ML323 attenuates VSV-induced IFN-β and promotes viral replication in vitro and in vivo. (A) Western blot analysis of UAF1 and USP1 expression in mouse peritoneal macrophages infected with VSV for the indicated time periods. Similar results were obtained in three independent experiments. (B–F) Mouse peritoneal macrophages were treated with 30 µM ML323 for 4 h and then infected with VSV for 12 h. This experiment was repeated twice with similar results. Western blot analysis of TBK1 expression (B). ELISA analyzed IFN-β production (C). IFN-β mRNA, ISG15 mRNA, and intracellular VSV RNA replicates were measured by RT-PCR (D–F). Data are representative of three experiments (mean ± SD of three samples; **, P < 0.01; by Student’s t test). (G and H) Serum levels of IFN-β and TNF-α from C57BL/6 mice pretreated with ML323 or vehicle control as measured by ELISA 2 h after i.p. LPS injection. This experiment was repeated twice with similar results. Data shown are means ± SD (n = 4; **, P < 0.01; by Student’s t test). (I and J) C57BL/6 mice were i.p. injected with thioglycolate to elicit peritoneal macrophages. After 3 d, the mice were treated with ML323 or DMSO and then infected with VSV i.p. for 12 h. This experiment was repeated twice with similar results. IFN-β expression in lavage was examined by ELISA (I). Data shown are means ± SD (n = 5; **, P < 0.01; by Student’s t test). TBK1 expression in peritoneal exudate cells was analyzed by Western blot (J). (K–M) C57BL/6 mice were pretreated with ML323 or DMSO and then infected with VSV i.p. for 8 h. This experiment was repeated twice with similar results. Serum level of IFN-β was measured by ELISA (K). IFN-β mRNA (L) and intracellular VSV RNA replicates (M) in the liver, lung, and spleen were measured by RT-PCR. Data shown are means ± SD (n = 4; *, P < 0.05; **, P < 0.01; by Student’s t test). (N) C57BL/6 mice were pretreated with ML323 or DMSO and then infected with VSV i.p. for 18 h. H&E staining of lung tissue sections. Bars, 100 µm.