High-throughput screen
design and execution: recombinant influenza
virus and high-throughput screen design and results. (a) Schematic
of the recombinant influenza A/WSN/33 virus expressing Renilla luciferase. The Renilla luciferase open reading
frame was inserted in the reverse orientation and complementary sense
between the 3′ and 5′ packaging sequences of the HA
segment, which contains the viral promoter and ensures correct packaging
of the recombinant segment. Due to the lack of HA ORF, this WSN-Renilla virus must be grown in an HA-complementing cell
line. Upon infection, the influenza virus polymerase recognizes the
promoter and the reporter gene is transcribed and expressed. (b) MDCK-HA
cells were plated in 1536-well plates and infected with WSN-Renilla virus (MOI = 0.05). Compounds were added 120 min
prior to infection, and expression of Renilla luciferase
was assayed 30 h later. A 50% reduction in luminescence signal was
employed as a cutoff. (c) Results from the HTS of 919,960 compounds
indicating the number of primary hits, the hits confirmed in dose–response,
and the selection of hits for revalidation.