Figure 14.

RT-PCR analysis of 3CL cleavage site mutants. Honey bee pupae transfected with synthetic RNA from DWV strain 1414 (rDWV 1414), a mutant genome of DWV with an exchange of the active cysteine within the 3CL protease (rDWV-C₂₃₀₇A), a mutant genome with an exchange of the 3BCL cleavage site (rDWV-Q₂₁₁₈A), a mutant genome with an exchange of the 3CDL cleavage site (rDWV-Q₂₃₉₃A), and a mutant genome with a deletion of the complete 3DL (rDWV-Δpol) were analyzed in RT-PCR. DWV RNA-transfected and mock-transfected control pupae were harvested three days post-transfection (p. t.). The pupae were homogenized, free synthetic RNA was digested with benzonase and total digestion protected RNA was prepared. A 450 bp amplicon of the DWV genome was amplified in a 40-cycles RT-PCR end-point assay. No amplification product became visible in the mock-transfected pupa, the N-terminal and the C-terminal 3CL cleavage site mutants and in the case of the mutant genome with a deletion of the RdRp gene. In contrast, the specific amplicon with a length of 450 bps appeared in the RT-PCR positive control and in the wild-type DWV genome-transfected pupa. Surprisingly, we also detected a weaker band of 450 bps in the 3CL cysteine mutant rDWV-C₂₃₀₇A indicating residual genome replication and/or RNA encapsidation.