Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxyterminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.The family Coronaviridae contains the causative agents of a number of significant respiratory and enteric diseases affecting humans, other mammals, and birds (55). One of the hallmarks of this family is that most of its members exhibit a very strong degree of host species specificity, the molecular basis of which is thought to reside in the particularity of the interactions of individual viruses with their corresponding host cell receptors.Coronaviruses have positive-stranded RNA genomes, on the order of 30 kb in length, that are packaged by a nucleocapsid protein (N) into helical ribonucleoprotein structures (31). The nucleocapsid is incorporated into viral particles by budding through the membrane of the intermediate compartment between the endoplasmic reticulum and the Golgi complex (26, 57). Subsequent to budding, it may acquire a spherical, possibly icosahedral superstructure (43,44). The virion envelope surrounding the nucleocapsid contains a minimal set of three structural proteins: the membrane glycoprotein (M), the small envelope protein (E), and the spike glycoprotein (S). In some coronaviruses, other proteins may also be present; these include a hemagglutinin-esterase (HE) (34, 54) and the product of the internal open reading frame of the N gene (I protein) (12, 53), neither of which is essential for virus infectivity.M is the most abundant of the virion structural proteins. It spans the membrane bilayer three times, having a short aminoterminal domain on the exterior of the virus and a large carboxy terminus, containing more than half the mass of the molecule, in the virion interior (48). By contrast, E is a minor structural protein, in both size and stoichiometry, and was only relatively recently identified as a constituent of viral particles (17,33,...
Coronavirus-like particles morphologically similar to normal virions are assembled when genes encoding the viral membrane proteins M and E are coexpressed in eukaryotic cells. Using this envelope assembly assay, we have studied the primary sequence requirements for particle formation of the mouse hepatitis virus (MHV) M protein, the major protein of the coronavirion membrane. Our results show that each of the different domains of the protein is important. Mutations (deletions, insertions, point mutations) in the luminal domain, the transmembrane domains, the amphiphilic domain, or the carboxy-terminal domain had effects on the assembly of M into enveloped particles. Strikingly, the extreme carboxy-terminal residue is crucial. Deletion of this single residue abolished particle assembly almost completely; most substitutions were strongly inhibitory. Site-directed mutations in the carboxy terminus of M were also incorporated into the MHV genome by targeted recombination. The results supported a critical role for this domain of M in viral assembly, although the M carboxy terminus was more tolerant of alteration in the complete virion than in virus-like particles, likely because of the stabilization of virions by additional intermolecular interactions. Interestingly, glycosylation of M appeared not essential for assembly. Mutations in the luminal domain that abolished the normal O glycosylation of the protein or created an N-glycosylated form had no effect. Mutant M proteins unable to form virus-like particles were found to inhibit the budding of assembly-competent M in a concentration-dependent manner. However, assembly-competent M was able to rescue assembly-incompetent M when the latter was present in low amounts. These observations support the existence of interactions between M molecules that are thought to be the driving force in coronavirus envelope assembly.
The coronavirus membrane (M) protein is the most abundant virion protein and the key component in viral assembly and morphogenesis. The M protein of mouse hepatitis virus (MHV) is an integral membrane protein with a short ectodomain, three transmembrane segments, and a large carboxy-terminal endodomain facing the interior of the viral envelope. The carboxy terminus of MHV M has previously been shown to be extremely sensitive to mutation, both in a virus-like particle expression system and in the intact virion. We have constructed a mutant, M⌬2, containing a two-amino-acid truncation of the M protein that was previously thought to be lethal. This mutant was isolated by means of targeted RNA recombination with a powerful host range-based selection allowed by the interspecies chimeric virus fMHV (MHV containing the ectodomain of the feline infectious peritonitis virus S protein). Analysis of multiple second-site revertants of the M⌬2 mutant has revealed changes in regions of both the M protein and the nucleocapsid (N) protein that can compensate for the loss of the last two residues of the M protein. Our data thus provide the first genetic evidence for a structural interaction between the carboxy termini of the M and N proteins of MHV. In addition, this work demonstrates the efficacy of targeted recombination with fMHV for the systematic genetic analysis of coronavirus structural protein interactions.The assembly of progeny virions of coronaviruses is brought about by cooperative interactions among a minimal set of four structural proteins, the large positive-stranded RNA genome, and a membrane envelope acquired from the intermediate compartment between the endoplasmic reticulum and the Golgi complex (15,17,49). The viral nucleocapsid is formed by the packaging of the genome by the nucleocapsid (N) protein into a helical ribonucleoprotein structure (19) that is further compacted into a core possibly having icosahedral symmetry (37,38). This nucleocapsid is incorporated into virions by intracellular budding through a membrane containing three envelope proteins: the membrane (M) glycoprotein, the small envelope (E) protein, and the spike (S) glycoprotein.Although the details of the coronavirus assembly process are not well understood, in recent years tremendous progress has been made in elucidating the molecular interactions that determine the formation of the virion envelope, particularly for the prototype murine coronavirus mouse hepatitis virus (MHV). Much of this progress has been realized by the study of virus-like particles (VLPs) formed by coexpression of the coronavirus M and E proteins, which has defined the minimal molecular interactions required for production of particles resembling authentic virions (3, 52). This system has provided an invaluable tool leading to detailed exploration of the roles of individual proteins and intermolecular interactions in the coronavirus assembly process (2-4, 6-8, 11, 52).One outcome of the MHV VLP work confirmed a previous conclusion, based on studies with the glycosylati...
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