Figure 15.

Western blot analysis of DWV mutants with an alanine exchange of residues potentially involved in 3CL catalysis. Honey bee pupae were transfected with synthetic RNA of DWV strain 1414 (rDWV 1414) and mutant genomes containing the amino acid exchanges H₂₁₉₀A, D₂₂₂₅A, H₂₁₇₀A, D₂₁₉₉A, and N₂₂₂₇A. Transfected and mock-transfected control pupae were harvested three days post-transfection (p. t.). The pupae were homogenized, and total bee protein was resolved via SDS-PAGE. A Western blot analysis using the anti-VP1 Mab VP1A1 showed no signal in the mock-transfected pupa and a typical VP1 pattern with bands at 47, 42, and 39 kDa in the wild-type DWV RNA-transfected pupa. The exchange of H₂₁₉₀, D₂₂₂₅, and D₂₁₉₉ against an alanine was tolerated by DWV in terms of RNA replication, virus growth, systemic infection, and detectable VP1 expression. However, rDWV-H₂₁₉₀A and also partially rDWV-D₂₂₂₅A showed an aberrant VP1 pattern with smaller bands at 28 and 25 kDa. In addition, rDWV-H₂₁₉₀A, rDWV-D₂₂₂₅A, and rDWV-D₂₁₉₉A showed weaker VP1 signals, indicating compromised viral growth. The mutations H₂₁₇₀A and N₂₂₂₇A were not tolerated by DWV, and no VP1 signal could be detected after transfection. The experiment demonstrates the importance of the identity of these two residues for 3CL function. The results of two different RNA syntheses and independent infection experiments are shown side by side in the analysis to allow for a more precise comparison to rDWV 1414 (wild-type) protein expression.