Fig. 2.

The effect of C547 alanine substitution on the recognition of S protein by neutralizing antibodies A2.1 and A2.3 and on viral recovery. (A) 293 T cells were transfected with the expression vector pCAGGS-S encoding the native spike protein or the amino acids substituted protein in positions 546–548 of the spike protein [pCAGGS-S (546–548)]. Twenty-four hours after transfection cells were metabolically labeled with [³⁵S]-methionine and cysteine for nine hours in 37 °C, a lysate was prepared and analyzed by immunoprecipitation with monoclonal neutralizing anti-S antibodies A2.1 and A2.3 or a polyclonal anti-S antibody B46. (B) 293 T cells were transfected with the expression vector pCAGGS-S or the expression vector encoding the amino acids substitutions in positions 546 and 548 [pCAGGS-S (546/548)], labeled with ³⁵S-methionine and cysteine and immunoprecipitated with anti-S monoclonal or polyclonal antibodies. DBT cells were infected with the recombinant MHV-A59 virus expressing the native spike protein [rA59/S (pMH54)] or the mutated spike protein [rA59/S (546/548)] and metabolically labeled with [³⁵S]- methionine and cysteine for one hour at 9 h post infection. Cell lysates were immunoprecipitated with the monoclonal neutralizing anti-S antibody A2.3. (C) Mean viral titers (left) and plaque diameters (right) of recombinant viruses expressing the native spike protein, rA59/S (pMH54), or the amino acid substituted protein and rA59/S (546/548), respectively. Error bars represent the standard deviations from the mean. S uncleaved denotes the 180 kDa uncleaved spike protein, and S cleaved denotes the 90 kDa cleaved S1 and S2 subunits.