Establishment of primary transgenic human airway epithelial cell cultures to study respiratory virus – host interactions

Hulda R. Jonsdottir; Sabrina Marti; Dirk Geerts; Regulo Rodriguez; Volker Thiel; Ronald Dijkman

Since we observed the same phenotype with two viruses with differential cell tropism, non-ciliated 529 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/694380 doi: bioRxiv preprint and ciliated cells respectively, there seems to be no cell-type bias for shRNA-mediated knockdown in 530 homogenously transgenic hAEC cultures. This is further corroborated by the uniform reduction of 531 GFP expression by shGFP1 in our double transgenic hAECs, in which the MFI of GFP fluorescence is 532 reduced by approximately 40%. Due to low inoculation dose (MOI 0.1), we hypothesize that the 533 amount of shRNA targeting the viral GFP mRNA transcript simply is not sufficient to achieve a 534 pronounced reduction of the logarithmic increase of viral replication. Adaptation of the inoculation 535 dosage or even the Polymerase III promoter in the lentiviral vector to achieve a higher cellular 536 concentration of shRNA might reveal a different phenotype. It has been reported that the viral 537 nucleoprotein of HCoV-229E counteracts RNA silencing [44] , which might also be the case for hRSV, 538 rendering our shRNA ineffective against these viruses. Further characterization and validation of this 539 phenomenon is fascinating but beyond the scope of this study.