Figure 4. RIP3 is required, and RIP1 is dispensable for MCMV-associated programmed necrosis.

(A) Viability of 3T3-SA cells expressing Sc, RIP3-A or RIP3-B shRNAs determined 18 hpi with WT or M45mutRHIM virus (MOI of 10). (B) Viability of SVEC4-10 cells using a subset of conditions described in (A). (C) IB of 3T3-SA cells infected with WT or M45mutRHIM virus (MOI of 5), harvested at indicated times for IP of RIP3 followed by detection of vIRA (M45) and RIP3. IB using ~5% of cell lysate to detect vIRA (M45) and β–actin. (D) IB of RIP3+/+, RIP3+/−, and RIP3−/− MEFs to detect RIP3, RIP1, and β–actin. (E) Replication of WT and M45mutRHIM viruses (MOI of 5) on RIP3+/+ (left panel), RIP3+/− (middle panel), and RIP3−/− (right panel) MEFs over a 72 h time course. Viral titers were determined by plaque assay with the first (0 h) time point representing the amount of virus in the inoculum. (F) IB analysis for FLAG-tagged proteins as well as β-actin (left panel) in RIP3−/− MEFs expressing FLAG-tagged RIP3, RIP3-KD, or RIP3-mRHIM and viability of reconstituted cells (right panel) infected with M45mutRHIM and WT virus. (G) Viability of RIP3+/+, RIP3+/−, and RIP3−/− MEFs infected with WT or M45mutRHIM virus in the presence or absence of Nec-1 (30 μM). (H) Viability of RIP3+/+ and RIP3−/− MEFs treated to induce necroptosis as described in Figure 2H in the presence or absence of Nec-1 (30 μM). (I) IB analysis for RIP1 as well as β–actin (top panel) and viability (bottom panel) of WT (RIP3+/+) MEFs stably expressing Sc, RIP1-A or RIP1-B shRNAs. Cell viability was determined for cells infected with WT or either of two independent isolates of M45mutRHIM virus. (J) IB analysis (top panel) and viability (bottom panel) of SVEC4-10 cells using a subset of conditions applied in (I). Error bars indicate SD of the mean. See also the related Figure S3.