Fig. 7: Loss of CMTR1 inhibits viral replication and up-regulates anti-viral genes.

a A549 cells transduced with gene-specific or non-targeting sgRNAs were transfected with PR8 PA, PB1, PB2, and NP plasmids together with green-Renilla and Luciferase reporter plasmids for 24 hours. Luciferase activity was measured and normalized to A549 cells transduced with non-targeting sgRNA. Error bars represent standard deviations from three independent experimental replicates. b A549 cells transduced with CMTR1 or non-targeting sgRNA were infected with PR8 virus, in vivo crosslinked, lysed and immunoprecipitated with either anti-eIF4E or anti-IgG antibody to extract capped cellular and viral RNA. qRT-PCR was performed to monitor the relative abundance of PR8 NP RNA. (Left): Fold change in NP RNA levels normalized to anti-IgG non-targeting sgRNA pulldown. (Right): Fold change in NP relative to GAPDH RNA levels normalized to non-targeting sgRNA. Error bars represent standard deviations from three independent experimental replicates. c Fold change in IFN- β relative to GAPDH mRNA levels measured by qRT-PCR. A549 cells transduced with CMTR1 or non-targeting sgRNA were either mocked treated, infected with PR8 virus or treated with 200 U/ml IFN- β for 16 h. Fold change was normalized to A549 cells transduced with non-targeting sgRNA. Error bars represent standard deviations from three independent experimental replicates. d Gene Ontology (GO) enrichment analysis showing the top 10 up-regulated gene categories in A549 cells transduced with CMTR1 sgRNA versus non-targeting sgRNA after PR8 virus infection. IFN-related and antiviral gene categories are colored in red. e Fold change in IFN- β relative to GAPDH mRNA levels measured by qRT-PCR. A549 cells were transduced with CMTR1 sgRNA alone or together with sgRNA targeting RIG-I, MAV or IRF3 and infected with PR8 virus. Fold change was normalized to cells transduced with non-targeting sgRNA. Error bars represent standard deviations from three independent experimental replicates. f A549 cells transduced with gene-specific or non-targeting sgRNA were pre-treated with 0, 1, 5 or 10 nM of Baloxavir followed by PR8 virus infection. Y-axis shows the percentage of HA-positive cells normalized to untreated cells. Error bars represent standard deviations from three independent experimental replicates. ****P = 0.0001, **P < 0.01, by one-way ANOVA test.