A single amino acid mutation in the spike protein of coronavirus infectious bronchitis virus hampers its maturation and incorporation into virions at the nonpermissive temperature

Abstract

The spike (S) glycoprotein of coronavirus is responsible for receptor binding and membrane fusion. A number of variants with deletions and mutations in the S protein have been isolated from naturally and persistently infected animals and tissue cultures. Here, we report the emergence and isolation of two temperature sensitive (ts) mutants and a revertant in the process of cold-adaptation of coronavirus infectious bronchitis virus (IBV) to a monkey kidney cell line. The complete sequences of wild type (wt) virus, two ts mutants, and the revertant were compared and variations linked to phenotypes were mapped. A single amino acid reversion (L294-to-Q) in the S protein is sufficient to abrogate the ts phenotype. Interestingly, unlike wt virus, the revertant grows well at and below 32 degrees C, the permissive temperature, as it carries other mutations in multiple genes that might be associated with the cold-adaptation phenotype. If the two ts mutants were allowed to enter cells at 32 degrees C, the S protein was synthesized, core-glycosylated and at least partially modified at 40 degrees C. However, compared with wt virus and the revertant, no infectious particles of these ts mutants were assembled and released from the ts mutant-infected cells at 40 degrees C. Evidence presented demonstrated that the Q294-to-L294 mutation, located at a highly conserved domain of the S1 subunit, might hamper processing of the S protein to a matured 180-kDa, endo-glycosidase H-resistant glycoprotein and the translocation of the protein to the cell surface. Consequently, some essential functions of the S protein, including mediation of cell-to-cell fusion and its incorporation into virions, were completely abolished.

Fig. 1

Analysis of the expression of the S protein from wt (wt6501), ts mutants (ts291602 and 282902) and revertant (rev-1) viruses. (a) Cells were infected with each virus at 32 °C and 40 °C as indicated on the top for 1 h and were maintained at the same temperatures. (b) Two dishes of cells were infected with each virus at 32 °C for 1 h. One of the duplicates was maintained at 32 °C (lanes 1, 3, 5, and 7) and the other one was shifted to 40 °C (lanes 2, 4, 6, and 8). Radiolabeled cell lysates were immunoprecipitated with anti-IBV antibodies. The proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. Numbers on the left indicate molecular mass in kilodalton and the position of the S protein is indicated on the right.

Fig. 2

Membrane fusion activity of the S protein expressed from wt and ts291602. (a) Vero cells were transfected with plasmids without insert (lanes 1 and 2) or with the S gene from the wt (lanes 3 and 4) and ts291602 (lanes 5 and 6), and were cultured at 32 °C (lanes 1, 3, and 5) or 40 °C (lanes 2, 4, and 6). The expression of the S protein was examined in Western blot using anti-IBV antibodies, and β-tubulin was immunostained as loading controls. (b) The membrane-fusion activity of the S protein from the wt (panels B, E, and H) and ts mutant (panels C, F, and I) at 32 °C (panels B and C) and 40 °C (panels C and F) 2 days post-transfection was compared. Panels H and I show induction of membrane fusion on cells transfected with the wt and ts291602 by shifting cells shown in panels E and F to 32 °C for 1 day. Panels A, D, and G show cells transfected with empty plasmids.

Fig. 3

Maturation defects of the S protein from the ts mutant. (a) Endo-H treatment of the S protein from rev-1 and ts291602. The S protein derived from rev-1 (lanes 5, 6, 7, and 8) and ts291602 (lanes 9, 10, 11, and 12) were expressed in Vero cells at 32 °C (lanes 5, 6, 9, and 10) and 40 °C (lanes 7, 8, 11, and 12), using a T7-vaccinia expression system. The radiolabeled proteins were immunoprecipitated with anti-IBV antibodies, the eluted proteins were Endo-H- (lanes 6, 8, 10, and 12) or mock-treated (lanes 5, 7, 9, and 11) and analyzed by SDS-PAGE. (b) Detection of the less matured S protein from ts291602 at 40 °C and the defect in cleavage of the mutant S protein. Cells were infected with ts291602 and rev-1 for 5 h at 32 °C, one of the duplicates was maintained at 32 °C (lanes 1, 3, 5, and 7), and the other one was shifted to 40 °C (lanes 2, 4, 6, and 8). Cells were radiolabeled and viral proteins were immunoprecipitated with anti-IBV antibodies and analyzed by SDS-PAGE. Lanes 1, 2, 3, and 4 refer to viral products detected from cell lysates and lanes 5, 6, 7, and 8 refer to viral proteins detected from virus particles released to the cultured media.

Fig. 4

Subcellular localization and translocation of the wt and ts mutant S protein in Vero cells at the permissive- and nonpermissive temperatures. Vero cells transiently expressing the wt and ts mutant S protein (as indicated on the top) were treated with cycloheximide at 3.5 h posttransfection for 30 min and were cultured at either 32 °C or 40 °C as indicated on the top. Cells were incubated with anti-IBV antibodies and then with the FITC-conjugated secondary antibodies at different time points as indicated on the left. The fluorescence was viewed using a confocal scanning Zeiss microscope.

Fig. 5

Quantitative analysis of cell surface expression of the wt and ts mutant S protein. HeLa cells expressing the empty plasmid (panels A and F), wt (panels B, D, G, and I) and ts mutant (panels C, E, H, and J) were stained directly with 1:100 diluted rabbit anti-IBV S protein polyclonal antibodies (panels A–E). The cells were also permeabilized with 0.1% saponin and stained with the same primary antiserum (panels F–J). Cells were then stained with 1:20 diluted FITC-conjugated swine anti-rabbit antibody (DAKO), fixed with 1% ice cold paraformaldehyde and analyzed by flow cytometry. Percentages indicate positive staining cells.

Fig. 6

Analysis of the structural proteins on purified ts291602 and rev-1 virions. Immunoprecipitations were performed using virions, purified from ts291602- (lanes 1 and 2) and rev-1-infected (lanes 3 and 4) Vero cells at 32 °C (lanes 1 and 3) and 40 °C (lanes 2 and 4). The viral proteins were separated on 12.5% polyacrylamide gels and detected by autoradiography. Numbers on the left indicate molecular masses in kilodalton and the positions of the three structural proteins are indicated on the right.