Genome-Wide RNAi Screening to Identify Human Host Factors Crucial for In fl uenza Virus Replication - RNA Interference from Biology to Therapeutics - page 245

Fig. 12.2 Outline of screening and validation procedure. A549 cells were reverse transfected with
a library of siRNAs and further incubated for 48 h to allow maximal gene knockdown. Subsequently,
cells were infected with IAV (either A/WSN/33 or the new pandemic H1N1 virus A/HH/04/09),
and 24 h (primary screen) or 36 h (secondary screen) later virus replication was quantified using
different techniques. The infection rate within transfected and infected A549 cells was detected
using immunofluorescence and automated microscopy. The numbers of infectious virus particles
were monitored by addition of A549 cell supernatants onto 293 T reporter cells that had been
transfected with an influenza-virus-specific luciferase construct ( FlaA ) [ 26 ] , followed by luciferase
measurement 16 h post-reinfection. For the validation of primary hits, the numbers of infectious
particles in the supernatants of A549 cells were quantified by reinfection of MDCK cells for 6 h,
followed by immuno fl uorescence and automated microscopy
In contrast, our group [ 4 ] set out to study the entire viral replication cycle from
viral entry to viral budding in a genome-wide RNAi screen. Human A549 lung
epithelial cells were first transfected with a genome-wide library of siRNA and
subsequently infected with influenza A/WSN/33 virus. The readout was then divided
into two parts: (1) Infected A549 cells were stained with a virus-specific antibody at
24 h postinfection to monitor cell infection rates, and (2) virus supernatants from
these transfected A549 cells were transferred onto 293 T human embryonic kidney
reporter cells, containing an inducible, influenza-virus-specific luciferase construct