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
). Subsequent to budding, it may acquire a spherical, possibly icosahedral superstructure (43
). 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
) and the product of the internal open reading frame of the N gene (I protein) (12
), 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 amino-terminal 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
). The most prominent virion protein, S, makes a single pass through the membrane envelope, with almost the entire molecule forming an amino-terminal ectodomain. Multimers of S make up the large peplomers, characteristic of coronaviruses, that recognize cellular receptors and mediate fusion to host cells.
Although the details of the coronavirus assembly process are not yet understood, major progress in elucidating the molecular interactions that determine the formation and composition of the virion envelope has been made in the past few years. Much of this has been driven by the demonstration that in the absence of viral infection, coexpression of the M, E, and S proteins results in the assembly of coronavirus-like particles (VLPs) that are released from cells (4
). The VLPs produced in this manner form a homogeneous population that is morphologically indistinguishable from normal virions. This finding, i.e., that coronavirus assembly does not require the active participation of the nucleocapsid, defined a new mode of virion budding. Furthermore, the coexpression system was used to show that S protein is also dispensable in the assembly process; only the M and E proteins are required for VLP formation (4
). This observation accorded well with earlier studies that noted the release of spikeless, noninfectious virions from mouse hepatitis virus (MHV)-infected cells treated with the glycosylation inhibitor tunicamycin (21
The VLP assembly system has provided a valuable avenue to begin exploring the roles of individual proteins in coronavirus morphogenesis (2
), leading to conclusions that, in some cases, have been complemented and extended by the construction of viral mutants (7
). One of many critical questions to be resolved is the nature of the apparently passive and optional participation of S protein in the budding process. Clearly, the S protein, although not required for virus assembly, is essential for virus infectivity. Abundant evidence points to the existence of specific interactions between the M and S proteins that are initiated after successful folding of the latter in the endoplasmic reticulum (36
). S multimers must somehow fit specifically into the interstices of the arrays of M (or M and E) monomers without contributing much to their overall stability.
To investigate which residues of S are involved in this association, VLPs were assembled from components of MHV and feline infectious peritonitis virus (FIPV) (15a
). MHV and FIPV belong to two different groups of coronaviruses, and each is highly specific for its corresponding host species. The S proteins of MHV and FIPV, with 1,324 and 1,452 residues, respectively, have only 26% overall amino acid identity, with their greatest divergence occurring in the amino-terminal half of each molecule (6
). They recognize different receptors: members of the murine biliary glycoprotein family for MHV (10
) and feline aminopeptidase N (fAPN) for FIPV (19
). Moreover, the locus of the receptor binding site varies for each, mapping in the amino-terminal 330 residues for the MHV S protein (29
) but within amino acids 600 to 676 for the FIPV S protein, by analogy to the highly conserved S protein of porcine transmissible gastroenteritis virus (16
). An additional point of difference is that during maturation the MHV S protein is proteolytically cleaved into two moieties of roughly equal size whereas the FIPV S protein remains intact. It was learned from experiments with the coexpression system that while the FIPV S protein could assemble into homologous FIPV VLPs, it could not be incorporated into heterologous VLPs formed by the MHV M and E proteins. By contrast, a chimeric S protein, composed of the entire ectodomain of FIPV S linked to the transmembrane domain and short carboxy-terminal cytoplasmic tail of MHV S, was fully able to be incorporated into MHV VLPs (15a
). In addition, the reciprocal construct, having the MHV S ectodomain linked to the FIPV transmembrane domain and cytoplasmic tail, was incorporated into FIPV VLPs. From these results, it could be concluded that the transmembrane and endodomains of a given S protein contain sufficient information for assembly into VLPs of the same species.
It remained to be resolved whether this principle would apply to the complete MHV virion and whether a heterologous S ectodomain in this context would still be functional in receptor binding and membrane fusion. To determine this, we sought to obtain a viable MHV mutant containing the equivalent FIPV-MHV chimeric S protein. Through targeted RNA recombination (13
) and selection on cells of the heterologous species, we were able to construct such a recombinant. The resulting chimeric virus (designated fMHV) had the host range characteristics that would be predicted for this type of mutant: it was able to grow in feline cells, and it was no longer able to grow in murine cells. The availability of fMHV is an important first step toward identification of the specific molecular interactions allowing S protein participation in the viral assembly process and toward our understanding of the principles governing viral particle formation.