouse macrophages are similar. Furthermore, the in vitro infection of both control and LPS-activated peritoneal macrophages with MHV3 did not cause any changes in the expression of glycosaminoglycans.     

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.     

Genetic analysis of determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain

The coronavirus spike protein (S) forms the distinctive virion surface structures that are characteristic of this viral family, appearing in negatively stained electron microscopy as stems capped with spherical bulbs. These structures are essential for the initiation of infection through attachment of the virus to cellular receptors followed by fusion to host cell membranes. The S protein can also mediate the formation of syncytia in infected cells. The S protein is a type I transmembrane protein that is very large compared to other viral fusion proteins, and all except a short carboxy-terminal segment of the S molecule constitutes the ectodomain. For the prototype coronavirus mouse hepatitis virus (MHV), it has previously been established that S protein assembly into virions is specified by the carboxy-terminal segment, which comprises the transmembrane domain and the endodomain. We have genetically dissected these domains in the MHV S protein to localize the determinants of S incorporation into virions. Our results establish that assembly competence maps to the endodomain of S, which was shown to be sufficient to target a heterologous integral membrane protein for incorporation into MHV virions. In particular, mutational analysis indicated a major role for the charge-rich carboxy-terminal region of the endodomain. Additionally, we found that the adjacent cysteine-rich region of the endodomain is critical for fusion of infected cells, confirming results previously obtained with S protein expression systems.     

Defective retrovirus-like 30S RNA species of rat and mouse cells are infectious if packaged by type C helper virus.

RNA species with properties of defective retrovirus-like 30S RNA genomes have previously been detected in both rats and mice and in some rat and mouse retroviruses. Using cell lines which express high levels of this retrovirus-like RNA, we formed pseudotypes of the 30S RNAs with helper-independent type C viruses. A pseudotype virus complex containing a mouse 30S subunit was transmitted to rat cells, and a pseudotype virus complex containing a rat 30S subunit was transmitted to bat cells. In other transmission experiments, a rat 30S subunit was isolated in nonproducer bat cells without detectable expression of the helper-independent type C virus used to pseudotype it. The results provide further support for the retrovirus-like nature of the rat 30S subunit and provide evidence which supports the protovirus hypothesis proposed by Temin.     

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.     

Suppression of immune response induction in Peyer's patch lymphoid cells from mice infected with mouse hepatitis virus

Multiple previous studies have demonstrated significant alterations of immunologic parameters associated with mouse hepatitis virus (MHV) infection, but effects of the virus on mucosal lymphoid cells have not been examined. Coincident with a natural outbreak of MHV at our institution, we noted alterations in immunoglobulin secretion by mature Peyer's patch B cells under an inductive stimulus provided by dendritic cells and mitogen-activated T cells (DC-T). MHV was isolated from mice affected during the outbreak, and experimental infection of mice with the isolate consistently resulted in failures of immunoglobulin secretion by cocultures of Peyer's patch DC-T and B cells. In subsequent experiments, MHV appeared to negatively affect DC-T more than B cells. Therefore, the effects of inapparent MHV infection on experimental mucosal immune responses can result from natural infection and can be experimentally reproduced.     

Murine coronavirus requires lipid rafts for virus entry and cell-cell fusion but not for virus release

Thorp and Gallagher first reported that depletion of cholesterol inhibited virus entry and cell-cell fusion of mouse hepatitis virus (MHV), suggesting the importance of lipid rafts in MHV replication (E. B. Thorp and T. M. Gallagher, J. Virol. 78:2682-2692, 2004). However, the MHV receptor is not present in lipid rafts, and anchoring of the MHV receptor to lipid rafts did not enhance MHV infection; thus, the mechanism of lipid rafts involvement is not clear. In this study, we defined the mechanism and extent of lipid raft involvement in MHV replication. We showed that cholesterol depletion by methyl beta-cyclodextrin or filipin did not affect virus binding but reduced virus entry. Furthermore, MHV spike protein bound to nonraftraft membrane at 4 degrees C but shifted to lipid rafts at 37 degrees C, indicating a redistribution of membrane following virus binding. Thus, the lipid raft involvement in MHV entry occurs at a step following virus binding. We also found that the viral spike protein in the plasma membrane of the infected cells was associated with lipid rafts, whereas that in the Golgi membrane, where MHV matures, was not. Moreover, the buoyant density of the virion was not changed when MHV was produced from the cholesterol-depleted cells, suggesting that MHV does not incorporate lipid rafts into the virion. These results indicate that MHV release does not involve lipid rafts. However, MHV spike protein has an inherent ability to associate with lipid rafts. Correspondingly, cell-cell fusion induced by MHV was retarded by cholesterol depletion, consistent with the association of the spike protein with lipid rafts in the plasma membrane. These findings suggest that MHV entry requires specific interactions between the spike protein and lipid rafts, probably during the virus internalization step.