FIG. 7.
Restoration or enhancement of the replication ability of DI9c NS5A mutants 16 to 19 cannot be explained by RNA recombination. (A) Detection level of quantitative RT-PCR. Applying primers B53 R and B42 (see Materials and Methods), RT-PCR was carried out with different amounts of in vitro-transcribed DI9c RNA. The standard reaction was carried out in parallel with the reactions shown in panel B. The detection limit was thus demonstrated to be 5 fg (corresponding 103 molecules) of DI9c RNA. (B) RT-PCR analysis of the DI9c ORF mutant RNAs after transfection into naive BHK or BHK/Hygbici cells. Mutants 15 to 19 (15 as a negative control) were transfected into normal BHK and BHK/Hygbici cells. The naive cells were harvested at 48 h p.t. (top right); the Hygbici cells were divided into two portions and harvested at 24 and 48 h p.t., respectively (bottom). RT-PCR was carried out on identical amounts of cytoplasmic RNA. Primers B49R (mutants 15 to 17) and B53R (mutants 18 and 19) were used for reverse transcription; primers B49R and B38 (mutants 15–17) and primers B53R and B42 (mutants 18 and 19) were used for the subsequent PCRs (Table 1). As a positive control, the RT-PCRs were performed on the in vitro-transcribed mutant RNAs (top left). The resulting DNA fragments, which were confirmed to comprise the expected portion of the NS5A-coding region by sequencing (data not shown), had sizes of 1.2 kb (mutants 15 to 17) and 1.1 kb (mutants 18 and 19). In addition, RT-PCR with primers B49R and B38 gave rise to an unspecific product of ca. 0.4 kb (see also control reactions). Replication of the mutant DI9c RNAs was confirmed via restriction digest of the RT-PCR products with MluI, because this restriction site was originally introduced into the mutant cDNA constructs (see Materials and Methods and control reactions). Digestion with MluI should yield the following fragments: mutant 15, 1.10 and 0.10 kb; mutant 16, 0.77 and 0.43 kb; mutant 17, 0.96 and 0.24 kb; mutant 18, 0.81 and 0.29 kb; mutant 19, 0.67 and 0.43 kb. Lanes: M, DNA length marker; italic numbers, assays performed on either in vitro-transcribed RNAs (top left) or cytoplasmic RNA fractions of BHK cells (top right) or Hygbici cells (bottom) which had been transfected with DI9c derivatives 15 to 19. Plus signs indicate that the PCR products were digested with MluI. Positions of the 0.5- and 1.0-kb marker fragments are given on the right. The MluI cleavage products are marked by dots.