Cell fusion induced by infection with mouse hepatitis virus strain A59 (MHV-A59) varied markedly in extent and time course in four different murine cell lines. When inoculated at a multiplicity of 3 to 5 PFU per cell, the Sac-, L2, and DBT cell lines began to fuse by 7 h, were fused into confluent syncytia by 9 to 12 h, and peeled from the substrate by 10 to 14 h. These virulent virus-cell interactions were in striking contrast to the moderate interaction of MHV-A59 with the 17 Cl 1 cell line, in which only small syncytia were observed 18 h postinoculation, and >50% of the cells remained unfused by 24 h. The yield of infectious virus produced by 17 Cl 1 cells was 10-fold higher than the yields from the other three cell lines. The processing of the nucleocapsid protein, the membrane glycoprotein El, and the peplomeric glycoprotein E2 were found to differ significantly in the four cell lines. Since the E2 glycoprotein is responsible for virus-induced cell fusion, we attempted to correlate differences in cellular processing of E2 with differences in fusion of infected cells. The predominant intracellular form of E2 in all cell lines was the 180K species. Pulse-chase experiments showed that a small portion of the 17 Cl 1 cell-associated 180K E2 was cleaved by 1 h after synthesis to yield 90K E2, shown in the preceding paper to consist of two different glycoproteins called 90A and 90B (L. S. Sturman, C. S. Ricard, and K. V. Holmes, J. Virol. 56:904-911, 1985). This cleavage occurred shortly before the release of virions from cells, as shown by pulse-chase experiments. After budding at intracellular membranes, virions released into the medium by the four cell lines contained different ratios of 180K to 90K E2. Virions from Saccells, which contained 100% 90K E2, fused L2 cells rapidly without requiring virus replication, whereas virions from 17 Cl 1 cells, which had 50% 90K E2, required trypsin activation to induce rapid fusion (Sturman et al., J. Virol. 56:904-911, 1985). The addition of protease inhibitors to the medium markedly delayed L2 cell fusion induced by MHV infection. The extent of coronavirus-induced cell fusion does not depend solely upon the percent cleavage of the E2 glycoprotein by cellular proteases, since extensive fusion was induced by infection of L2 and DBT cells but not 17 Cl 1 cells, although all three cell lines cleaved E2 to the same extent. Differences observed between the molecular weights of the E2 cleavage products in several cell lines could result from host cell-dependent differences in glycosylation or cleavage of E2. Such changes in E2 processing could affect the cell-fusing activity of the glycoprotein. Cell lines also differ in susceptibility to the immediate cell-fusing effects of concentrated MHV (Sturman et al., J. Virol. 56:904-911, 1985). Thus, host-dependent differences in the precise location of the cleavage site of E2, the rate of transport of cleaved E2 to the cell membrane, or the response of the cell membranes to the fusing effects of cleaved E2 may also determine the extent...

The receptor for mouse hepatitis virus strain A59 (MHV-A59) is a 110to 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 48to 58-kDa protein from livers of MHVsusceptible 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 48to 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 MIHV-A59 may be due to absence or mutation of the virus-binding domain in the nonfunctional receptor homolog in SJL/J mice. Mouse hepatitis virus (MHV) is a murine coronavirus which causes inapparent enteric or respiratory infection, diarrhea, hepatitis, and acute and chronic demyelinating diseases of the central nervous system (33). Classic studies by Bang and co-workers showed that mouse strains differ markedly in their susceptibility to MHV and that mouse strain susceptibility correlates with permissiveness for MHV replication in peritoneal macrophages cultured from the different mouse strains (2). A single recessive gene, hv-1, was associated with resistance of C3H mice to MHV-2 (34). A wide variety of mouse strains has been tested for susceptibility to several of the many strains of MHV which differ in virulence and tissue tropism (1, 3, 8-10, 16, 18, 19, 26, 27). SJL/J mice and/or peritoneal macrophages cultured from them are highly resistant to infection with the neurotropic strain MHV-JHM and the hepatotropic strain MHV-A59. This resistance trait has been mapped to a single recessive gene at the hv-2 locus near the svp-2 and albino loci on mouse chromosome 7 (15-17, 26). A solid-phase virusbinding assay was used to show that a receptor for MHV-A59 is present on the brush border membranes of enterocytes and hepatocytes from MHV-susceptible...

RNA-binding proteins of coronavirus MHV-A59 were identified using an RNA overlay-protein blot assay (ROPBA). The major viral RNA-binding protein in virions and infected cells was the phosphorylated nucleocapsid protein N (50K). A new 140K virus structural protein was identified as a minor RNA-binding protein both in virions and in infected cells. The 140K protein was antigenically related to N, and upon reduction, yielded only 50K N. Thus, the 140K protein is probably a trimer of N subunits linked by intermolecular disulfide bonds. Several cellular RNA-binding proteins were also detected. RNA-binding of N was not nucleotide sequence specific. Single-stranded RNA of MHV, VSV, or cellular origin, a DNA probe of the MHV leader sequence, and double-stranded bovine rotavirus RNA could all bind to N. Binding of MHV RNA was optimal between pH 7 and 8, and the RNA could be eluted in 0.1 M NaCl. The ROPBA is a useful method for the initial identification of RNA-binding proteins, such as N and the 140K protein of murine coronavirus.

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Coronaviruses, like virtually all single-stranded RNA viruses, contain r nucleocapsid (N) protein in close structural association with their genomes. Knowledge of the structure and functions of N proteins would be fundamental to an understanding of the viral life cycle and pathogenesis. We have undertaken a study of the N protein of mouse hepatitis virus (MHV) in an attempt to learn the roles of this protein in viral assembly, transcription and translation. Specifically, we would like to examine: (i) the nature of the interaction between N protein and the genome RNA; (ii) the nature of the interactions between adjacent and more distant pairs of N monomers in the helically symmetric viral nucleocapsid; and (iii) the

Spiroplasma sp. strain GNAT3597T was isolated from the biting midge genus Atrichopogon (Diptera: Ceratopogonidae). It was serologically distinct from other Spiroplasma species, groups or subgroups. Dark-field microscopy of the cells revealed the classical helical shape and subsequent transmission electron microscopy revealed cells surrounded by only a cell membrane (i.e. lacking a cell wall). Growth of strain GNAT3597T occurred in M1D medium at 30 °C. Strain GNAT3597T catabolized both glucose and arginine, but did not hydrolyse urea. The DNA G+C content of strain GNAT3597T was 29±1 mol%. Only one strain, SMCAT (Spiroplasma mirum), is serologically related to strain GNAT3597T, although the relationship is weak (positive reaction to only a 1 : 80 dilution). It is therefore proposed that strain GNAT3597T (=ATCC BAA-520T=NBRC 100390T) represents a novel species, Spiroplasma atrichopogonis sp. nov. (class Mollicutes: order Entomoplasmatales: family Spiroplasmataceae).

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