DOI: 10.6084/m9.figshare.13826429.v1
title: Genome-wide siRNA screen in THP-1 cells infected with Influenza A virus

description: To identify host cell factors that impact viral replication, the human monocytic cell line THP-1 was treated with an arrayed library of siRNA pools and subsequently infected with influenza A/Wyoming/03/03 (H3N2) or A/Vietnam/1203/04 (H5N1) HALo virus in the presence or absence of interferon beta. The Influenza A/Vietnam/1203/04 (H5N1) HALo mutant virus is an attenuated H5N1 virus generated from wild-type Influenza A/Vietnam/1203/04 (H5N1) virus as described in Steel, J., et al. J Virol. 2009 Feb; 83(4):1742-53.<br> Genome-wide siRNA screenA genome-wide siRNA screen was carried out in human macrophage THP-1 cells to identify novel host cell factors that affect the replication of influenza A virus. The screen was performed using the arrayed genome-wide ON-TARGETplus SMARTpool siRNA library (Dharmacon), where each pool contains 4 unique siRNAs targeting a given gene. In addition, two non-targeting siRNAs (scr76, scr77) were added to each plate as negative controls, and siRNAs targeting IRF9 and the influenza A virus protein NP were included as positive controls. THP-1 cells were differentiated using 10 ng/ml phorbol-12-myristate-13-acetate (PMA) for 72 hours at 37C, 5% CO2. Pooled siRNAs were arrayed in 384-well plates at a concentration of 12.5 nM siRNA per well. To enable the formation of siRNA-transfection reagent complexes, 0.075ul Lipofectamine RNAiMAX transfection reagent diluted in 9.925 ul Opti-MEM media (both reagents Thermo Fisher Scientific) were added to each well. Following a 20 min incubation period at room temperature, 15,000 differentiated THP-1 cells diluted in 20 ml RPMI media supplemented with 10% FBS, 1% PenStrep and 1% HEPES were seeded on top of the complexes and incubated for 48 hours at 37C, 5% CO2. Cells were then mock-treated or treated with 100 IU/ml universal interferon beta (IFN) diluted in 10 ul of serum free RPMI media. After 6 hours of incubation at 37C, 5% CO2, media was removed, and cells were infected with the influenza A virus strain A/Wyoming/3/2003 H3N2 or A/Vietnam/1203/2004 H5N1 HALo at a multiplicity of infection (MOI) of 0.50 (H3N2) or 0.25 (H5N1 HALo) diluted in in 20 ul serum free RPMI media. After a 1-hour incubation at room temperature, the inoculum was removed and replaced with 40 ul of serum-free RPMI media and cells were incubated for 24 hours at 37C, 5% CO2. Cells were then fixed with 4% PFA for 30 min at room temperature and washed twice with PBS. Cells were permeabilized with 0.5% Triton X-100 for 20 min, followed by blocking with 3% BSA for 1 hour at room temperature. Primary anti-NP mouse monoclonal antibody (HT103, provided by Dr. Adolfo García-Sastre, Icahn School of Medicine at Mount Sinai) was added for 2 hours at room temperature, followed by three washes with PBS and a 1-hour incubation with Alexa Fluor 488-conjugated anti-mouse secondary antibody (Thermo Fisher Scientific, Cat# A-11001) diluted in 3% BSA. Following three washes with PBS, cells were stained with DAPI (4,6-diamidine-2-phenylindole), and plates were sealed and stored at 4C until imaging.<br> Imaging and data analysisReplication of influenza A virus in the assay plates was assessed using high-throughput microscopy (high-content screening, HCS). The assay plates were analyzed by high-content imaging using a Vala Sciences IC200 microscope. Using automated analysis software Columbus v2.5 (Perkin Elmer), the number of nuclei per well was counted and the number of cells positive for Alexa 488 staining was used to calculate the percentage of infected cells. Infection data from each well was normalized to the plate median, and the Z score was calculated. Cytotoxicity resulting from siRNA transfections was evaluated by normalizing the total number of cells per well to the average value of wells treated with the non-targeting control siRNA scr76 in the same plate. Screens were performed in duplicate and replicate data was averaged. siRNA pools with a cell viability &lt;70% were excluded from hit selection. Genes with a corresponding Z score &lt; -1.5 were considered host dependency factor hits, and genes with a Z score &gt; 1.5 host restriction factors hits.