Retargeting of Coronavirus by Substitution of the Spike Glycoprotein Ectodomain: Crossing the Host Cell Species Barrier

Abstract

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,...

Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides

“…Reaction was developed with 3-amino-9-ethylcarbazole (AEC; Sigma) according to the manufacturer's instructions. Inhibition of MHV by HR peptides was tested as described above but using LR7 cells (19) rather than VERO 118 cells. Immunoperoxidase (IPOX) detection of MHV-positive cells was carried out by using a rabbit polyclonal antibody against MHV (1:300) (20) in combination with a HRP swine-anti rabbit antibody (1:300) (Dako).…”

Coronavirus avian infectious bronchitis virus

“…It has been shown for some coronaviruses that the S protein is a determinant of tissue tropism [60]. Rottier and colleagues exchanged the S protein gene of murine hepatitis virus (MHV) with that of feline coronavirus (FCoV).…”

“…The S2 polypeptides differ by less than half that amount, to an extent similar to that found in the other structural proteins (10 to 15%; (reviewed by [23])). However, some serotypes differ by approximately 50% of S1 amino acids [17,39,60]. The differences between the S1 proteins undoubtedly have a selective advantage; generally speaking, the immunity induced by inoculation with one serotype protects poorly against infection with heterologous serotypes (reviewed by Cavanagh [20]), as it is the S protein that is the major inducer of virus neutralizing (VN) antibody and protection [13,14,51,56,57,96].…”

Protection Against CTL Escape and Clinical Disease in a Murine Model of Virus Persistence

“…Targeted recombination was used to generate recombinant virus, as described elsewhere (25,26). In brief, a plasmid containing genes 3-7 of JHM was used as the substrate for synthesis of donor RNA (Fig.…”