C67-03 Identification of Druggable Host Dependency Factors in RSV Infection With Crispr-cas9 in A549 Airway Epithelial Cells

Rationale Infants and young children are susceptible to severe Respiratory Syncytial Virus(RSV) infection and developing long-term sequelae such as recurrent wheezing. Host Dependency Factors(HDFs) are host proteins that support the viral lifecycle. Thus, identification of HDFs offers avenues to developing targeted antiviral therapies against RSV. We utilized CRISPR-Cas9 to screen genes with available FDA approved pharmacological inhibitors to identify druggable HDFs for RSV infection. Methodology CRISPR knockouts were generated in A549 cells through non-homologous end-joining. Knockout(KO) cells were exposed to an RSV-A2 strain encoding GFP (RSV-GFP) and sorted to identify infected (GFP-positive) and uninfected cells (GFP-negative). Genomic DNA of uninfected and infected cells was isolated and sequenced. MAGeCK-RRA determined significant hits in uninfected cells. Pharmacological inhibitors were used to target the top hits in A549 cells and primary airway epithelial cells(AECs) derived from high-risk donors (N = 3, median age 4.71 weeks). Flow cytometry was used to assess the percentage of RSV infected cells. Confocal microscopy was utilized at select timepoints post RSV infection to evaluate viral entry in the presence of pharmacological inhibitors. Results We found 14 significant (p-value < 0.001) candidate targets, with the top three candidates being components of V-ATPase. Pharmacological inhibitors were used to evaluate V-ATPase as a hit. Pharmacological inhibition of V-ATPase with Bafilomycin and Concanamycin A showed a dose-dependent reduction in A549 cells up to 90% and 60% in primary AECs. We have performed confocal microscopy at multiple timepoints post-exposure to assess the effect of V-ATPase inhibition on RSV localization and intracellular trafficking. Conclusions We identified V-ATPase as a HDF for RSV in A549 cells through CRISPR-Cas9 KO screening with a druggable sgRNA library. Our methodology demonstrates that CRISPR KO screening can be utilized to identify significant HDFs relevant to viral infection for other viruses, which will enable more high throughput studies into the underlying mechanisms of those HDFs, resulting in the development of more effective antiviral therapies. Our results also show significant clinical impact, as the V-ATPase inhibition shows the potential to inhibit RSV infection even in cells from vulnerable populations. Further CRISPR screening using the primary cells could reveal more personalized treatments through the identification of HDFs specific to each donor.