FIGURE 4.

Revertant analysis of cavity B mutant viruses, L328A and K330A. The mutant viruses were continuously cultured on Vero cells for seven rounds (P1 to P7). The plaque morphology and sequencing chromatogram for P2 and P7 are presented for the L328A (A) and K330A (B) viruses. The engineered mutant nucleotides are underlined. C, validation of the K330A adaptive mutation. Revertant analysis of the P2 K330A virus revealed a second site mutation G840R in the RdRp domain (data not shown). For validation of G840R in restoring the replication of K330A mutation, two mutant genome-length RNAs (G840R and K330A/G840R) were transfected into BHK-21 cells. The transfected cells were monitored for viral E protein expression at 24, 48, and 72 h post-transfection. Plaque morphologies were shown for recovered viruses.