Acquisition of cell-cell fusion activity by amino acid substitutions in spike protein determines the infectivity of a coronavirus in cultured cells

Abstract

Coronavirus host and cell specificities are determined by specific interactions between the viral spike (S) protein and host cell receptor(s). Avian coronavirus infectious bronchitis (IBV) has been adapted to embryonated chicken eggs, primary chicken kidney (CK) cells, monkey kidney cell line Vero, and other human and animal cells. Here we report that acquisition of the cell-cell fusion activity by amino acid mutations in the S protein determines the infectivity of IBV in cultured cells. Expression of S protein derived from Vero- and CK-adapted strains showed efficient induction of membrane fusion. However, expression of S protein cloned from the third passage of IBV in chicken embryo (EP3) did not show apparent syncytia formation. By construction of chimeric S constructs and site-directed mutagenesis, a point mutation (L857-F) at amino acid position 857 in the heptad repeat 1 region of S protein was shown to be responsible for its acquisition of the cell-cell fusion activity. Furthermore, a G405-D point mutation in the S1 domain, which was acquired during further propagation of Vero-adapted IBV in Vero cells, could enhance the cell-cell fusion activity of the protein. Re-introduction of L857 back to the S gene of Vero-adapted IBV allowed recovery of variants that contain the introduced L857. However, compensatory mutations in S1 and some distant regions of S2 were required for restoration of the cell-cell fusion activity of S protein carrying L857 and for the infectivity of the recovered variants in cultured cells. This study demonstrates that acquisition of the cell-cell fusion activity in S protein determines the selection and/or adaptation of a coronavirus from chicken embryo to cultured cells of human and animal origins.

Figure 1. Summary of amino acid substitutions in the IBV S genes from chicken embryo- and cell culture-adapted IBV Beaudette strains.

a. Schematic diagram of the IBV S gene structure and functional domains. Also shown are amino acid substitutions in the S protein derived from IBV strains EP3 (accession No. AAY24433), p7 (accession No. AAY21245), p65 (accession No. AAY24433) and CK (accession No. CAC39114). Black dots indicate the identified amino acid positions that affect the cell–cell fusion activity of S protein in this study. b. Comparison of amino acid sequences in the heptad repeat I region of S proteins from coronavirus feline infectious peritonitis virus (FIPV), transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), human coronavirus 229E (HCoV 229E), human coronavirus NL63 (HCoV NL63), SARS-CoV, human coronavirus HKU1 (HCoV HKU1), bovine coronavirus (BCoV), mouse hepatitis virus (MHV) A59, MHV JHM, IBV strains EP3, p65 and Beau-CK.

Figure 2. Induction of cell–cell fusion by S(EP3) and S(CK) constructs.

a. Western blot analysis of cells expressing IBV N (lane 1), S(EP3) (lane 2) and S(CK) (lane 3). Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated constructs. Cells were harvested at 12 hours post-transfection and lysates prepared. The viral protein expression was analyzed by Western blot with rabbit anti-IBV S and anti-IBV-N polyclonal antibodies. The same membrane was also probed with anti-β-tubulin monoclonal antibody as a loading control. b. Detection of cell–cell fusion by indirect immunofluorescence. Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 12 hours post-transfecion, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-IBV S and anti-IBV N polyclonal antibodies. c. Cell surface expression of S(EP3) and S(CK) constructs. Vero cells were infected with the vaccinia/T7 recombinant virus and transfected with the indicated constructs. At 12 hours post-transfecion, cells were either stained directly with 1∶100 diluted rabbit anti-IBV S polyclonal antibodies (panels A, B and C), or permeabilized with 0.1% saporin following by staining with the same antibodies (panels D, E and F). The cells were then incubated with 1∶20 diluted FITC-conjugated swine anti-rabbit antibody, fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry.

Figure 3. Acquisition of the cell–cell fusion activity by L857-F mutation in the heptad repeat 1 region and mutational analysis of the L857 residue.

a. Schematic diagram of various wild type and mutant S constructs as well as several chimeric constructs used in this study. b. Western blot analysis of cells expressing wild type, mutants, and chimeric IBV S constructs. Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated constructs. Cells were harvested at 12 hours post-transfection and lysates prepared. The viral protein expression was analyzed by Western blot with rabbit anti-IBV S antibodies. The same membrane was also probed with anti-β-tubulin monoclonal antibody as a loading control. c. Detection of cell–cell fusion by indirect immunofluorescence. Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated S constructs. At 12 hours post-transfecion, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-IBV S polyclonal antibodies.

Figure 4. Analysis of the effect of L857 residue on IBV infectivity and growth properties by re-introduction of F857-L back into the genome of Vero-adapted IBV p65.

a. Comparison of the plaque sizes of wild type IBV (IBV), wild type recombinant IBV (rIBV) and variant 4 (rFLv4). Confluent monolayers of Vero cells grown on 6-well plates were infected with wild type IBV (IBV), wild type recombinant IBV (rIBV) and variant 4 (rFLv4), and incubated in the presence of 0.5% carboxymethy cellulose. At 2 days post-infection, cells were washed, fixed with 4% formaldehyde, and stained with 0.1% toluidine blue. b. Comparison of the growth curves of wild type IBV (IBV), wild type recombinant IBV (rIBV) and variant 4 (rFLv4). Vero cells were infected with viruses, harvested at 0, 4, 8, 12, 18, 24, 30, 36 and 48 hours post-infection, and TCID50 was determined. c. Analysis of S protein expression in cells infected with wild type IBV (IBV), wild type recombinant IBV (rIBV) and variant 4 (rFLv4). Vero cells infected with the indicated viruses with the same MOI were harvested at 16 hours post-infection. The viral protein expression was analyzed by Western blot with rabbit anti-IBV S antibodies. The same membrane was also probed with anti-β-tubulin monoclonal antibody as a loading control.

Figure 5. Induction of cell–cell fusion by various L857-containing S constructs.

a. Western blot analysis of cells expressing L857-containign S constructs, S(FL), S(FLv1), S(FLv2), S(FLv3) and S(FLv4). Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated constructs. Cells were harvested at 12 hours post-transfection and lysates prepared. The viral protein expression was analyzed by Western blot with rabbit anti-IBV S antibodies. The same membrane was also probed with anti-β-tubulin monoclonal antibody as a loading control. b. Detection of cell–cell fusion by indirect immunofluorescence. Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated S constructs. At 12 hours post-transfecion, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-IBV S polyclonal antibodies.

Figure 6. Further enhancement of the cell–cell fusion activity by G405-D mutation in the S1 region.

a. Western blot analysis of cells expressing S(EP3), S(CK), S(p7), S(p65), S(EP3(G405D)) and S(CK(G405D)) constructs. Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated constructs. Cells were harvested at 12 hours post-transfection and lysates prepared. The viral protein expression was analyzed by Western blot with rabbit anti-IBV S antibodies. The same membrane was also probed with anti-β-tubulin monoclonal antibody as a loading control. b. Detection of cell–cell fusion by indirect immunofluorescence. Vero cells were infected with vaccinia/T7 recombinant virus and transfected with the indicated S constructs. At 12 hours post-transfecion, cells were fixed with 4% paraformaldehyde and stained with rabbit anti-IBV S polyclonal antibodies.

Figure 7. Diagram showing a two-step adaptation process of chicken embryo-adapted IBV to Vero cells.

Also shown are the numbers of amino acid changes during each adaptation process. ^aThe accession No. for S genes from EP3 is DQ001338, p7 is DQ001337, p65 is DQ001339. ^bThe accession No. for this Vero-adapted strain is AAV98206. ^cThe accession No. for these two strains are CAC39114 and CAC39300.