CRISPR editing of candidate host factors that impact influenza A virus infection

Influenza A virus (IAV) is a respiratory pathogen with a segmented negative-sense RNA genome that is capable of causing epidemics and pandemics. The error-prone nature of the IAV polymerase results in antigenic drift and antigenic shift which contribute to low vaccine efficacy and escape from antivirals. The host factors required for the complete IAV infectious cycle have not been fully identified. Here, we examined select host factors that were identified by independent CRISPR screens as candidate contributors to IAV infectivity. We performed CRISPR-mediated knockout of cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS) as well as CRISPR-mediated overexpression of beta-1,4 N-acetylgalactosaminyltransferase 2 (B4GALNT2) and adenosine deaminase acting on RNA 1 (ADAR1) in the human bronchial epithelial A549 cell line and evaluated for reduction of IAV infection. We confirmed that the knockout of CMAS or overexpression of B4GALNT2 restricts IAV infection by diminishing binding to the cell surface. While ADAR1 overexpression did not significantly inhibit IAV replication, it had a pro-viral effect with coxsackie B virus infection. Taken together, these data provide insight into how host factors identified in CRISR screens can modulate IAV infection and be utilized to further understand the IAV life cycle and for developing antiviral strategies. A549 Cas9 and A549-dCas9-VP64 cells were generated via lentiviral transduction of SpCas9 or the VP64 activator domain (dCas9-VP64) into A549 cells. Clonal cell lines with high expression of Cas9 or dCas9-VP64 proteins were established by limiting dilution after selection with 10 ug/mL blasticidin S HCl (ThermoFisher #A1113903) for 14 d. For A549 CalB4GALNT2 and CalADAR1 overexpressing lines, sgRNA sequences for the CRISPRa candidate genes were cloned into expression vector pXPR-502 as described previously (28, 37). sgRNA targeting the B4GALNT2 promoter region or ADAR1 and complementary oligos with appropriate nucleotide overhangs were ordered from ThermoFisher and annealed with a thermocycler to form dsDNA duplex following established protocol. Briefly, 1 uM of oligos were mixed with T4 Ligase Buffer (NEB) and T4 PNK enzyme. Annealing of the oligos was performed with the thermocycler following the program: 37 °C for 30 min, 95 °C for 5 min, then decreasing the temperature by 5 °C/min until 25 °C was reached. 90 uL of water was added to each reaction and 2 uL of duplexed oligos were ligated to 25 ng of linearized pXPR-502 lentiviral expression vector in 20 uL ligation reaction at 16 °C overnight. 7 uL of the ligated material was used to transform Stbl3 E. coli, which was plated on LB plates containing 100 ug/mL ampicillin. At least three bacterial colonies were sent out for Sanger sequencing and plasmids with the correct sgRNA sequences were expanded for lentiviral transduction (37). Confluent HEK293T cells in a 10 cm dish were transfected with plasmids expressing VSV-G envelope protein (pMD2.G), HIV structural proteins (psPAX2), and pXPR-502 expressing the specific sgRNA from a U6 promoter and a puromycin resistance gene from an EF-1a promoter at the ratio of 1:1.5:3 (4, 6, and 12 ug, respectively) using TransIt-293T. Supernatants were collected every 24 h for 3 d and pooled. The supernatant was concentrated with LentiX-concentrator and added onto A549-dCas9-VP64 cells. Lentivirus-transformed cells were then selected with 2 ug/mL puromycin for 7 d. The survivor cells were then expanded for maintained in complete DMEM supplemented with 2 ug/mL puromycin. Supernatants were collected for 3 d and passed through a 0.45 uM filter to remove debris before adding onto target cell lines. Media was replaced with DMEM + 10 ug/mL blasticidin, 2 ug/mL puromycin (A1113803, Invitrogen) at 24 h post-transduction and cells were cultured for 7 d. Clonal knockdown and overexpression cell lines were established by limiting dilution and with passage in selective media. RNA was purified from cell lines by TRIzol Reagent. Strand-specific total RNA with ribosomal RNA depletion was used for libraries generated with 1 µg of input RNA using the TruSeq Stranded mRNA Sample Prep Kit (Illumina) and sequenced on an Illumina NextSeq 2000 machine. Paired-end sequence reads were aligned to the human reference genome (hg38_v44) using STAR (38) and transcript quantification was performed with RSEM (39) through the UMass Chan Medical School DolphinNext (40). Downstream analysis was performed in R version 4.4.0 using the RStudio interface. Relative transcript abundance was estimated using DESeq2 based on normalized counts of alignments, three replicates of parental A549-dCas9-VP64 were compared to clones containing the ADAR and B4GALNT2 constructs. Cutoff for significance was p < 0.1 adjusted for multiple comparisons. Volcano plots were constructed using the EnhancedVolcano package.