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Coronaviruses have a marked tropism for epithelial cells. In this paper the interactions of the porcine transmissible gastroenteritis virus (TGEV) and mouse hepatitis virus (MHV-A59) with epithelial cells are compared. Porcine (LLC-PK1) and murine (mTAL) epithelial cells were grown on permeable supports. By inoculation from the apical or basolateral side both TGEV and MHV-A59 were found to enter the polarized cells only through the apical membrane. The release of newly synthesized TGEV from LLC-PK1 cells occurred preferentially from the apical plasma membrane domain, as evidenced by the accumulation of viral proteins and infectivity in the apical culture fluid. In contrast, MHV was released preferentially from the basolateral membrane of mTAL cells. The apical release of TGEV and the basolateral release of MHV may explain the in vivo establishment of a local and systemic infection, respectively.

The M and S envelope glycoproteins of mouse hepatitis virus associate in the process of virus assembly. We have studied the intrinsic properties of M/S heterocomplexes by coexpressing M and S in the absence of other coronaviral proteins. The formation of M/S complexes under these conditions indicates that M and S can interact independently of other coronaviral factors. Pulse-chase analysis revealed that M and S associate in a pre-Golgi compartment. M/S complexes are efficiently transported beyond the coronavirus budding compartment to the Golgi complex. The failure to detect complexes at the surface of coexpressing cells demonstrated that they are retained intracellularly. Thus, coexpression of the envelope glycoproteins drastically affects the intracellular transport of the S protein: instead of being transported to the cell surface, S is retained intracellularly by its association with M.

The genomic organization at the 3' end of canine coronavirus (CCV) and feline enteric coronavirus (FECV) was determined by sequence analysis and compared to that of feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) of swine. Comparison of the latter two has previously revealed an extra open reading frame (ORF) at the 3' end of the FIPV genome, lacking in TGEV, now designated ORF 6b. Both CCV and FECV possess 6b-related ORFs. The CCV ORF 6b is colinear with that of FIPV, but the predicted amino acid sequences are only 58% identical. The FECV ORF 6b contains a large deletion compared to that of FIPV, reducing the colinear part to 60%. The sequence homologies were highest between CCV and TGEV on the one hand and between FECV and FIPV on the other. The expression product of the CCV and the FECV ORF 6b can be detected in infected cells by immunoprecipitation.

A new protein of the feline infectious peritonitis virus (FIPV) was discovered in lysates of infected cells. Expression of the gene encoding open reading frame (ORF) 6b of FIPV in recombinant vaccinia virus infected cells was used to identify it as the 6b protein. It is a novel type of viral glycoprotein whose function is not clear. It is a soluble protein contained in microsomes; its slow export from the cell is caused by the presence of an ER-retention signal at the C-terminus. This amino acid sequence, KTEL, closely resembles the consensus KDEL-signal of soluble resident ER proteins. A mutant 6b protein with the C-terminal sequence KTEV became resistant to digestion by endo-beta-N-acetylglucosaminidase H with a half-time that was reduced threefold. In contrast, a mutant with the sequence KDEL was completely retained in the ER. The FIPV 6b protein is the first example of a viral protein with a functional KDEL-like ER-retention signal.

At the April 1992, mid-term meeting of the International Committee on Taxonomy of Viruses (ICTV) a proposal from the Coronaviridae Study Group (CSG) to include the torovirus genus in the Coronaviridae was accepted. Following another proposal, the arterivirus genus was removed from the Togaviridae but not assigned to another family. The arteriviruses have some features in common with the Coronaviridae but also have major differences. After much debate, culminating in September 1992, it was decided that the CSG would not recommend inclusion of arterivirus in the Coronaviridae. It was agreed that (a) the nomenclature used for coronavirus genes, mRNAs and polypeptides (Cavanagh et al., 1990) should be used for toroviruses, (b) that the small (about 100 amino acids) membrane-associated protein, which is distinct from the integral membrane glycoprotein M, associated with virions of infectious bronchitis (Liu & Inglis, 1991) and transmissible gastroenteritis (Godet et al., 1992) coronaviruses would be referred to by the acronym sM (lower case 's') and (c) that 'pol' (polymerase) should be used as a working term for gene 1, which comprises open reading frames (ORFs) 1a and 1b in both genera of the Coronaviridae.

Using different approaches we have demonstrated the formation of a complex between the S protein and the M protein in the process of mouse hepatitis virus (MHV) assembly. Preservation of the M/S heterocomplexes was critically dependent on the solubilization conditions. Pulse-chase labeling of MHV-infected cells followed by a co-immunoprecipitation assay revealed that newly synthesized S and M engage in complex formation with different kinetics, the S protein reacting much slower. Sedimentation experiments showed the M/S heteromultimer complexes to be very large. A model for the role of the complex formation in MHV assembly is presented.

The gene encoding the fusogenic spike protein of the coronavirus causing feline infectious peritonitis (FIPV) was recombined into the genome of vaccinia virus, strain WR. The recombinant induced spike protein specific, in vitro neutralizing antibodies in mice. When kittens were immunized with the recombinant, low titers of neutralizing antibodies were obtained. After challenge with FIPV, these animals succumbed earlier than the vWR-immunized control group ("early death syndrome").

In this paper we demonstrate that the organization of the polymerase gene of toroviruses and coronaviruses is similar. The polymerase gene of both virus families consists of at least two large ORFs (1a and 1b). Four domains of conserved amino acid sequences have been identified in nearly identical positions in the 3' ORF of the pol gene of toroviruses and coronaviruses. The most 3' conserved domain which is still unique for these viruses encodes a 33-kDA protein in MHV-A59, which is cleaved from a precursor protein. Expression of ORF1b of the pol gene of both virus families occurs by ribosomal frameshifting. A predicted stem-loop structure and pseudoknot are conserved in the ORF1a/ORF1b overlap of toro- and coronaviruses. On the basis of these results we postulate that toro- and coronaviruses are ancestrally more related to each other than to other families of positive stranded RNA viruses.