FIG 6.

RNA Pol mutations. (A) Mutations in RPO147 and RPO132 that occurred during passages of MYXA8 were mapped onto the S. cerevisiae RNAP II crystal structure. Green, RPB1; blue, RPB2. (B) Single nucleotide mutations were inserted by homologous recombination into RPO147 and RPO132 of MYXA8 P0 viruses. Plaques formed by the cloned recombinant viruses and a MYXA8 P0 virus are shown. (C) Virus yields at 2, 24, 48, and 72 h after infection with 0.01 PFU/cell of WT, MYXA8 P0 clone A (MYX-A8-A), MYXA8 P0 clone C (MYX-A8-C), and the recombinant viruses with single nucleotide mutations in RPO147 (M424I) or RPO132 (K1046Q). (D) Replication competition between the MYXA8 P0 clone A virus and the recombinant virus with mutation in RPO147. BS-C-1 cells were infected in triplicate with 0.01 PFU/cell of a mixture comprised of 90% MYXA8 P0 and 10% MYXA8 with the M424I mutation in RPO147. After each of three rounds of passaging, an aliquot was removed for Sanger sequencing of the region including the M424I mutation. Graphs of the sequence are shown with a dotted oval around the mutated nucleotide. The approximate percentages of M424I after the third passages are indicated at the right.