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. 2000 Feb;74(3):1393-406.
doi: 10.1128/jvi.74.3.1393-1406.2000.

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

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Free PMC article

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

L Kuo et al. J Virol. .
Free PMC article

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 carboxy-terminal 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.

Figures

FIG. 1
FIG. 1
Construction and composition of the donor RNA template for incorporation of the FIPV S gene ectodomain into MHV. Transcription vector pFM1 was derived from parent plasmid pFV1 (13) via six intermediates, including pMH49 and pMH54, as described in Materials and Methods. The chimeric FIPV-MHV S gene was shuttled into pFM1 from the subclone pGTFMS. MHV and FIPV sequences are indicated, respectively, by open and shaded rectangles. The arrow at the left end of each vector indicates the T7 promoter; the solid circle represents the polylinker between the 5′-end segment of the MHV genome (denoted 5′/1) and the 3′ region containing the structural genes, the 3′ untranslated region (denoted 3′), and the polyadenylated segment (denoted A). Restriction sites relevant to plasmid construction are shown and, unless enclosed in parentheses, are unique in the plasmid in which they appear. At the bottom are shown the sequences in pFM1: 1, between the polylinker and the HE gene fragment; 2, at the MHV-FIPV junction in the signal peptide-encoding portion of the chimeric S gene (with signal peptide residues boxed); 3, at the FIPV-MHV junction in the transmembrane domain-encoding portion of the chimeric S gene; and 4, in the region immediately downstream of the S gene. Nucleotides mutated to create restriction sites are underlined. The boundaries between MHV and FIPV sequence are indicated by short vertical lines; thicker horizontal bars between these indicate nucleotides or amino acids common to both the MHV and FIPV sequences.
FIG. 2
FIG. 2
Scheme for construction of fMHV by targeted recombination between the MHV N gene deletion mutant, Alb4 (27), and donor RNA transcribed from the plasmid pFM1. The deletion in the Alb4 N gene is shown as a discontinuity. A single crossover event anywhere within the HE gene fragment of the donor RNA should generate a recombinant, fMHV, containing both the ectodomain-encoding region of the FIPV S gene (shaded) and the wild-type MHV N gene. The recombinant should simultaneously lose the ability to infect murine cells and gain the ability to infect feline cells.
FIG. 3
FIG. 3
Growth of fMHV in feline cells. (A) Plaque-forming ability of fMHV. Monolayers of murine L2 cells or feline FCWF cells were mock infected or infected with wild-type MHV or either of two independent isolates of fMHV. Plaques were visualized at 66 h postinfection, after staining with neutral red. (B) Single-step growth kinetics of fMHV-C and FIPV in FCWF cells. Viral infectivity in culture medium at different times postinfection was determined by a quantal assay on FCWF cells, and 50% tissue culture infective doses (TCID50) were calculated.
FIG. 3
FIG. 3
Growth of fMHV in feline cells. (A) Plaque-forming ability of fMHV. Monolayers of murine L2 cells or feline FCWF cells were mock infected or infected with wild-type MHV or either of two independent isolates of fMHV. Plaques were visualized at 66 h postinfection, after staining with neutral red. (B) Single-step growth kinetics of fMHV-C and FIPV in FCWF cells. Viral infectivity in culture medium at different times postinfection was determined by a quantal assay on FCWF cells, and 50% tissue culture infective doses (TCID50) were calculated.
FIG. 4
FIG. 4
PCR analysis of fMHV recombinants. In each experiment, RT-PCR was used to amplify regions of RNA isolated from cells infected with each of four independent isolates of fMHV or two MHV controls. The controls, Alb129 (13) and Alb203 (7), are MHV mutants that were also obtained by targeted recombination between Alb4 and pFV1-related donor RNAs; both are phenotypically wild type and are isogenic with wild-type MHV in the region under analysis. PCR products were analyzed by electrophoresis in 0.8% agarose gels stained with ethidium bromide. Sizes of relevant standard (std) marker DNA fragments are indicated on the right or left of each gel. PCR primers (Table 1) used in each experiment, their loci in the MHV or fMHV genomes, and the predicted sizes of the PCR products or restriction fragments of the PCR products are indicated on the right.
FIG. 4
FIG. 4
PCR analysis of fMHV recombinants. In each experiment, RT-PCR was used to amplify regions of RNA isolated from cells infected with each of four independent isolates of fMHV or two MHV controls. The controls, Alb129 (13) and Alb203 (7), are MHV mutants that were also obtained by targeted recombination between Alb4 and pFV1-related donor RNAs; both are phenotypically wild type and are isogenic with wild-type MHV in the region under analysis. PCR products were analyzed by electrophoresis in 0.8% agarose gels stained with ethidium bromide. Sizes of relevant standard (std) marker DNA fragments are indicated on the right or left of each gel. PCR primers (Table 1) used in each experiment, their loci in the MHV or fMHV genomes, and the predicted sizes of the PCR products or restriction fragments of the PCR products are indicated on the right.
FIG. 5
FIG. 5
RNA sequence of the FIPV-MHV S gene junctions in fMHV. RNA isolated from cells infected with independent recombinants fMHV-A and fMHV-C was sequenced with a primer complementary to nt 118 to 141 of the FIPV S gene (left set, upstream junction) or a primer complementary to nt 3817 to 3837 of the MHV S gene (right set, downstream junction). For each junction, both the directly read negative-strand cDNA sequence and the inferred positive-strand RNA sequence are shown.
FIG. 6
FIG. 6
Viral proteins in fMHV-infected cells. FCWF cells infected with fMHV and, for comparison, FIPV-infected FCWF cells and MHV-infected LR7 cells were labeled for 1 h with 35S-amino acids. Immunoprecipitations were performed on aliquots of cleared lysates of these cells by using the following antibodies (Ab.): K134 rabbit serum against purified MHV-A59 (αMHV); serum G73 from a FIPV-infected cat (αFIPV); and MAb WA3.10 and 23F4.5, recognizing the ectodomains of MHV S (αSm) and FIPV S (αSf), respectively. As indicated, proteins were heated at 95°C (+) or analyzed without heating (−) in SDS–12.5% polyacrylamide gels. The positions of the S, M, and N proteins in the gel are indicated on the left for MHV and on the right for FIPV.
FIG. 7
FIG. 7
Protein composition of purified fMHV. 35S-labeled fMHV and, for comparison, similarly labeled FIPV and MHV were prepared and purified by floatation in sucrose gradients. Virus particles were subsequently affinity purified with specific antibodies and analyzed in an SDS–12.5% polyacrylamide gel. Indications are as described in the legend to Fig. 6.
FIG. 8
FIG. 8
Blocking of spike-receptor interactions. (A) Neutralization of viral infectivity. fMHV, FIPV, and MHV were preincubated with anti-MHV serum (K134) or anti-FIPV serum (G73) before being inoculated on LR7 (MHV) or FCWF cells (fMHV and FIPV). Infection was visualized at 6 h postinfection by immunofluorescence microscopy. (B) Receptor dependence of infection. mTAL and MKFA cells (mTAL cells expressing fAPN), the latter without or with treatment with antibodies to the fAPN receptor, were inoculated with fMHV, MHV, and FIPV, and infection was visualized by immunofluorescence analysis.
FIG. 8
FIG. 8
Blocking of spike-receptor interactions. (A) Neutralization of viral infectivity. fMHV, FIPV, and MHV were preincubated with anti-MHV serum (K134) or anti-FIPV serum (G73) before being inoculated on LR7 (MHV) or FCWF cells (fMHV and FIPV). Infection was visualized at 6 h postinfection by immunofluorescence microscopy. (B) Receptor dependence of infection. mTAL and MKFA cells (mTAL cells expressing fAPN), the latter without or with treatment with antibodies to the fAPN receptor, were inoculated with fMHV, MHV, and FIPV, and infection was visualized by immunofluorescence analysis.
FIG. 9
FIG. 9
Amino acid sequence alignment of the carboxy-terminal ends of the MHV and FIPV S proteins.

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