Encephalitis and poor neuronal death-mediated control of herpes simplex virus in human inherited RIPK3 deficiency

Abstract

Inborn errors of TLR3-dependent type I IFN immunity in cortical neurons underlie forebrain herpes simplex virus-1 (HSV-1) encephalitis (HSE) due to uncontrolled viral growth and subsequent cell death. We report an otherwise healthy patient with HSE who was compound heterozygous for nonsense (R422*) and frameshift (P493fs9*) RIPK3 variants. Receptor-interacting protein kinase 3 (RIPK3) is a ubiquitous cytoplasmic kinase regulating cell death outcomes, including apoptosis and necroptosis. In vitro, the R422* and P493fs9* RIPK3 proteins impaired cellular apoptosis and necroptosis upon TLR3, TLR4, or TNFR1 stimulation and ZBP1/DAI-mediated necroptotic cell death after HSV-1 infection. The patient's fibroblasts displayed no detectable RIPK3 expression. After TNFR1 or TLR3 stimulation, the patient's cells did not undergo apoptosis or necroptosis. After HSV-1 infection, the cells supported excessive viral growth despite normal induction of antiviral IFN-β and IFN-stimulated genes (ISGs). This phenotype was, nevertheless, rescued by application of exogenous type I IFN. The patient's human pluripotent stem cell (hPSC)-derived cortical neurons displayed impaired cell death and enhanced viral growth after HSV-1 infection, as did isogenic RIPK3-knockout hPSC-derived cortical neurons. Inherited RIPK3 deficiency therefore confers a predisposition to HSE by impairing the cell death-dependent control of HSV-1 in cortical neurons but not their production of or response to type I IFNs.

Figure 1.. Compound heterozygous RIPK3 mutations in a patient with HSE

A. Family pedigree with allele segregation of the two RIPK3 mutations. The proband (patient 1, P1), in black, is compound heterozygous for the p.Arg422* (R422*) and p. Pro493fs9* (P493fs9*) mutations. Each parent is heterozygous for one mutant allele. B. Images of the brain of P1, showing lesions affecting the left insula. C. Graph showing the CADD scores of all homozygous RIPK3 nonsynonymous or essential-splicing variants reported by the gnomAD database, and their minor allele frequency (MAF). MSC 95%: mutation significance cutoff for the 95% confidence interval. D. Schematic representation of the structure of the RIPK3 protein and the impact of the two mutations.

Figure 2.. In vitro production and function of the RIPK3 variants upon transient transfection

A. Confocal microscopy imaging of HeLa cells 24 h after transfection with wild-type (WT) and mutant RIPK3. Cells were stained with an antibody directed against the N-terminus (N-ter) of RIPK3 and the corresponding Alexa Fluor 488-conjugated secondary antibody (green). The nuclei were stained with DAPI (blue). Scale bar, 10 μm. The results shown are representative of three independent experiments. B. RIPK3 mRNA levels were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in HEK293T cells 24 h after transfection with a luciferase-expressing vector (Luci), or WT and mutant RIPK3 constructs. We used two probes, targeting exons 2–3 (upper panel) and exons 9–10 (lower panel) of RIPK3. The data shown are the means from two biological replicates from one experiment representative of three independent experiments. C. Immunoblotting analysis of total RIPK3 and autophosphorylated RIPK3 (p-RIPK3, Ser227) levels in HEK293T cells 24 h after transfection with C-terminally Myc-tagged RIPK3 WT and mutant constructs. RIPK3 proteins were detected with antibodies against the N terminus (N-ter) or C terminus (C-ter) of RIPK3, p-RIPK3 (Ser227) and Myc-tag. The results shown are representative of three independent experiments. D. RIPK3 overexpression-mediated NF-κB promoter-driven reporter assay in HEK293T cells, 24 h after transfection with the NF-κB reporter plasmid, along with various doses of empty vector (EV), WT and mutant RIPK3 constructs; analysis of luciferase activity. The data shown are the means of at least three biological replicates from three independent experiments. E. Myc-tagged WT and mutant RIPK3 constructs were co-expressed with FLAG-tagged WT and mutant RIPK3 constructs in HEK293T cells, which were then subjected to immunoprecipitation (IP) with anti-Myc antibody-conjugated agarose beads, and immunoblotting with anti-FLAG or anti-Myc antibodies. The results shown are representative of three independent experiments. F. HEK293T cells cotransfected with Myc-tagged RIPK1 and FLAG-tagged WT and mutant RIPK3 WT plasmids, subjected to IP and immunoblotting as in (E). The results shown are representative of three independent experiments.

Figure 3.. In vitro production and function of the RIPK3 variants after stable transduction

A. RIPK3 mRNA levels were determined by RT-qPCR in parental HT29 cells, or RIPK3 knockout (KO) HT29 cells left non-transfected (NT) or stably transfected with a mock vector (Luci) or with WT or mutant RIPK3 constructs in a lentiviral system. The data shown are the means from three biological replicates from three independent experiments. B. Immunoblotting analysis of RIPK3 protein levels in the parental HT29 cells, and in RIPK3 KO HT29 cells, as in (A). The results shown are representative of three independent experiments. C. Pulse-chase analysis to measure WT and mutant RIPK3 protein degradation. HEK293T cells were transfected with FLAG-tagged WT and mutant RIPK3 plasmids for 24 h. They were then treated with 100 μg/ml cycloheximide (CHX) for the indicated times, and subjected to western blotting (lower panel). The relative amounts of RIPK3 were calculated after normalization against GAPDH and are shown in the graph (upper panel). The results shown are representative of three independent experiments. D. Immunoblotting analysis of RIPK3 levels in RIPK3 KO HT29 cells stably expressing WT and mutant RIPK3, treated with protein degradation inhibitors (5 nM bortezomib (BTZ) for 12 h, 10 mM MG132, 50 mM chloroquine diphosphate (CQ), or 10 mg/mL E64d plus 10 mg/mL pepstatin) for 6 h. The red asterisk indicates the bands corresponding to RIPK3. The results shown are representative of three independent experiments. E. Immunoblot analysis of phosphorylated MLKL (p-MLKL, Ser358) in RIPK3 KO HT29 cells stably expressing Luci, WT or mutant RIPK3, treated with DMSO solvent as a control or with PBZ complex (consisting of poly(I:C), BV6 and Z-VAD), or TBZ complex (containing TNF, BV6 and Z-VAD) for the indicated times. The results shown are representative of three independent experiments. F. Viability of RIPK3^−/− HT29 cells stably expressing WT and mutant RIPK3 constructs, treated with DMSO solvent (control), or with poly(I:C), TNF, PB (poly(I:C) + BV6), TB (TNF + BV6), PBZ or TBZ complex for the indicated times. The results shown are the means ± SD from three biological replicates from one experiment, representative of three independent experiments. P values were obtained by one-way ANOVA and Tukey’s multiple comparison tests, and P values are indicated for the comparison of R422* or P493fs9* RIPK3 transduced cells with WT RIPK3 transduced cells. ns-not significant, *P<0.05, **P<0.01, ****P<0.0001. G. Viability of RIPK3^−/− HT29 cells stably expressing WT and mutant RIPK3 constructs, treated with DMSO solvent (control), or with L (LPS), LB (LPS + BV6) or LBZ (LPS + BV6 + Z-VAD) complex for 24 h. The results shown are the means ± SD from three biological replicates from one experiment, representative of two independent experiments. P values were obtained by one-way ANOVA and Tukey’s multiple comparison tests, and the corresponding P values are indicated. ****P<0.0001. H. Viability of RIPK3 KO HT29 cells stably co-expressing FLAG-tagged DAI with WT and mutant RIPK3, left non-infected (NI), or after infection with HSV-1 FmutRHIM (MOI=5) for 24 h. The data shown are means ± SEM from two independent experiments, with three biological replicates per experiment. P values were obtained by one-way ANOVA and Tukey’s multiple comparison tests, and the corresponding P values are indicated. *P<0.05, ****P<0.0001.

Figure 4.. Impaired RIPK3 production and function in P1’s cells

A. RIPK3 mRNA levels were measured by RT-qPCR in EBV-B cells (EBV-B), SV40-fibroblasts (SV40-F) and primary fibroblasts (Primary-F) from healthy controls (Ctrls) and P1, with two probes targeting exons 2–3 (upper panel) and exons 9–10 (lower panel) of RIPK3. The results data shown are the means of four biological replicates from two independent experiments. B. Relative abundance (in percentages) of the RIPK3 cDNA generated from mRNA extracted from SV40-F from P1, assessed by TOPO-TA cloning. C. Immunoblot analysis of endogenous RIPK3 levels in EBV-B, SV40-F and Primary-F from healthy controls (C1, C2, C3) and P1, with antibodies against the N-terminus and C-terminus of RIPK3. The results shown are representative of more than three independent experiments. D. Immunoblot analysis of endogenous RIPK3 levels in SV40-F from healthy controls (C1, C2) and P1 treated with protein degradation inhibitors (5 nM BTZ for 12 h, 10 mM MG132, 50 mM CQ, or 10 mg/mL E64d plus 10 mg/mL pepstatin) for 6 h. The red asterisks indicates the bands corresponding to RIPK3. The results shown are representative of three independent experiments. E. Immunoblot analysis of p-MLKL levels in SV40-F from a healthy control (C1) and P1, either left non-transfected (NT) or transiently transfected with Luci or WT RIPK3 for 24 h, and then stimulated with PBZ or TBZ for 4 h. The red asterisks indicate the bands corresponding to RIPK3. The results shown are representative of three independent experiments. F. Immunoblot analysis of full-length and cleaved caspase 3 in SV40-F from healthy controls and P1, treated for 8 h with PB complex containing poly(I:C) and BV6, or TB complex containing TNF and BV6. The results shown are representative of three independent experiments. G. Viability of primary fibroblasts from healthy controls (Ctrls, n=3) and P1, either non- transduced or transduced with Luci, WT, variants with P1’s mutations or D142N RIPK3 lentiviruses for 48 h, then treated with DMSO solvent (D), or with poly(I:C), PB or PBZ complex for the times indicated. The results shown are the means ± SEM from three independent experiments (transduction with WT or P1 mutant RIPK3) and two independent experiments (for D142N RIPK3), with three biological replicates per experiment. H. Viability of primary fibroblasts from healthy controls (Ctrls, n=3) and P1, either non-transduced or transduced with Luci, WT, variants with P1’s mutations or D142N RIPK3 lentiviruses for 48 h, and treated with DMSO solvent (D), TNF, TB or TBZ complex for the times indicated. The results shown are the means ± SEM from three independent experiments (for WT or P1 mutant RIPK3) and two independent experiments (for D142N RIPK3), with three biological replicates per experiment. In G and H, P values were obtained by one-way ANOVA with Tukey’s multiple comparison tests, and the corresponding P values are indicated. ns-not significant, ***P<0.001, ****P<0.0001. I-J. Immunoblot analysis of p-MLKL in SV40-F from a healthy control (C1), P1, other HSE patients (TLR3^−/−, TRIF^−/−, UNC93B1^−/−), and a TBK1^−/− patient, after stimulation with PBZ (I) or TBZ (J) for 4 h. The results shown are representative of three independent experiments. K-L. Immunoblot analysis of full-length and cleaved caspase 3 in SV40-F from a healthy control, P1, TLR3^−/−, TRIF^−/−, UNC93B1^−/−, or TBK1^−/− patients, after stimulation with PB (K) or TB (L) for 8 h. The results shown are representative of three independent experiments.

Figure 5.. Intact signaling via the TLR3- and TNFR1-dependent NF-κB, IRF3 and MAPK pathways in P1 fibroblasts

A. Immunoblot analysis of total and phosphorylated P65, IRF3, ERK1/2 and JNK1/2 in SV40-F from healthy controls (C1, C2), P1, TLR3^−/− and NEMO^−/− patients, after stimulation with 25 μg/ml poly(I:C) for the times indicated. The results shown are representative of three independent experiments. B. Immunoblot analysis of total and phosphorylated P65, ERK1/2 and JNK1/2 in SV40-F from healthy controls, P1 and a NEMO^−/− patient, after stimulation with 20 ng/ml TNF for the times indicated. The results shown are representative of three independent experiments. C. Immunoblot analysis of total and phosphorylated IRF3 in SV40-F from healthy controls, P1, TLR3^−/− and NEMO^−/− patients, after stimulation with 20 ng/ml TNF for 24 h. The results shown are representative of three independent experiments. D. SV40-F from healthy controls (Ctrls, n=3), P1 and TLR3^−/− and NEMO^−/− HSE patients were left unstimulated (NS) or were stimulated with various doses of poly(I:C) alone, Lipofectamine alone (Lipo), or both (poly(I:C)+Lipo), for 24 h. The amounts of IFN-β, IFN-λ1, IL-6 and CCL3 in culture supernatants were determined with Legendplex cytometric bead arrays. The results shown are the means ± SEM from two independent experiments, with three biological replicates per experiment. Each dot represents one biological replicate. P values were obtained by one-way ANOVA with Tukey’s multiple comparison tests, and the corresponding P values are indicated. ns-not significant, *P<0.05, **P<0.01, ****P<0.0001.

Figure 6.. Enhanced susceptibility of RIPK3-deficient fibroblasts and hPSC-derived cortical neurons to HSV-1

A. Principal component analysis of the transcriptome of human primary fibroblasts without (NS) or with HSV-1 infection for 24 hours, in cells from healthy controls (Ctrls, n=3), P1 and other patients with recessive TLR3, IFNAR1 or NEMO deficiency. B. Genes differentially expressed between HSV-1 and NS in human primary fibroblasts, as in (A). Heatmap including 3060 genes with relative absolute fold-changes in expression > 2 (in all three healthy controls) in response to HSV-1 relative to NS samples. C. SV40-F from healthy controls (Ctrls, n=3), P1 and other patients with recessive TLR3, IFNAR1 or NEMO deficiencies were left untreated or were treated with IFN-β for 24 h, and then infected with HSV-1 (MOI = 0.001). Virus replication was then evaluated at the indicated timepoints post infection. HSV-1 replication was quantified by the TCID₅₀ virus titration method. The data shown are the means ± SEM of two independent experiments with two biological replicates per experiment. P values were obtained by one-way ANOVA with Tukey’s multiple comparison tests, and the P values for the 24 h and 48 h time points are indicated for the comparison of P1’s cells with control cells. *P<0.05. D-G, Virus replication levels for VSV (D), measles virus (MeV) (E), influenza A virus (IAV) (F), and EMCV (G) in SV40-F, as in (C), at the indicated times post infection with VSV (MOI = 0.1), MeV (MOI = 0.5), IAV (MOI = 10), or EMCV (MOI = 0.01), as assessed by the TCID₅₀ virus titration method (VSV and MeV), plaque assay (IAV), or expression levels of the three-dimensional region of the EMCV genome, as measured by RT-qPCR. The data shown are the means of two to four biological replicates from two (IAV and EMCV) or four (VSV, MeV) independent experiments. H. RIPK3 mRNA levels, as measured by RT-qPCR, in cortical neurons differentiated from the hPSCs of healthy controls and P1. We used two probes, targeting exons 2–3 (left) and exons 9–10 (right) of RIPK3. The data shown are the means of two biological replicates from one experiment. I. Relative abundance (in percentages) of the RIPK3 cDNA generated from mRNA extracted from hPSC-derived cortical neurons from a healthy control and P1, assessed by TOPO-TA cloning. J. hPSC-derived cortical neurons from a healthy control, P1 and other HSE patients with AR TLR3 or IFNAR1 deficiencies, with or without IFN-β pretreatment for 24 h, were infected with HSV-1 (MOI=0.001) and virus replication levels were measured at the indicated timepoints post infection. HSV-1 replication was quantified by the TCID₅₀ virus titration method. The data shown are means ± SEM for four independent experiments (a healthy control, P1 and TLR3-deficienct cells) and two independent experiments (IFNAR1-deficiency cells). P values were obtained by one-way ANOVA with Tukey’s multiple comparison tests, and the P values for the 72 h time points are indicated for the comparison of P1’s cells with control cells. ****P<0.0001. K. Viability of hPSC-derived cortical neurons from a healthy control, P1 and a TLR3^−/− HSE patient, left non-infected (NI), or infected with HSV-1 (MOI=0.001) for the times indicated. The data shown are means ± SEM for two independent experiments, with three biological replicates per experiment. P values were obtained by one-way ANOVA with Tukey’s multiple comparison of P1’s cells with control cells, and the corresponding P values are indicated. *P<0.05, ***P<0.001. L. RIPK3 mRNA levels, as measured by RT-qPCR, in cortical neurons from parental and RIPK3 KO hPSCs. We used two probes, targeting exons 2–3 (left) and exons 9–10 (right) of RIPK3. The data shown are the means of three biological replicates from one experiment. M. hPSC-derived cortical neurons derived from parental healthy control cells, RIPK3 KO cells and HSE patients with AR TLR3 were infected with HSV-1 (MOI=0.001) for the times indicated. HSV-1 replication was quantified by the TCID₅₀ virus titration method. The data shown are means ± SEM for three independent experiments (the parental cells and RIPK3 KO cells) and two independent experiments (TLR3-deficient cells). P values were obtained by one-way ANOVA with Tukey’s multiple comparison tests, and the P value for the 48 h time point is indicated for the comparison of RIPK3 KO cells with control parental cells. **P<0.01. N. Viability of hPSC-derived cortical neurons from healthy parental control cells, RIPK3 KO cells and a TLR3^−/− HSE patient, left non-infected, or infected with HSV-1 (MOI=0.001) for the times indicated. The data shown are means ± SEM for two independent experiments, with three biological replicates per experiment. P values were obtained by one-way ANOVA with Tukey’s multiple comparison tests, and the P values for the indicated timepoints are indicated for the comparison of RIPK3 KO cells with control parental cells. The corresponding P values are indicated. ns - not significant, *P<0.05.