DAI/ZBP1/DLM-1 complexes with RIP3 to mediate virus-induced programmed necrosis that is targeted by murine cytomegalovirus vIRA

Abstract

Programmed necrosis, like apoptosis, eliminates pathogen-infected cells as a component of host defense. Receptor-interacting protein kinase (RIP) 3 (also called RIPK3) mediates RIP homotypic interaction motif (RHIM)-dependent programmed necrosis induced by murine cytomegalovirus (MCMV) infection or death receptor activation and suppressed by the MCMV-encoded viral inhibitor of RIP activation (vIRA). We find that interferon-independent expression of DNA-dependent activator of interferon regulatory factors (DAI, also known as ZBP1 or DLM-1) sensitizes cells to virus-induced necrosis and that DAI knockdown or knockout cells are resistant to this death pathway. Importantly, as with RIP3(-/-) mice, vIRA mutant MCMV pathogenesis is restored in DAI(-/-) mice, consistent with a DAI-RIP3 complex being the natural target of vIRA. Thus, DAI interacts with RIP3 to mediate virus-induced necrosis analogous to the RIP1-RIP3 complex controlling death receptor-induced necroptosis. These studies unveil a role for DAI as the RIP3 partner mediating virus-induced necrosis.

Figure 1. RIP3 levels differentially confer susceptibility to necroptosis and virus-induced programmed necrosis

(A) Immunoblot (IB) analysis to detect RIP3, RIP1 and β-actin from NIH3T3 cells stably expressing 3XFLAG-tagged RIP3, RIP3mRHIM, or control. (B) Viability of RIP3-expressing NIH3T3 stable cell lines infected with M45mutRHIM MCMV at a multiplicity of infection (MOI) of 10. (n=4) (C) Viability of RIP3-expressing NIH3T3 stable cell lines after treatment with TNF (25 ng/ml) and zVAD-fmk (50 μM) to induce necroptosis (n=3–4). (D) Viability of NIH3T3, 3T3-SA and SVEC4-10 cell lines infected with M45mutRHIM MCMV (MOI=10). (n=3–7) (E) IB analysis of uninfected NIH3T3, 3T3-SA, and SVEC4-10 cells to detect DAI, RIP3, RIP1, and β-actin. A non-specific (*ns) band is characteristic for this antibody (Karayel et al., 2009). **p<0.001, #not significant (p>0.05). See also related Figure S1.

Figure 2. Increased DAI expression sensitizes resistant cells to virus-induced necrosis

(A) IB analysis to detect FLAG and β-actin of NIH3T3 stable cells expressing FLAG-tagged WT DAI, FLAG-tagged DAImRHIM, or control. (B) Viability of DAI-expressing NIH3T3 stable cell lines infected with M45mutRHIM MCMV (MOI=10) and treated with DMSO or Nec-1 (30 μM). (n=4) (C) IB analysis to detect FLAG, RIP3, RIP1 and β-actin in stable NIH3T3 cell lines expressing FLAG-tagged WT DAI together with either a scramble control (Sc) or one of two RIP3-specific shRNAs (RIP3-A or RIP3-B). (D) Viability of DAI-expressing, RIP3 knockdown NIH3T3 stable cell lines following infection with M45mutRHIM MCMV (MOI=10)(n=3). **p<0.001, #not significant (p>0.05). See also related Figure S2.

Figure 3. DAI and RIP3 cooperate in virus-induced necrosis

(A) RIP3 and DAI interaction in the absence of vIRA. IB of immunoprecipitations (IP) and whole cell lysates (WCL; 5% total) showing DAI, M45, RIP3 and β-actin in 3T3-SA cells infected with M45mutRHIM MCMV (MOI=5) harvested at the indicated times (h.p.i.). (B) IB analysis to detect DAI, RIP3, RIP1 and β-actin in 3T3-SA cells transfected with non-targeting (NT) control, DAI, or RIP3 siRNAs (C) Viability of 3T3-SA cells transfected with the indicated siRNA and infected with M45mutRHIM (MOI=10) and treated with DMSO control or Nec-1 inhibitor (30μM). (n=4) (D) IB analysis to detect DAI, RIP3, RIP1, and β-actin in C57BL/6 control, DAI^−/−, and RIP3^−/− murine embryonic fibroblasts (MEFs) (E) Viability of C57BL/6 control, DAI^−/−, and RIP3^−/− MEFs infected with M45mutRHIM MCMV (MOI=10)(n=3). (F) Single-step replication of WT and M45mutRHIM MCMV (MOI of 5) on control C57BL/6 (left) and DAI^−/− (right) MEFs. Viral titers determined by plaque assay with the first (0 h) time point representing amount of virus in the inoculum. Error bars, SD. *p<0.01, **p<0.001, #not significant (p>0.05). See also related Figure S3.

Figure 4. M45mutRHIM virus attenuation in vivo is reversed in DAI^−/− mice

(A) Footpad swelling induced by parental WT or M45mutRHIM MCMV infection. Footpad thickness of C57BL/6 control (top) and DAI^−/− (bottom) mice inoculated with 10⁶ pfu was measured with a digital caliper, and mean values plotted at the indicated times over a 14 day time course (n=7–9 animals/group). (B–D) Viral titers from spleen (B), liver (C), and SGs (D) of control (C57BL/6), RIP3^−/−, and DAI^−/− mice infected via intraperitoneal inoculation with 10⁶ pfu of indicated virus and harvested at the indicated time (n= 5–10 animals/group). Error bars, SEM. See also related Figure S4.