Figure 4. Impacts of top-ranking host factors on virion entry and replication pathways.

(a-b) Effects of perturbation of top-ranking host factors on CPE caused by SARS-CoV-2 infection at different infection conditions. For gene-specific knockdown (KD) effect (a), two pro-viral factors (ATP6V0D1, DPAGTT) and three anti-viral factors (DAZAP2, VTA1, KLF5) were selected. For gene-specific overexpression effect (b), two anti-viral factors (DAZAP2, VTA1) were selected. Genetically modified A549-AC cells were infected with recombinant SARS-CoV-2 at MOI=0.5, 2.5, and 5 for 48 hours. A549-AC cells expressing a non-targeting gRNA (NC) or the GFP vector were served as control cells for the KD and overexpression experiments, respectively. Data were normalized using the viability of corresponding cells at mock condition. (c-f) Effects of perturbation of top-ranking host factors on SARS-CoV-2 attachment and entry. Genetically modified A549-AC cells were infected with recombinant SARS-CoV-2 at MOI =1. To evaluate the changes in the viral attachment (c and d), the infection was performed at 4°C for 1 h; whereas to evaluate the changes in viral entry (e and f), the infection was performed at 37°C for 1 h. The levels of RNAs encoding the viral N protein and ACTB mRNAs were determined by RT-PCR. Viral RNA levels were normalized using the expression of ACTB mRNA. (g-h) Effects of perturbation of top-ranking host factors on SARS-CoV-2 replication. A549-AC cells with gene-specific KD (g) or overexpression (h) were infected with SARS-CoV-2-Nluc at MOI= 0.02, 0.1, and 0.5. The luciferase signals were measured at 24 hours post-infection. Statistical significances between cells with gene-specific overexpression and control cells at each infection condition were determined by one-way ANOVA with repeated measurements. At least two independent experiments were performed, and samples were triplicated in each independent experiment. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. n.s., not significant.