Reduced incorporation of SARS-CoV spike protein into viral particles due to amino acid substitutions within the receptor binding domain.

Cell clone #21 is a long-term producer of the infectious SARS-coronavirus, although the incorporation rate of spike (S) protein into virions is significantly lower. Sequencing analysis of the viral structural proteins revealed four and one amino acid substitutions in the S and membrane (M) proteins, respectively. We demonstrated, using a viral-like particle formation system, that the S mutations were involved in the lower incorporation of the S protein into virions, although the M mutation that disrupts the glycosylation was not present in this phenotype. Further mutational experiments identified two substitutions, Y442C and L472F, within the receptor binding domain that could be critical for the reduced S incorporation, as well as reduced binding affinity between the S protein and ACE2 receptor. Thus, these two amino acid substitutions might lead to a conformational change in the S protein, resulting in reduced incorporation into viral particles.