FIG. 2.

Failure of the NS2Aα QST mutant to produce infectious virus. (A) Infectious-center and infectivity assays. WT or NS2Aα QST RNA transcripts were electroporated into BHK cells. Serial 10-fold dilutions were seeded on 35-mm-diameter dishes in the presence of the respective untransfected cells and overlaid with agarose. After 3 days, cells were fixed and crystal violet staining was performed. Supernatants from parallel liquid cultures were used to perform a plaque assay on new BHK cells. In addition, total cellular RNA was extracted from parallel cultures of reinfected cells and used for RT-PCR analysis. (B) RNA analysis. The region containing the NS2Aα cleavage site sequence was amplified from total cellular RNAs from the experiment described in panel A. As a control for DNA carryover, parallel reactions without reverse transcriptase (RT) were performed (lanes 2 and 4). Amplifications from the WT full-length plasmid (pWT) or the mutant full-length plasmid (pQST) served as positive controls (lanes 6 and 7). Lane M, DNA size markers (molecular sizes are in kilobases on the left). Control, no-template control.