Structure-based identification of functional residues in the nucleoside-2'-O-methylase domain of Bluetongue virus VP4 capping enzyme

Stewart, Meredith E.; Roy, Polly

The mutation of non-catalytic residues either singly or in combination indirectly affected 2 0 -O MTase activity in vitro, therefore, we investigated the biological significance of these residues on BTV replication. The same mutations as described were introduced at each position (N311, Y334 or R367) either singly or in combination (NYR) into the S4 genomic clone and reverse genetics system used to recover each mutant virus. Unlike the mutants of the catalytic residue (D265E or D265V), all other mutant viruses were recovered from normal BSR cells (Fig. 7A) . The plaque morphology of the viruses with Y334A and R367A mutations were comparable to WT virus, while N311A and NYR mutation viruses exhibited smaller plaque sizes (Fig. 6A) . Development of plaques was slowest in the virus with a triple mutation (NYR) taking up to 5 days p.i. to yield pinhead sized plaques (data not shown), significantly smaller than the normal plaque size of WT at 3 days p.i. (Fig. 7A) . Although, there was a distinct difference in the plaque size, sequence analysis of the genomic S4 segment of the mutant viruses after 10 passages demonstrated that the mutant viruses were stable.