Fig 3.

Mutant 7 is severely temperature sensitive and exhibits a RNA replication defect at the nonpermissive temperatures. (A) Growth phenotypes of mutant 7 (E₁₄₈A/R₁₄₉A/E₁₅₀A) and its partial revertant variant 7r (E₁₄₈A/R₁₄₉A). The titers of lysates of the Wt and mutant 7 from the 33°C transfections (Tf) and of plaque-purified variant 7r were determined at 33°C, 37°C, and 39.5°C by plaque assay. Virus titers are shown on the left and plaque sizes on the right of the panel. For mutant 7, plaque pictures were taken from different dilutions (10⁻⁵, 10⁻², and 10⁻²) at 33°C, 37°C, and 39.5°C, respectively. (B) Analysis of RNA replication phenotype of mutant 7. The genome structure of the R-Luc reporter virus (R-PPP) is shown on top of the panel. RNA replication and encapsidation by the Wt and mutant 7 were assayed at different temperatures using the luciferase reporter virus. Transfection of RNA transcripts and luciferase assays were performed as described in Materials and Methods. R-PPP, Renilla luciferase gene fused to three domains of the PV polyprotein; Mock, no viral RNA added.