Fig 4.
Quasi-infectious mutants 1 and 11 are defective in RNA replication. (A) Growth phenotypes of mutants 1 and 11. At the time of full CPE (passage 2 with mutant 11; passage 1 with mutant 1), the transfection lysates at 33°C were collected and the virus titers were determined by plaque assay at 33°C, 37°C, and 39.5°C. Virus titers are illustrated on the left side and plaque sizes on the right side of the panel. For mutants, plaque pictures are taken from different dilutions at different temperatures as follows: for mutant 1, 10−5, 10−6, and 10−5 at 33°C, 37°C, and 39.5°C, respectively; and for mutant 11, 10−5, 10−3, and 10−3 at 33°C, 37°C, and 39.5°C, respectively. (B) HeLa R19 cells were transfected with 5 μg of Wt or mutant Renilla luciferase reporter virus RNA transcripts in both the presence and absence of 2 mM GnHCl, and then they were subjected to passage once at 33°C on HeLa cells. Luciferase activities were determined as described in Materials and Methods. Mock, no viral RNA added.