Modulation of cellular macromolecular synthesis by coronavirus: implication for pathogenesis

S Kyuwa, M Cohen, G Nelson, S M Tahara, S A Stohlman
1994 Journal of Virology  
Infection with the murine coronavirus strain JHM decreases cell surface expression of major histocompatibility complex class I antigens. Northern blots showed that JHM virus infection rapidly reduced the level of actin mRNA, whereas the levels of major histocompatibility complex class I and tubulin mRNAs were reduced only slightly. By contrast, the mRNA levels of interleukin 1ij, colony-stimulating factor 1 receptor, and tumor necrosis factor alpha increased following infection. JHM virus
more » ... on. JHM virus (JHMV), a neurotropic strain of murine coronavirus consists of three glycoproteins designated the spike (S), membrane (M), and hemagglutinin-esterase (HE) proteins and a nucleocapsid (N) protein (16). Although wild-type JHMV induces fatal encephalomyelitis and primary demyelination in mice, a small percentage of survivors show paralytic disease with chronic demyelination (15). This system provides a valuable animal model of demyelinating diseases in the central nervous system (CNS) such as multiple sclerosis. In addition, isolation of monoclonal antibody (MAb) neutralization-resistant viral variants, which induce demyelinating disease without fatal encephalitis, have extended the usefulness of this model (5, 8, 40) . A number of laboratories, including our own, have been pursuing the immunopathology of this model (15). These studies suggest that virus clearance from the CNS is mediated by CD8+ cytotoxic T lymphocytes (CTL) (31, 32, 35, 41, 42) . However, CD4+ T cells are also required, perhaps as helper T cells for the induction of virus-specific CTL or as effector cells themselves (15, 35) . The CD8+ T-cell population might be involved not only in the clearance of virus but also in the development of demyelination following JHMV infection (9, 39, 42). CD8+ CTL recognize viral antigen in the context of major histocompatibility complex (MHC) class I molecules. Accumulating evidence suggests that octapeptides or nonapeptides with allele-specific motifs bind in the groove of the MHC class I molecules within the endoplasmic reticulum (26, 38). These complexes are transported to the cell surface for presentation to CD8+ CTL with appropriate T-cell receptors (37). Since the CD8+ CTL-mediated immune response is essential in JHMV infection, the expression of MHC class I antigens during JHMV infection is an important consideration in pathogenesis. Murine coronavirus infection inhibits cellular macromolecular synthesis in L-2 cells (13), DBT cells (36), and J774.1 cells (data not shown). By contrast, the expression of MHC class I antigen on astrocytes, oligodendroglia (17, 34), and brain endothelial cells (14) increases after murine coronavirus infection. Therefore, the effect of JHMV infection on cell surface expression of MHC class I molecules on J774.1 cells, a target cell line used for analysis of JHMV-specific CTL (31, 32, 43), was examined by radioimmunoassay. J774.1 cells grown in 96-well plates were infected with the DL variant of JHMV at a multiplicity of infection of 4 and incubated at 37°C for 10 h. Cells were washed with chilled phosphate-buffered saline (PBS) and incubated with 50 pLl of MAb (1 ,ug/ml) per well at 4°C for 4 h. Cells were then washed five times with chilled PBS and incubated with 50 [lI of 125I-labeled protein A (approximately 105 cpm) at 4°C overnight. Unbound radioactivity was removed with five washes of chilled PBS, and the bound fraction was quantitated with a gamma counter. Table 1 shows decreased expression of MHC class I and increased expression of the JHMV S glycoprotein 10 h postinfection (p.i.). No expression of I-Ad was detected. In vivo treatment with anti-N MAb protects mice from lethal coronavirus infection, perhaps by antibody-dependent cellular cytotoxicity response (18, 23, 24) . Consistent with the binding of anti-N antibody to the surface of infected DBT cells (24), a determinant defined by MAb J.3.3 (7) was detected on JHMV-infected J774.1 cells, while binding of another anti-N MAb (J.3.5) was not detected (Table 1) . We have recently mapped the binding domains for a panel of anti-N MAbs, including J.3.3 and J.3.5 (30). J.3.3 binds to the acidic carboxy terminus of the N protein, while J.3.5 binds to a more basic internal domain (amino acids 249 to 277) (30). The detailed mechanism of J.3.3 binding is not clear; however, the rapid cleavage of the carboxy-terminal portion of the N protein during infection (unpublished data) and the expression of this epitope on the surface of infected cells provide a possible explanation for the protection of mice from coronavirus infection mediated by anti-N MAb (18, 23, 24) . To examine the down regulation of MHC class I during the 6to 10-h incubation used for CTL assays (31, 32, 43) , the kinetics of down regulation of MHC class I was compared with the increased expression of the JHMV S glycoprotein on the cell surface. There was no detectable loss of MHC expression for the first 6 h p.i. (Fig. 1) . However, expression of all three H-2d MHC class I molecules was decreased by 8 h p.i. In contrast to decreased MHC class I expression, the expression of the JHMV S protein on the cell surface of infected J774.1 cells was clearly detectable at 8 h p.i. and increased with time following JHMV infection. These data contrast with results showing up regulation of MHC class I expression on primary astrocytes, oligodendroglia, and brain endothelial cells (14, 17, 34) . Differences among cell types might account for this 6815
doi:10.1128/jvi.68.10.6815-6819.1994 fatcat:mb7b2u4ayjhrvcwdb2jixqm37a