siRNA (5 L of 5 M/well) and DharmaFECT Reagent 4 (2 L/well) were diluted in 200 L of serum-free MEM. terminus of both E3 and DAI encode Z-NA binding domains, E3 protein may function as a competitor of Z-form nucleic acid sensing or signaling. Results The N Terminus Is Required for Type I IFN Resistance in L929 Cells. The VACV E3 protein plays an essential role in counteracting the host innate immune system. While the C-terminal dsRNA BT-11 binding domain has been extensively characterized, the role of the N-terminal Z-NA BD in innate immune evasion has been difficult to characterize, due to the lack of a cell culture system where the phenotype of N-terminal E3 mutants in mice can be reproduced. Virulence of VACV in mice is dependent on the presence of a full-length E3 protein. A mutant virus encoding an N-terminal Z-NA BD truncation (VACV-E3L83N) is highly attenuated in WT mice (1, 4, 6) but not in mice, implicating the N terminus in subverting type I IFN signaling (6). While characterizing VACV mutants in several mouse cell lines, we identified L929 cells as having a phenotype consistent with the IFN-sensitive (IFNS) phenotype seen in vivo. L929 cells were pretreated with increasing doses of mouse IFN, then infected with equivalent plaque forming units (pfu) of WTVACV or VACV-E3L83N. As shown in Fig. 1mice. Open in a separate window Fig. 1. E3 N-terminal truncations result in IFN sensitivity BT-11 and rapid cell death in L929 cells. (and < 0.001. We began characterizing IFN sensitivity of VACV-E3L83N in L929 cells by performing a [35S]-methionine labeling experiment to determine if viral protein translation was altered in IFN-treated cells. Viral protein synthesis appeared reduced in IFN-treated, VACV-E3L83NCinfected cells (Fig. S1). However, visualization of the Coomassie blue-stained gel revealed a strong reduction in total protein on the gel compared with controls (Fig. 1and Fig. S1), suggesting that protein was lost from the mutant virus-infected cells. This pattern suggested that VACV-E3L83N virus-infected cells, but not WTVACV-infected cells, might leak their contents, leading to a reduced recovery of proteins from VACV-E3L83NCinfected cells. Open in a separate window BT-11 Fig. S1. (and Movies S1 and S2). Starting at 4 h postinfection (HPI), VACV-E3L83NCinfected cells underwent progressive cytoplasmic enlargement and plasma membrane disruption, patterns that were not observed in cells infected with WTVACV, irrespective of IFN treatment (Fig. 1and Movies S1 and S2). Such a pattern of cellular swelling and membrane disruption suggests that a Rabbit Polyclonal to CDK2 rapid death occurs in cells infected with VACV-E3L83N, where leakage may underlie the global loss of protein recovery seen in Fig. 1and Fig. S1. IFN Sensitivity Results in a Rapid Death Characterized by Membrane Permeability. To establish that leakage was occurring in VACV-E3L83NCinfected cells, we evaluated cellular permeability using a membrane-impermeable nuclear stain. This assay revealed that L929 cells pretreated with IFN and infected with VACV-E3L83N became permeable, while the uninfected control cells or cells infected with WTVACV did not (Fig. 1 and and < 0.001. Necroptosis occurs independently of caspase activity and depends on the protein kinase, RIPK3. Thus, we asked if a RIPK3-specific inhibitor, GSK872, could reverse the cell death induced in IFN-treated VACV-E3L83NCinfected L929 cells. Treatment with GSK872 inhibited E3L83N-induced cell death in IFN-treated cells (Fig. 2 and < 0.001. NS, no significance (>0.05). Open in a separate window Fig. S3. VACV is not a direct inhibitor of necroptosis. (or WT C57BL/6 mice were inoculated by intranasal route with 106 pfu of the indicated viruses (five mice BT-11 per group). (or WT C57BL/6 mice were infected by intranasal route with 106 pfu of the indicated viruses (five mice per group). **< 0.01. N.S., no significance (>0.05). Deficiency of RIPK3 or ZBP1 Rescues VACV-E3L83N Virulence in Mice. Given the importance of mouse studies that have defined the N terminus in subverting type I IFN signaling and virulence (6), we sought to pursue in vivo studies in WT C57BL/6, mice. Mice were infected intranasally with 106 pfu of either WTVACV or VACV-E3L83N [in the mouse-adapted, neurovirulent Western Reserve (WR) strain and monitored for clinical symptoms]. WTVACV infections resulted in significant pathology in WT, mice. As previously described, at this dose the VACV-E3L83N mutant was apathogenic.