RNA VIRUS VECTORS CARRYING DAI AND RIPK3

Statement Of Government Support

This invention was made with government support under Grant Nos. CA168621 and

All 13469 awarded by the National Institutes of Health. The government has certain rights in the invention.

Field

The present disclosure relates generally to the field of virotherapy. More particularly, the present disclosure relates to RNA viruses as vectors for delivery of at least one of DNA- dependent activator of interferon-regulatory factors (DAI) and receptor-interacting

serine/threonine-protein kinase 3 (RIPK3) to infected cells or tumor cells to facilitate death of such cells as well as to enhance an immune response against the underlying infection or tumor.

Background

Various publications, including patents, published applications, accession numbers, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference, in its entirety and for all purposes, in this document.

Influenza A virus (IAV), a member of the family Orthomyxoviridae and primary causative agent of influenza in birds and mammals, is an enveloped virus with a negative-sense, single- stranded, segmented RNA genome. Acute IAV infection is accompanied by lysis of infected primary epithelial cells and fibroblasts in culture, and by destruction of airway epithelia in vivo, indicative of an integral role for cell death in the virus life cycle, the immune response to this virus, or both. It is believed that cell death may represent a host defense mechanism that limits both virus spread and host immunopathology early in an infection.

In murine fibroblasts and airway epithelial cells, IAV lysis is driven by receptor- interacting serine/threonine-protein kinase 3 (RIPK3). Upon IAV infection, RIPK3 nucleates a necrosome complex, that minimally also includes receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL), which then activates both apoptosis, via a RIPK1 + Fas-Associated protein with Death Domain (FADD) + Caspase 8 axis, and necroptosis, mediated by MLKL. Formation of the RIPK3 necrosome and consequent activation of cell death both require active IAV replication. DNA-dependent activator of interferon-regulatory factors (DAI) senses IAV genomic RNA and links replicating IAV to RIPK3 activation. It is believed that DAI recognizes IAV RNA by a mechanism requiring the second of its Za domains, and nucleates a RHIM-dependent RIPK3 -containing necrosome. DAI also mediates IAV-induced RIPK3 -independent apoptosis. Consequently, cells lacking DAI are remarkably resistant to IAV-triggered lysis, and DAI-deficient mice are hyper-susceptible to lethal infection by this virus.

Summary

The present disclosure provbides RNA virus vectors. These RNA virus vectors may be used for treating tumors, and also for treating virus infections, particularly for human patients. These RNA virus vectors may be used in the manufacture of a medicament, including a medicament for treating tumors as well as a medicament for treating viral infections.

In general, an RNA virus vector comprises an RNA virus that comprises at least a gene encoding the human DNA-dependent activator of interferon-regulatory factors (DAI) protein. The RNA virus may further comprise a gene encoding the human receptor-interacting serine/threonine-protein kinase 3 (RIPK3) protein. In some embodiments, the RNA virus is a positive sense RNA virus, non-limiting examples of which include an attenuated Kunjin virus, Polio virus, Semliki Forest virus, Venezuelan Equine Encephalitis virus, or Sinbis virus. In some embodiments, the RNA virus is a negative sense RNA virus, non-limiting examples of which include an attenuated Rabies virus, Influenza virus, Vesicular Stomatitis virus,

Respiratory Syncytial virus, Sendai virus, Measles virus, New Castle Disease virus, or Simian Virus 5 (SV5) virus. In some embodiments, the RNA virus is Influenza virus. In some embodiments, the RNA virus is a retrovirus, such as an attenuated Lentivirus.

The gene encoding the human DAI protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12. The gene may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human DAI protein may comprise the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:4, or comprise the complement thereof.

The gene encoding the human RIPK3 protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 13. The gene may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human RIPK3 protein may comprise the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:5, or comprise the complement thereof.

In some embodiments, the RNA virus further comprises a gene encoding the human mixed lineage kinase domain- like (MLKL) protein. The gene encoding the human MLKL protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:14. The gene may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human MLKL protein may comprise the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6, or comprise the complement thereof.

The RNA virus vectors may be used in a method for treating a tumor in a subject in need thereof. In general, such methods comprise comprising infecting cells of the tumor with the RNA virus vector that comprises a gene encoding the human DAI protein and, otionally, a gene encoding the human RIPK3 protein and, optionally, a gene encoding the human MLKL protein. Such genes may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human DAI protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12, for example, the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:4. The gene encoding the human RIPK3 protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 13, for example, the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:5. The gene encoding the human MLKL protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:14, for example, the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6. The methods may be used to treat any tumor. In some embodiments, the tumor is a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood.

As part of the tumor-treating method, the step of infecting cells of the tumor may comprise administering the RNA virus vector to the subject. The RNA virus vector may be actively targeted to the tumor, or may travel via the blood or other biologic fluid to the tumor location.

The RNA virus vectors may be used in methods for treating a viral infection in a subject in need thereof. In general, such methods comprise comprising infecting cells infected with the viral infection with the RNA virus vector that comprises a gene encoding the human DAI protein and, optionally, a gene encoding the human RIPK3 protein and, optionally, a gene encoding the human MLKL protein. Such genes may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human DAI protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12, for example, the nucleic acid sequence of SEQ ID NO:l or SEQ ID NO:4. The gene encoding the human RIPK3 protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 13, for example, the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:5. The gene encoding the human MLKL protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14, for example, the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6. The methods may be used to treat any viral infection, including a negative sense viral infection. The methods may be used to treat an influenza virus infection, including influenza Type A, Type B, and Type C infections.

As part of the infection-treating methods, the step of infecting cells infected with a virus infection may comprise administering the RNA virus vector to the subject. The RNA virus vector may be actively targeted to infected cells, or may travel via the blood or other biologic fluid to the site of the infection.

An RNA virus vector may be used for the treatment of a tumor. The RNA virus vector may be used for the treatment of a tumor of the head and neck. The RNA virus vector may be used for the treatment of a tumor of the esophagus. The RNA virus vector may be used for the treatment of a tumor of the lung. The RNA virus vector may be used for the treatment of a tumor of the breast. The RNA virus vector may be used for the treatment of a tumor of the pancreas. The RNA virus vector may be used for the treatment of a tumor of the kidney. The RNA virus vector may be used for the treatment of a tumor of the liver. The RNA virus vector may be used for the treatment of a tumor of the stomach. The RNA virus vector may be used for the treatment of a tumor of the colon. The RNA virus vector may be used for the treatment of a tumor of the ovary. The RNA virus vector may be used for the treatment of a tumor of the uterus. The RNA virus vector may be used for the treatment of a tumor of the prostate gland. The RNA virus vector may be used for the treatment of a tumor of the bladder. The RNA virus vector may be used for the treatment of a tumor of the blood.

An RNA virus vector may be used for the treatment of a viral infection. The RNA virus vector may be used for the treatment of a negative sense RNA virus infection. The RNA virus vector may be used for the treatment of an influenza Type A infection. The RNA virus vector may be used for the treatment of an influenza Type B infection. The RNA virus vector may be used for the treatment of an influenza Type C infection.

Brief Description Of The Drawings

Figures 1A through 1G show RIPK3 is required for IAV- induced lysis of MEFs and alveolar epithelial cells. Figure 1A shows Ripk3+/+ and ripk3-/- MEFs were infected with the indicated strains of influenza virus at m.o.i.=2 or treated with TNF-a (50ng/ml) in the presence of cycloheximide (250ng/ml) and zVAD (50μΜ) and cell viability was determined at 24 h.p.i. Figure IB shows photomicrographs of ripk3+/+ and ripk3-/- MEFs infected with PR8 or treated with TCZ for 24 hours. Figure 1C shows FACS analysis of ripk3 +/+and ripk3-/- MEFs infected with PR8-GFP (m.o.i.=2). The y-axis shows side scatter. Figure ID shows Ripk3+/+ and ripk3-/- MEFs infected with PR8 were examined for virus replication by immunoblotting with antiserum raised against PR8 or a monoclonal antibody to NS1. A non-specific band detected in uninfected lysates by the anti-PR8 antiserum is indicated with an asterisk (*). Molecular weights (in kDa) are shown to the left. Figure IE shows kinetics of cell death after PR8 infection of ripk3+/+ and ripk3-/- MEFs at the indicated m.o.i.s. Figure IF shows Ripk3-/- (in the presence or absence of 50 μΜ zVAD) , ripk3-/-casp8-/-, or ripk3-/-fadd-/- MEFs were infected with PR8 and cell viability was determined 36 h.p.i. Figure 1G shows parental LET1 lung epithelial cells, or LET1 cells in which RIPK3 expression was ablated by CRISPR/Cas9 targeting of the sequence 5'-TGAGAACGTTCTGCTCCTGC-3' (SEQ ID NO:21) in the murine ripk3 gene, were infected with PR8 and viability (left) or progeny virion output (right) was determined 12 h.p.i. Inset: Immunoblot showing RIPK3 levels in these cells, with β-actin included as a loading control. Error bars represent mean +/- S.D. * p <0.05; ** p <0.005.

Figures 2A through 2C show the role of TNF-a and TRAIL in IAV-activated RIPK3- independent apoptosis. Figure 2 A shows reconstitution of ripk3 ^~/~ MEFs with full-length murine RIPK3 restores susceptibility to IAV-induces cell death. Immortalized ripk3 ^'A MEFs were retro virally reconstituted with full-length wild-type murine RIPK3 , or with a control vector (Vec), infected with PR8 (m.o.i.=5) and examined for viability by Sytox Green positive (dead) cells were expressed as object counts per well. Figure 2B shows DNA microarray analysis profiles expression patterns of TNF superfamily members in IAV- infected ripk3 ^~/~ MEFs. For this study, we used the PR8-ANS1 mutant, to avoid NS1 -mediated suppression of RLR responses and allow full expression of the IAV-activated transcriptome in MEFs. From this analysis, only genes encoding deposited into GEO were identified; Series Number GSE80740. Figure 2C shows Ripk3 ^'A MEFs were infected with PR8 (m.o.i.=2) in the presence or absence of neutralizing antibodies to murine TNF-a (5 μg/ml) or TRAIL-R2 (10 μg/ml), and viability was determined 36 h.p.i Equivalent amount of rabbit (rlgG) and goat (glgG) immunoglobulins were used as controls. At these doses, anti-TNF-a and anti-TRAIL-R2 antibodies were able to robustly protect against apoptosis activated by the combinations of TNF-a (0.5 ng/niL) and SMAC mimetic (5 μΜ), or TRAIL (2 μg/ml) and cycloxehimide (CHX, 250 ng/ml),

respectively. Error bars represent mean +/- S.D. NS = not significant; *p <0.005.

Figure 3 shows the levels of effector proteins in cells used. Levels of key cell death effector proteins were examined in the indicated murine cell lines by immunoblot analysis following treatment with mIFN-β (1000 U/ml) for up to 24 hours. Figures 4 A through 4E show IAV activates formation of a RIPK3 -containing necrosome complex. Figure 4A shows wild type MEFs were infected with PR8 (m.o.i.=2) in the presence or absence of zVAD (25 μΜ), or treated with TCZ (right). Cells were lysed at the indicated time points, and anti-RIPK3 immunoprecipitates were examined for necrosome formation. IAV- or TCZ- activated RIPK3-RIPK1 necrosomes additionally contain FADD and MLKL. Whole-cell extract (5% input) was examined in parallel for RIPK1, RIPK3, FADD, MLKL, and IAV NS1 proteins. Figure 4B shows time course of RIPK1/RIPK3 necrosome formation demonstrates that necrosome assembly succeeds virus replication. Figure 4C shows RIPK3 immunoprecipitations performed on extracts from PR8-infected MEFs that were pre- treated with the indicated doses of Actinomycin D (ActD), cycloxehimide (CHX) or Nucleozin (Nuc), before infection indicates that the IAV-induced necrosome formation requires ongoing transcription, translation, and viral replication. Figure 4D shows RIPK3 immunoprecipitations were performed on extracts from MEFs infected with PR8 or treated with TCZ in the presence or absence of the indicated doses of the RIPK3 kinase inhibitors GSK'872 or GSK'843 (left) or the RIPK1 kinase inhibitors GSK'963 or GSK'481 (right). Figure 4E shows primary early passage wild-type (WT) and the indicated knock-out MEFs were infected with PR8 in the presence of zVAD (50 μΜ) with or without the RIPK3 inhibitor GSK'843 (5 μΜ). As controls, ripk3-/- MEFs retro virally reconstituted with full- length murine RIPK3, or with an empty vector, were used. Viability was determined 24 h.p.i. Error bars represent mean +/- S.D. ** p <0.005.

Figures 5A through 5E show RIPK3 activates parallel pathways of necroptosis and apoptosis upon IAV infection. Figure 5A shows Ripk3+/+ MEFs were infected with PR8 (m.o.i.=2) in the presence or absence of zVAD (50 μΜ), RIPK1 kinase inhibitors (GSK'963 (5 μΜ), GSK'481 (5 μΜ), and Nec-1 (50 μΜ)), or RIPK3 kinase inhibitors (GSK'872 (5 μΜ), and GSK'843 (5 μΜ)) and cell viability was determined 24 h.p.i. TCZ-treated wild-type MEFs exposed to the same doses of RIPK1 or RIPK3 inhibitors were used as controls. Figure 5B shows LETl lung epithelial cells were infected with PR8 in the presence or absence of zVAD (50 μΜ) or GSK'843 (5 μΜ), and viability was determined 12 h.p.i. LETl cells treated with TCZ were included as controls. Figure 5C shows human HT-29 cells were infected with PR8 (m.o.i.=5) in the presence or absence zVAD (50 μΜ) or GSK'840 (3 μΜ) and viability was determined 14 h.p.i. HT-29 cells treated with human TNF-a (0.5 ng/mL), SMAC mimetic LCL161 (5 μΜ), and zVAD (50 μΜ) were included as controls (TSZ). Figure 5D shows kinetics of cell death after wild-type or UV- inactivated (PR8 UV) PR8 infection in ripk3+/+ MEFs in the presence or absence of caspase inhibitor QvD (40 μΜ) or GSK'872 (5 μΜ) was analyzed for up to 36 h.p.i. Cell death was determined Sytox Green uptake, and quantified as number of Sytox Green positive cells over time. Figure 5E shows wild-type MEFs infected with PR8 in the presence of GSK' 843 (5 μΜ), zVAD (50 μΜ) or both inhibitors together were examined for phosphorylated MLKL or cleaved caspase 8 by immunoblot analysis at the indicated times p.i. Error bars represent mean +/- S.D. * p <0.05; ** p <0.005.

Figure 6 shows IAV-induces necrosome formation is independent of type 1 IFNs, TNF- a, or known RNA sensing pathways. RIPK3-RIPK1 necrosome formation was evaluated on lysates from the indicated PR8-infected wild-type or knock-out MEFs. PR8 (m.o.i.=2) in the presence of zVAD (25 μΜ) was used to stimulate necrosome formation.

Figures 7A and 7B show activation of apoptosis and necroptosis is intrinsic to the IAV- infected cell. Figure 7A shows wild-type MEFs were infected with PR8-GFP (m.o.i.=2) for 18 hours and sorted by GFP-positive and GFP-negative populations, representing cells harboring actively-replicating virus and cells without detectable virus replication, respectively. Figure 7B shows GFP-positive and GFP-negative cells were examined for phosphorylation of MLKL and cleavage of caspase 8 by immunoblot analysis.

Figures 8 A and 8B show MLKL drives necroptosis and FADD mediates apoptosis following IAV infection. Figure 8A shows wild type, fadd -/-, rnlkl-/-, and mlkl-/-fadd-/-double knock-out MEFs were infected with PR8 in the presence or absence of pan-caspase inhibitor zVAD (50 μΜ), the caspase 8 inhibitor zIETD (50 μΜ) and/or 5 μΜ of RIPK3 kinase inhibitors (GSK' 872 and GSK' 843). Cell viability was determined 24 h.p.i. Figure 8B shows wild type, fadd -/-, rnlkl-/-, rnlkl-/- fadd-/-, and ripk3-/- MEFs were infected with PR8 for 16 hours, and examined for phosphorylated MLKL or cleaved caspase 8 by immunoblot analysis. Error bars represent mean +/- S.D. * p <0.05; ** p <0.005.

Figures 9 A through 9D show IAV activates both RIPK3 -dependent early apoptosis and RIPK3 -independent late apoptosis in MLKL-deficient cells. Figure 9A shows zIETD is a weaker inhibitor of IAV-activated apoptosis than zVAD. Mlkl ^~ ' ^~ MEFs were infected with PR8 in the presence or absence of zVAD (50 μΜ), zIETD (50 μΜ) added once, or zIETD added once and supplemented at 12 h.p.i (two last bars), and cell viability was determined 24 h.p.i. Figure 9B shows kinetics of cell death after PR8 (m.o.i.=5) infection of ripk3 ^+/+ , ripk3 ^~ ' ^~ , and rnlkl ' ^' fadd ^' ' ^' double knock-out MEFs. Figure 9C shows wild types (ripk3 ^+/+ ) and rnlkl ^' ' ^' fadd ^' ' ^' double- knockout MEFs infected with PR8 (m.o.i.=5) for the indicated times and examined for virus replication by immunoblotting with antiserum to PR8 or antibodies to NSl. A non-specific band detected in uninfected lysates by the anti-PR8 antiserum is indicated with an asterisk (*).

Molecular weights (in kDa) are shown to the left. Figure 9D shows Mlkl ^~ ' ^~ , ripk3 ^~ ' ^~ , and ripk3 ^~ ' ^~ rnlkl ' ^' MEFS were infected with PR* (m.o.i. =5) in the presence or absence of zVAD (50 μΜ) and cell viability was determined at 24 and 36 h.p.L, to evaluate PIPK3 -dependent early apoptosis and RIPK3 -independent late apoptosis, respectively. Both early and late apoptosis proceed normally in cells with MLKL-deficiency. Error bars represent mean +/- S.D. NS= not significant; * p <O.05; **p <0.005; ***p <0.0005.

Figures 10A through 10D show RIPK1 mediates RIPK3 -dependent apoptosis in IAV- infected cells. Figure 10A shows Ripkl-/-, ripkl k45/a/k45a, and ripkl dl38n/dl38n MEFs were infected with PR8 (m.o.i.=2) in the presence or absence of zVAD (50 μΜ) or 5 μΜ of the RIPK3 inhibitors GSK'872 or GSK'843, and viability was determined 24 h.p.i. Figure 10B shows Ripkl-/-, ripkl k45/a/k45a, and ripkldl38n/dl38n MEFs were treated with TCZ and viability was determined 24 h.p.i. Figure IOC shows Ripkl-/-, ripkl dl38n/dl38n (referred to as ripklkd/kd), and ripk3-/- MEFs were infected with PR8 and examined for phosphorylated MLKL or cleaved caspase 8 by immunoblot analysis at the indicated times p.i. Figure 10D shows Ripkl +/+ and ripkl-/- MEFs were infected with PR8 in the presence of zVAD (25 μΜ). Cells were lysed at the indicated time points, and RIPK3-immunoprecipitated lysates were examined for presence of FADD. Error bars represent mean +/- S.D. * p <0.05; ** p <0.005; *** p <0.0005.

Figures 11A through 11G show RIPK3-activated necroptosis and apoptosis pathways are both required for protection against IAV in vivo. Figure 11A shows survival analysis of age- and sex-matched ripk3+/+ and ripk3-/- mice infected with PR8 (4000 EID50/mouse by i.n.). Figure 11B shows survival analysis of age- and sex-matched mlkl+/+, mlkl-/-, and mlkl-/- fadd-/- double knock-out mice with PR8 (4000 EID50/mouse by i.n.). Data are pooled from two independent experiments. Figure 11C shows virus titers were determined on infected lungs 3 or 9 d.p.i. by plaque assay (n=3-5). Figure 11D shows paraffin embedded sections of ripk3+l+ and ripk3-l- mouse lungs 6 d.p.i. were stained with anti-IAV antibodies, and slides were scanned and virus spread was quantified using Aperio ImageScope. Representative images of anti-IAV stained 11 ripk3+l+ (top) and 10 ripk3-l- (bottom) lungs are shown to the left, and quantification of virus spread from lungs of individual mice are shown to the right. Figure 1 IE shows representative images of H&E- stained 11 ripk3+l+ and 10 ripk3-l- lungs 6 d.p.i at two magnifications reveal severe edema and alveolar damage in ripk3-l- pulmonary tissue (left). Quantification of alveolar fibrin deposition from lungs of individual mice is shown to the right. The extent and severity of pulmonary damage was determined from blinded sections examined by a pathologist and scored for the distribution of alveolar fibrin deposition on a severity scale of 1-5. These scores were converted to a semi-quantitative scale). Figure 11F shows representative images of CD3+ staining in 11 ripk3+/+ and 10 ripk3-/- lungs 6 d.p.i. Quantification of CD3+ staining from lungs of individual mice are shown to the right. Figure 11G shows quantification of frequencies of IAV-specific (DbPA224) CD8+ T cells and percentage of IAV PA224 peptide- stimulated poly- functional IFN-Y+TNF-a+CD8+ T cells in BAL fluid from ripk3+l+ and ripk3- I- animals (n=3-5) at 9 d.p.i. The low pathogenic HKx31 reassortant of PR8 was used in these experiments to avoid 9 d.p.i. lethality effects seen with PR8. Error bars represent mean +/- S.D. * /? <0.05; *** p <0.0005.

Figures 12A through 12G show DAI is essential for IAV-induced cell death in MEFs and airway epithelial cells. Figure 12A shows wild type (WT) or zbpl ' ^' MEFs from two separately-maintained colonies (MEF 1 and MEF 2) were infected with PR8 (MOI=2 and 5), or treated with the combination of TNF-a (50 ng/ml) + cycloheximide (250 ng/ml) + zVAD (50 μΜ) (TCZ) and cell viability was determined 24 h.p.i. Figure 12B shows photomicrographs of WT and zbpl ' ^' MEFs infected with PR8 (MOI=2) or treated with TCZ for 24 hours. Figure 12C shows FACS analysis of WT and zbpl ' ^' MEFs infected with PR8-GFP (MOI=2) for 18 hours. The y-axis shows side scatter. Figure 12D shows lysates from WT and zbpl ' ^' MEFs infected with PR8 (MOI=2) for the indicated times were examined for expression of NS1 and DAI. β- actin was used as a loading control. Figure 12E shows immortalized zbpl ' ^' MEFs reconstituted with empty vector (EV), wild-type full-length murine DAI (DAI), or murine DAI with mutant RHIM domain (amino acids 192-195 IQIG to AAAA) (DAI mutRHIM) were infected with PR8 (MOI=2) and cell viability was determined 24 h.p.i. Expression of WT or mutant DAI in these cells is shown to the right. Figure 12F shows WT MEFs in which DAI expression was ablated by CRISPR/Cas9 targeting of the sequences 5 ' -TCTGGAGTCACACAAGAGTCCCCT-3 ' (CRISPR 1) (SEQ ID NO:16) or 5 ' -GCTCAGTACATCTACATGGACAAGTCCTTG-3 ' (CRISPR 2) (SEQ ID NO: 17) in the murine zbpl gene were infected with PR8 (MOI =2) and cell viability was determined 24 h.p.i. Ablation of DAI expression was confirmed by immunoblotting (right). Figure 13G shows LETl cells in which DAI expression was ablated by CRISPR/Cas9 targeting of murine zbpl gene using sequences shown in 12E, were infected with PR8 (MOI=2) and cell viability was determined 12 h.p.i. In parallel, progeny virion output from these cells was determined 30 h.p.i. (right). Ablation of DAI expression was confirmed by immunoblotting.

Figures 13 A through 13F show DAI is required for IAV induced formation of RIPK3- containing necrosome complex. Figure 13 A shows wild-type MEFs were infected with PR8 (MOI=2) and anti-RIPK3 immunoprecipitates were examined for DAI, RIPKl and MLKL at the indicated times p.i. Figure 13B shows wild-type (WT), zbpl ' ^' , and ripk3 ^'A MEFs infected with PR8 (MOI=2) (MOI=2) and examined for phosphorylated (p) MLKL and cleaved caspase-8 (CC8 pl8). In parallel, wild-type (WT), zbpl ' ^' , and ripk3 ^A MEFs were treated with TCZ hours and phosphorylated MLKL. Figure 13C shows Zbpl ' ^' MEFs reconstituted with empty vector (Vec), full-length murine DAI (DAI), or DAI with a mutated RHIM domain (DAI mutRHIM) were infected with PR8 (MOI=2) for 14 or 18 hours and examined for phosphorylated MLKL and cleaved caspase-8. In parallel, reconstituted cells were treated with TCZ and examined for expression of phosphorylated p MLKL. Figure 13D shows anti-RIPK3 immunoprecitates were examined from zbpl ^~ ' ^~ MEFs reconstituted with full-length murine DAI (DAI) or DAI with a mutated RHIM domain (DAI mutRHIM) infected with PR8 (MOI=2) for DAI and RIPK3. Whole-cell extract (5% input) was examined in parallel for RIPK3, DAI and IAV NS1 proteins. Figure 13E shows kinetics of cell death after PR8 infection (MOI=2) of WT, zbpl ' ^' , ripk3 ^'A , ripkl ^~/~ ripk3 ^~/~ double knockout and mlkl' ^~ fadd ^~ ' ^~ double knockout MEFs. Figure 13F shows whole cell extracts from WT, zbpl ' ^' , ripk3 ^~ ' ^~ and ripkl ' ripkS ^' ' ^' double knockout MEFs infected with PR8 at MOI=2 or MOI=5 were examined for expression of cleaved caspase-8 (CC8).

Figures 14A through 14E show DAI senses IAV RNA. Figure 14A shows a schematic of mutants used in studies (see, Figure 14B) Zbpl ' ^' MEFs reconstituted with WT DAI or with the indicated mutants were infected with PR8 (MOI=2) and cell viability was measured 24 h.p.i. Figure 14C shows Zbpl ' ^' MEFs reconstituted with WT DAI or with the indicated mutants were infected with PR8 (MOI=2, top) or TCZ (bottom) and examined for phosphorylated MLKL. Figure 14D shows similarity of Zal and Za2 domains bound to either Z-DNA and Z-RNA substrates. The known structures of human DAI-Za2 bound to Z-DNA (PDB code 3EYI, first panel) and ADAR1 Zal bound to Z-RNA (PDB code 2GXB, second panel). Homology model of murine DAI Za2 bound to Z-RNA is shown in the third panel, with the location of N 122 and Y126 depicted. A superposition of the mDAIZa2 bound to zRNA compared to the known structure of hDAIZa2 bound to zDNA. The conserved N and Y residues of the mDAIZa2:Z- RNA complex are shown as ball-and-stick representation, while the corresponding residues in hDAI-Za2:zDNA complex are shown as sticks. Figure 14E shows Integrated Genome Viewer representation of captured sequencing reads of negative polarity from FLAG- immunoprecipitations of PR8-infected 293T cells expressing either FLAG-DAI (top) or FLAG- RIG-I (bottom). Horizontal bars represent a single 150 nt read and the position where it aligned relative to each IAV gene segment, depicted at the top of each sample. The histogram is a synopsis of total coverage in any given position.

Figures 15 A through 15E show DAI is required for protection against IAV in vivo. Figures 15A and 15B show survival and weight loss, respectively, analysis of age- and sex- matched WT and zbpl ' ^' mice infected with PR8 (1000 EIDso/mouse i.n.). Dead mice are represented by black circles. Figure 15C shows virus titers from WT and zbpl ' ^' mice infected with PR8 (750 EIDso/mouse i.n.) were determined by plaque assay (n=3-5 mice/condition). Figure 15D shows representative images of lungs from WT and zbpl ' ^' mice stained with anti- IAV antibodies 9 d.p.i. Figure 15E shows model of DAI- induced cell death following IAV infection. DAI dimers sense IAV vRNA in a manner requiring the Za2 domain, leading to RHIM-based interaction with, and activation of, RIPK3. Downstream of RIPK3, parallel pathways of MLKL-driven necroptosis and RIPKl/FADD/caspase-8-mediated apoptosis cooperate to eliminate the infected cell. DAI also activates apoptosis independently of RIPK3, via a putative RIPKl/FADD-caspase-8 axis.

Figures 16A through 16E show DAI is selectively required for IAV- and IBV induced cell death in MEFs and airway epithelial cells. Figure 16A shows wild-type (WT) or zbpl ' ^' MEFs were infected with IAV strains PR8, Brisbane/10/2007, Brisbane/59/2007, and IBV strains Brisbane/60/2008 and Florida/4/2006 (MOI=2) and cell viability was measured 24 h.p.i. Figure 16B shows whole cell extracts from WT, zbpl ^'1' and ripk3 ^~ ' ^~ MEFs treated with IFN-b for 18 or 24 hours were examined for FADD, Caspase-8, RIPK1, RIPK3, MLKL and DAI. Beta- actin was used as a loading control. Figure 16C shows WT MEFs or WT MEFs in which DAI expression was ablated by CRISPR/Cas9 targeting of the sequences CRISPR 1 CRISPR 2 in the murine zbpl gene were treated with CZ or TCZ and cell viability was measured 24 h.p.i. Figure 16D shows Zbpl ' ^' MEFs reconstituted with empty vector (Vec), full length murine DAI (DAI) and full-length DAI with a mutated RHIM domain (DAI mutRHIM) were treated with CZ or TCZ and cell viability was measured 24 h.p.i. Figure 16E shows LET1 cells in which DAI expression was ablated by CRISPR/Cas9 targeting of murine zbpl gene using sequences described in C were treated with CZ or TCZ and cell viability was measured 24 h.p.i.

Figure 17 shows DAI is required for activation of RIPK3 in LET1 AECs. Whole cell extracts of LET1 cells expressing empty vector (Vec), or LET1 cells in which with a zbpl or ripk3 were deleted by CRISPR/Cas9 targeting were infected with PR8 (MOI=2, top) or TCZ (bottom) were examined for phosphorylated MLKL and cleaved caspase-8.

Figures 18 A through 18E show DAI senses IAV RNA in a manner requiring its Za2 domain. Figure 18A shows kinetics of cell death in PR8-infected immortalized zbpl ' ^' MEFs reconstituted with WT DAI or its mutants. Figure 18B shows expression levels of FLAG-tagged constructs in immortalized ibpl ' ^' MEFs reconstituted with WT DAI or its mutants. Figure 18C shows expression of FLAG-tagged DAI constructs in FLAG immunoprecipitates from IAV- infected 293T cells 12 h.p.i. Figure 18D shows quantification of RNA eluted from anti-FLAG immunoprecipitate of the indicated FLAG-DAI constructs from PR8-infected 293T cells 12 h.p.i. Figure 18E shows PCR detection of PB2 vRNA in eluted RNA from FLAG-DAI immunoprecipitates, using primers specific for the vRNA ends of the PB2 segment. RNA+: A549 cells infected (MOI=3) with PR8. Description Of Embodiments

Various terms relating to embodiments of the present disclosure are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided in this document.

As used throughout, the singular forms "a," "an," and "the" include plural referents unless expressly stated otherwise.

A molecule such as a polynucleotide has been "isolated" if it has been removed from its natural environment and/or altered by the hand of a human being.

Nucleic acid molecules include any chain of at least two nucleotides, which may be unmodified or modified RNA or DNA, hybrids of RNA and DNA, and may be single, double, or triple stranded.

Inhibiting includes, but is not limited to, interfering with, reducing, decreasing, blocking, preventing, delaying, inactivating, desensitizing, stopping, knocking down (e.g., knockdown), and/or downregulating the biologic activity or expression of a protein or biochemical pathway.

The terms "subject" and "patient" are used interchangeably. A subject may be any animal, such as a mammal. A mammalian subject may be a farm animal (e.g., sheep, horse, cow, pig), a companion animal (e.g., cat, dog), a rodent or laboratory animal (e.g., mouse, rat, rabbit), or a non-human primate (e.g., old world monkey, new world monkey). In some embodiments, the mammal is a human, such as a MIBC patient.

It has been observed in accordance with the present disclosure that the protein DAI (also known as ZBPl/DLM-1) was required for RIPK3 activation and cell death in IAV-infected murine cells. It was observed that DAI senses IAV RNA via its Za2 domain and, in turn, participates in activation of RIPK3. DAI also mediates RIPK3 -independent apoptosis, by activating a RIPKl-FADD-caspase 8 pathway. These findings identify DAI as a mediator of IAV-driven cell death, and implicate this protein as a new sensor of RNA viruses. Thus, while DAI is clearly important in host defense against DNA viruses, it also participates in the immune response to RNA viruses. Influenza virus is a negative sense RNA virus. The virus encodes an RNA polymerase (RNA-dependent RNA polymerase) that constructs a positive sense template of viral RNA, which effectively serves as an mRNA for translation of viral proteins during an infection.

Without intending to be limited to any particular theory or mechanism of action, it is believed that during construction of the plus sense RNA template by the virus RNA polymerase, at least a transient double stranded RNA (dsRNA) molecule is produced (including the original negative sense strand and the newly synthesized positive sense strand). Without intending to be limited to any particular theory or mechanism of action, it is further believed that this dsRNA molecule interacts with the DAI protein (e.g., at the Za2 domain that senses double stranded DNA), thereby activating DAI, which in turn activates RIPK3 to induce either or both of RIPK3- dependent apoptosis and RIPK3 -independent necroptosis via the necrosome complex.

Nevertheless, it is possible that single stranded RNA can interact with and activate DAI.

Accordingly, the present disclosure features compositions and methods that take advantage of the therapeutic capacity of DAI to recognize RNA, and facilitate cell death by way of apoptosis and/or necroptosis, with necroptosis further facilitating an immune response, including an inflammatory response.

In some embodiments, the present disclosure provides virus vectors, comprising a gene encoding the DNA-dependent activator of interferon-regulatory factors (DAI) protein. In some embodiments, the present disclosure provides a virus vector, comprising a gene encoding the DAI protein and a gene encoding the receptor-interacting serine/threonine-protein kinase 3 (RIPK3) protein. In some embodiments, the present disclosure comprises a virus vector comprising a gene encoding the RIPK3 protein, but not a gene encoding the DAI protein. The virus vector may be a DNA virus or an RNA virus. An RNA virus vector may be a retrovirus vector, a positive sense RNA virus, or a negative sense RNA virus, and the RNA may comprise single stranded RNA. Negative sense RNA comprises a nucleic acid sequence that is complementary to the mRNA that it encodes, and which is produced by RNA-dependent RNA polymerase. Positive sense RNA is similar to mRNA and may be translated accordingly.

Retroviruses suitable for use as a vector include, but are not limited to a Lentivirus. Positive sense RNA viruses suitable for use as a vector include, but are not limited to, the Kunjin virus, Polio virus, Semliki Forest virus, Venezuelan Equine Encephalitis virus, and Sinbis virus. Negative sense RNA viruses suitable use as a vector include, but are not limited to, the Rabies virus, Influenza virus, Vesicular Stomatitis virus, Respiratory Syncytial virus, Sendai virus, Measles virus, New Castle Disease virus, or Simian Virus 5 (SV5) virus. Negative sense RNA viruses are suitable insofar as their production of dsRNA induces DAI to sense the presence of dsRNA and facilitate RIPK3 and necrosome-mediated cell death. In any case, it may be desired that the virus vector is in a live, but attenuated form, such that the virus vector does not substantially induce untoward effects in the host during use as a therapeutic agent, for example, the virus' s normal virulence factors are removed or altered/lessened such that the virus itself does not substantially cause illness in the host during use.

In some embodiments, the DAI protein encoded by the gene is the human DAI protein. The gene may be in DNA or RNA form, and may comprise the sense sequence or antisense sequence form of the gene. In some embodiments, the gene encodes a DAI protein having the amino acid sequence of SEQ ID NO: 12. In some embodiments, the gene may comprise the nucleic acid sequence of SEQ ID NO: l (DNA) or SEQ ID NO:4 (RNA), or may comprise the complementary nucleic acid sequence thereof (e.g., SEQ ID NO:7 (DNA) or SEQ ID NO: 10 (RNA)).

In some embodiments, the RIPK3 protein encoded by the gene is the human RIPK3 protein. The gene may be in DNA or RNA form, and may comprise the sense sequence or antisense sequence form of the gene. In some embodiments, the gene encodes a RIPK3 protein having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the gene may comprise the nucleic acid sequence of SEQ ID NO:2 (DNA) or SEQ ID NO:5 (RNA), or may comprise the complementary nucleic acid sequence thereof (e.g., SEQ ID NO:8 (DNA) or SEQ ID NO: 1 1 (RNA)).

In some embodiments, the virus vector further comprises a gene encoding the human mixed lineage kinase domain- like (MLKL) protein. The gene may be in DNA or RNA form, and may comprise the sense sequence or antisense sequence form of the gene. In some embodiments, the gene encodes a MLKL protein having the amino acid sequence of SEQ ID NO: 14. In some embodiments, the gene may comprise the nucleic acid sequence of SEQ ID NO:3 (DNA) or SEQ ID NO:6 (RNA), or may comprise the complementary nucleic acid sequence thereof (e.g., SEQ ID NO:9 (DNA) or SEQ ID NO: 12 (RNA)).

In some embodiments, the virus vector further comprises a gene encoding a caspase inhibitor protein. The caspase inhibitor protein may be a viral caspase inhibitor protein. In some embodiments, the caspase inhibitor protein inhibits caspase 8. Non-limiting examples of viral caspase inhibitors that may be encoded by such a gene include the viral inhibitor of caspase 8- induced apoptosis (vICA) protein, cytokine response modifier A (CrmA), and Vaccinia virus serpin SPI-2/B 13R. The gene may be in DNA or RNA form, and may comprise the sense sequence or antisense sequence form of the gene. In some embodiments, for example, where the virus vector comprises an influenza virus (e.g., Type A), the virus may activate DAI-RIPK3 cell death without a need for concurrent caspase inhibition.

In some embodiments, the virus vector further comprises a surface (e.g., envelope) protein or glycoprotein that facilitates tropism between the virus vector and the target cells or tissue. In some embodiments, the virus vector has broad tropism such that the virus vector is capable of infecting a wide variety of host cells. In some embodiments, the virus may be engineered to express particular proteins or glycoproteins to extend the natural tropism of the virus vector to additional cell types, or to otherwise facilitate viral interaction with host cell receptors toward enhancing infection efficiency. Thus, the virus vector may be targeted to particular tissues in order to achieve tissue tropism, for example, to a tumor. Targeting may comprise pseudotyping, for example, where cell surface glycoproteins on the virus are added, modified, or replaced to alter specificity for the virus vector' s receptor(s) and, hence, permit the virus to different cell types and tissues than it otherwise would in an unaltered state.

In some embodiments, the virus vector is capable of targeting, binding to, and infecting tissue or cells that are infected with another, pathogenic virus, or tissue or cells that are cancerous. In some embodiments, the virus vector is capable of targeting, binding to, and infecting tissue or cells that are infected with a pathogenic strain of an influenza virus (e.g., Type A, Type B, or Type C influenza virus). The pathogenic influenza virus may comprise a seasonal or pandemic strain of the influenza virus. In some embodiments, the virus vector is capable of targeting, binding to, and infecting cancerous or precancerous tissue or cells. The cancerous or precancerous tissue or cells may comprise any tissue or cells capable of expressing the DAI and RIPK3 proteins encoded by the virus vector, as well as capable of expressing the MLKL protein and/or caspase inhibitor protein if such are encoded by the virus vector. The cancerous or precancerous tissue may be deficient in endogenous DAI protein.

Thus, the virus vectors may be used therapeutically to treat viral infections or to treat tumors. In some embodiments, methods for treating a tumor comprise infecting cells of the tumor with any of the virus vectors described or exemplified herein. The cells may be infected by administering the virus vector to the tumor, e.g., by directly contacting the tumor cells with the virus vector, or by administering the virus vector to the tumor patient, e.g., to the blood, followed by delivery of the vector to the tumor by the body. Delivery to the tumor may be targeted, and/or may be facilitated.

The tumor may be any tumor that includes the biochemical machinery capable of causing RIPK3 -mediated cell death. The tumor may be a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood. The tumor may have reduced expression or no expression of endogenous RIPK3.

In some embodiments, methods for treating a viral infection comprise infecting cells infected with a virus with any of the virus vectors described or exemplified herein. The virally- infected cells may be further infected with the virus vector by administering the virus vector to the cells, e.g., by directly contacting the virally- infected cells with the virus vector, or by administering the virus vector to the virally- infected patient, e.g., to the blood, followed by delivery of the vector to the site of the infection by the body. Delivery to the site of the virus infection may be targeted, and/or may be facilitated. In some embodiments, the virus infection to be treated is a pathogenic virus infection, for example, an influenza virus infection. Thus, for example, in some embodiments, virus vectors based on the influenza virus may be used to treat cells that are infected with a strain of influenza virus, including influenza type A, type B, or type C.

Any virus vector described or exemplified herein may be used in the manufacture of a medicament, for example, in the manufacture of a medicament for the treatment of a virus infection such as an influenza virus infection (e.g., influenza type A infection, influenza type B infection, or influenza type C infection). The virus vectors may also be used in the manufacture of a medicament for the treatment of a tumor such as a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood. Thus, a virus vector may be used in the treatment of a viral infection, or may be used in the treatment of a tumor.

The combination of DAI and RIPK3 may be used therapeutically to kill tumor cells or virally- infected cells. Nevertheless, the cell death induced by DAI and RIPK3 may be undesired in some contexts. For example, without intending to be limited to any particular theory or mechanism of action, it is believed that the activity of DAI and/or RIPK3 may, in some cases such as pandemic strains of the influenza virus may contributes to the virulence of the virus. Thus, it is believed that inhibiting DAI or RIPK3-induced cell death may benefit patients infected with a pandemic or virulent strain of the influenza virus. In such cases, it is believed that keeping host-infected cells alive longer will reduce the virulence of the virus. Accordingly, a patient infected with a pandemic or virulent strain of virus such as a pandemic strain of the influenza virus (type A, type B, or type C) may be treated by inhibiting one or more of DAI or RIPK3 in virally- infected cells. Thus, a subject infected with a pandemic strain of the influenza virus may be treated by administering to the subject an effective amount of a DAI or a RIPK3 inhibitor. The inhibitor may be potaninib or may be debrafenib, or any derivative thereof that inhibits DAI or RIPK3. Accordingly, potaninib or debrafenib, or derivative thereof, may be used in the treatment of influenza virus infection, particularly for a pandemic or virulent strain of influenza virus, where inhibition of necrosis may be desired.

The following representative embodiments are presented:

Embodiment 1. An RNA virus vector, comprising an RNA virus comprising a gene encoding the human DNA-dependent activator of interferon-regulatory factors (DAI) protein and a gene encoding the human receptor- interacting serine/threonine-protein kinase 3 (RIPK3) protein.

Embodiment 2. The RNA virus vector according to embodiment 1, wherein the RNA virus is a positive sense RNA virus.

Embodiment 3. The RNA virus vector according to embodiment 1 or embodimen t2, wherein the positive sense RNA virus is an attenuated Kunjin virus, Polio virus, Semliki Forest virus, Venezuelan Equine Encephalitis virus, or Sinbis virus.

Embodiment 4. The RNA virus vector according to embodiment 1, wherein the RNA virus is a negative sense RNA virus.

Embodiment 5. The RNA virus vector according to embodiment 1 or embodiment 4, wherein the negative sense RNA virus is an attenuated Rabies virus, Influenza virus, Vesicular Stomatitis virus, Respiratory Syncytial virus, Sendai virus, Measles virus, New Castle Disease virus, or Simian Virus 5 (SV5) virus.

Embodiment 6. The RNA virus vector according to embodiment 1, wherein the RNA virus is a retrovirus.

Embodiment 7. The RNA virus vector according to embodiment 1 or embodiment 6, wherein the retrovirus is an attenuated Lentivirus.

Embodiment 8. The RNA virus vector according to any one of embodiments 1 to 7, wherein the gene encoding the human DAI protein comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12.

Embodiment 9. The RNA virus vector according to any one of embodiments 1 to 8, wherein the gene encoding the human DAI protein comprises RNA.

Embodiment 10. The RNA virus vector according to any one of embodiments 1 to 9, wherein the gene encoding the human DAI protein comprises a negative sense RNA.

Embodiment 11. The RNA virus vector according to any one of embodiment 1 to 7, wherein the gene encoding the human DAI protein comprises the nucleic acid sequence of SEQ ID NO:l, SEQ ID NO:4, or the complement of SEQ ID NO:l or SEQ ID NO:4. Embodiment 12. The RNA virus vector according to any one of embodiments 1 to 11, wherein the gene encoding the human RIPK3 protein comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 13.

Embodiment 13. The RNA virus vector according to any one of embodiments 1 to 12, wherein the gene encoding the human RIPK3 protein comprises RNA.

Embodiment 14. The RNA virus vector according to any one of embodiments 1 to 13, wherein the gene encoding the human RIPK3 protein comprises a negative sense RNA.

Embodiment 15. The RNA virus vector according to any one of embodiments 1 to 11, wherein the gene encoding the human RIPK3 protein comprises the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:5, or the complement of SEQ ID NO:2 or SEQ ID NO:5.

Embodiment 16. The RNA virus vector according to any one of embodiments 1 to 15, wherein the RNA virus further comprises a gene encoding the human mixed lineage kinase domain-like (MLKL) protein.

Embodiment 17. The RNA virus vector according to embodiment 16, wherein the gene encoding the human MLKL protein comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 14.

Embodiment 18. The RNA virus vector according to embodiment 15 or embodiment 16, wherein the gene encoding the human MLKL protein comprises RNA.

Embodiment 19. The RNA virus vector according to any one of embodiments 15 to 18, wherein the gene encoding the human MLKL protein comprises a negative sense RNA.

Embodiment 20. The RNA virus vector according to embodiment 15, wherein the gene encoding the human MLKL protein comprises the nucleic acid sequence of SEQ ID NO:3, SEQ ID NO:6, or the complement of SEQ ID NO:3 or SEQ ID NO:6.

Embodiment 21. A method for treating a tumor in a subject in need thereof, comprising infecting cells of the tumor with the RNA virus vector according to any one of embodiments 1 to 20.

Embodiment 22. The method according to embodiment 21, wherein the tumor is a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood.

Embodiment 23. The method according to embodiment 21 or embodiment 22, wherein infecting cells of the tumor comprises administering to the subject the RNA virus vector.

Embodiment 24. The method according to any one of embodiments 21 to 23, wherein the RNA virus vector is actively targeted to the tumor. Embodiment 25. The method according to any one of embodiments 21 to 24, wherein the subject is a human being.

Embodiment 26. A method for treating a viral infection in a subject in need thereof, comprising infecting cells infected with the viral infection with the RNA virus vector according to any one of embodiments 1 to 20.

Embodiment 27. The method according to embodiment 26, wherein the viral infection comprises a negative sense RNA virus infection.

Embodiment 28. The method according to embodiment 27, wherein the negative sense RNA virus infection is an influenza virus infection.

Embodiment 29. The method according to embodiment 28, wherein the influenza virus infection is an influenza type A virus infection.

Embodiment 30. The method according to embodiment 27, wherein the influenza virus infection is an influenza type B virus infection.

Embodiment 31. The method according to embodiment 27, wherein the influenza virus infection is an influenza type C virus infection.

Embodiment 32. The method according to any one of embodiments 26 to 31, wherein infecting cells infected with the viral infection comprises administering to the subject the RNA virus vector.

Embodiment 33. The method according to any one of embodiments 26 to 32, wherein the RNA virus vector is actively targeted to the cells infected with the viral infection.

Embodiment 34. The method according to any one of embodiments 26 to 33, wherein the subject is a human being.

Embodiment 35. Use of the RNA virus vector according to any one of embodiments 1 to 20 in the manufacture of a medicament.

Embodiment 36. Use of the RNA virus vector according to any one of embodiments 1 to 20 for the treatment of a tumor.

Embodiment 37. The use according to embodiment 36, wherein the tumor is a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood.

Embodiment 38. Use of the RNA virus vector according to any one of embodiments 1 to 20 for the treatment of a viral infection.

Embodiment 39. The use according to embodiment 38, wherein the RNA virus vector is used for the treatment of a negative sense RNA virus infection. Embodiment 40. The use according to embodiment 38 or 39, wherein the RNA virus vector is used for the treatment of an influenza type A, influenza type B, or influenza type C infection.

Embodiment 41. An RNA virus vector, comprising an RNA virus comprising a gene encoding the human DNA-dependent activator of interferon-regulatory factors (DAI) protein.

Embodiment 42. The RNA virus vector according to embodiment 41, wherein the RNA virus is a positive sense RNA virus.

Embodiment 43. The RNA virus vector according to embodiment 41 or embodiment 42, wherein the positive sense RNA virus is an attenuated Kunjin virus, Polio virus, Semliki Forest virus, Venezuelan Equine Encephalitis virus, or Sinbis virus.

Embodiment 44. The RNA virus vector according to embodiment 41, wherein the RNA virus is a negative sense RNA virus.

Embodiment 45. The RNA virus vector according to embodiment 41 or embodiment 44, wherein the negative sense RNA virus is an attenuated Rabies virus, Influenza virus, Vesicular Stomatitis virus, Respiratory Syncytial virus, Sendai virus, Measles virus, New Castle Disease virus, or Simian Virus 5 (SV5) virus.

Embodiment 46. The RNA virus vector according to embodiment 41, wherein the RNA virus is a retrovirus.

Embodiment 47. The RNA virus vector according to embodiment 41 or embodiment 46, wherein the retrovirus is an attenuated Lentivirus.

Embodiment 48. The RNA virus vector according to any one of embodiments 41 to 47, wherein the gene encoding the human DAI protein comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 12.

Embodiment 49. The RNA virus vector according to any one of embodiments 41 to 48, wherein the gene encoding the human DAI protein comprises RNA.

Embodiment 50. The RNA virus vector according to any one of embodiments 41 to 49, wherein the gene encoding the human DAI protein comprises a negative sense RNA.

Embodiment 51. The RNA virus vector according to any one of embodiment 41 to 47, wherein the gene encoding the human DAI protein comprises the nucleic acid sequence of SEQ ID NO:l, SEQ ID NO:4, or the complement of SEQ ID NO:l or SEQ ID NO:4.

Embodiment 52. A method for treating a tumor in a subject in need thereof, comprising infecting cells of the tumor with the RNA virus vector according to any one of embodiment 41 to 51. Embodiment 53. The method according to embodiment 51, wherein the tumor is a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood.

Embodiment 54. The method according to embodiment 52 or embodiment 53, wherein infecting cells of the tumor comprises administering to the subject the RNA virus vector.

Embodiment 55. The method according to any one of embodiments 52 to 54, wherein the RNA virus vector is actively targeted to the tumor.

Embodiment 56. The method according to any one of claims 52 to 55, wherein the subject is a human being.

Embodiment 57. A method for treating a viral infection in a subject in need thereof, comprising infecting cells infected with the viral infection with the RNA virus vector according to any one of embodiments 41 to 51.

Embodiment 58. The method according to embodiment 57, wherein the viral infection comprises a negative sense RNA virus infection.

Embodiment 59. The method according to embodiment 58, wherein the negative sense

RNA virus infection is an influenza virus infection.

Embodiment 60. The method according to embodiment 59, wherein the influenza virus infection is an influenza type A virus infection.

Embodiment 61. The method according to embodiment 58, wherein the influenza virus infection is an influenza type B virus infection.

Embodiment 62. The method according to embodiment 58, wherein the influenza virus infection is an influenza type C virus infection.

Embodiment 63. The method according to any one of embodiments 57 to 62, wherein infecting cells infected with the viral infection comprises administering to the subject the RNA virus vector.

Embodiment 64. The method according to any one of embodiments 57 to 63, wherein the RNA virus vector is actively targeted to the cells infected with the viral infection.

Embodiment 65. The method according to any one of embodiments 57 to 64, wherein the subject is a human being.

Embodiment 66. Use of the RNA virus vector according to any one of embodiments 41 to 51 in the manufacture of a medicament.

Embodiment 67. Use of the RNA virus vector according to any one of embodiments 41 to 51 for the treatment of a tumor. Embodiment 68. The use according to embodiment 67, wherein the tumor is a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood.

Embodiment 69. Use of the RNA virus vector according to any one of embodiments 41 to 51 for the treatment of a viral infection.

Embodiment 71. The use according to embodiment 69, wherein the RNA virus vector is used for the treatment of a negative sense RNA virus infection.

Embodiment 72. The use according to embodiment 69 or embodiment 70, wherein the RNA virus vector is used for the treatment of an influenza type A, influenza type B, or influenza type C infection.

The following examples are provided to describe the present disclosure in greater detail. They are intended to illustrate, not to limit, the present disclosure.

Examples

Example 1: RIPK3-mediated apoptosis in antiviral immunity: Materials and Methods

Mice and cells. Ripk3-/-, ripk3-/-casp8-/-, fadd-/-, ripk3-/-fadd-/-, mlkl-/-,

ripk3-/-mlkl-/-, mlkl-/-fadd-/-, ripkl-/-, ripkl k45/a/k45a , ripkldl38n/dl38n, ifnarl-/-, statl-/- , eif2ak2-/-, mavs-/-, and tnfrl-/- MEFs were generated in-house from E14.5 embryos and used within five passages in experiments. Early passage ddx58-/- and myd88-/-trif-/- MEFs were purchased from Oriental BioService Inc. (Osaka, Japan). HT-29 cells were obtained from the American Type Culture Collection (Manassas, VA).

Reagents. Biological and chemical reagents were from the following sources:

Necrostatin l(Bio Vision), Nucleozin (Sigma), Q-VD-OPh (Apexbio), zIETD.fmk (Calbiochem), zVAD.fmk (Bachem), mIFN-β (PBL), murine and human TNF-a (R&D systems), mTRAIL (R&D systems), SMAC mimetic LCL161 (Chemietek). Inhibitors of RIPK1 (GSK'481 ,

GSK'963) and RIPK3 (GSK'840, GSK'872, GSK'843) from GlaxoSmithKline. Antibodies for immunoblot analysis of β-actin (Sigma), caspase 8 (Cell Signaling), cleaved caspase 8 (Cell Signaling), FADD (Millipore), IAV NP (BioRad), IAV NS1 (Santa Cruz), MLKL (Abgent or Millipore), p-MLKL (Abeam), RIPK1 (BD Transduction Labs), RIPK3 (ProSci or Santa Cruz), were obtained from the indicated commercial sources. Neutralizing antibodies to mTNF-a (Cell Signaling) and mTRAIL-R2 (R&D systems) were obtained from the indicated sources.

Antiserum to PR8 was produced in-house. For detection of IAV and CD3 in paraffin-embedded tissue, anti-IAV antibodies (US Biologicals) and anti-CD3 antibodies (Santa Cruz) were used. For FACS, antibodies to CD8a (clone 53-6.7), anti-CD 16/CD32 (clone 2.4G2), anti-TNF-a (clone MP6-XT22), anti-IFN- γ (clone XMG1.2), and anti-CD28 were obtained from BD PharMingen.

Viruses. IAV strains PR8 and A/HKx31 were generated by reverse genetics. All IAV and IBV strains were propagated by allantoic action of embryonated hen's eggs with diluted (1 :106) seed virus. Virus titers were determined as 50% egg infectious dose (EID ₅₀ ) and by plaque assay on Madin-Darby Canine Kidney (MDCK) cells.

Virus infection and titration. Mice were anesthetized with Avertin (2,2,2- tribromoethanol) or isoflurane and infected intranasally with virus inoculum diluted in endotoxin-free phosphate-buffered saline. Mice were either monitored for survival and weight loss over a period of 18 days or sacrificed at defined time points for analysis of histology and virus replication. Mice losing >35% bodyweight were considered moribund and euthanized by CO ₂ asphyxiation. To determine progeny virus production in infected mice, lung homogenates were titered by plaque assay on MDCK cells. For cell culture experiments, near-confluent monolayers of cells were infected with virus in serum-free DMEM for 1 hour, with occasional gentle rocking, in a humidified tissue culture incubator maintained at 37 °C and 5% CO ₂ .

Following infection, the inoculum was removed and replaced with growth medium. In conditions involving small-molecule inhibitors, cells were pre-incubated for 1 hour with inhibitors before infection; after removal of inoculum and washing, inhibitors were added back to the medium. Titration of virus from supernatants of infected LETl cells was determined by a chicken red blood cell-based hemagglutination assay. Cell viability was determined by Trypan Blue exclusion or on an Incucyte Kinetic Live Cell Imaging System (Essen Bioscience).

Example 2: RIPK3-mediated apoptosis in antiviral immunity: Results

RIPK3 is required for IAV-induced cell death in murine fibroblasts and lung epithelial cells. To examine the role of RIPK3 in IAV induced cell death, early-passage murine embryo fibroblasts (MEFs) from ripk3-/- and littermate-control ripk3+/+ mice were infected with IAV strains Puerto Rico/8/1934 (PR8), Brisbane/59/2007, Brisbane/ 10/2007, and Perth/16/2009 and these cells were observed over a time course of 36 hours. PR8 (H1N1) is a commonly-used natural isolate of IAV, while Brisbane/59/2007 (H1N1), Brisbane/ 10/2007 (H3N2), and Perth/16/2009 (H3N2) and are seasonal strains of IAV. Two strains of influenza B virus (IBV), Brisbane/60/2008 and Florida/4/2006, were also included.

Each of these strains induced extensive cytopathic effect (CPE) and cell death in infected wild-type MEFs within 24 hours (see, Figures 1A and IB). Similarly-infected ripk3-/- MEFs were almost completely resistant to IAV- and IBV- induced CPE and cell death at this time point (>85% viability, see, Figures 1A and IB), indicating that activating RIPK3- mediated cell death is a common cytopathic feature of influenza viruses. Focusing on IAV for the rest of this study, it was observed that the capacity of ripk3-/- MEFs to withstand IAV-induced lysis paralleled their resistance to cell death induced by the combination of TNF-a, cycloheximide, and the pan-caspase inhibitor zVAD (TCZ), an established trigger of necroptosis in MEFs (see, Figures 1A and IB), and was largely reversed by the re-introduction of RIPK3 expression (see, Figure 2A). Using recombinant PR8 expressing GFP (PR8-GFP) it was observed that ripkJ-/- MEFs were neither defective in viral entry nor any less permissive to IAV than ripk3+/+ MEFs: both genotypes displayed equivalent levels GFP-positivity 24 h.p.i. with PR8- GFP (see, Figure 1C). In line with these observations, immunoblot analysis of viral protein expression in lysates prepared from ripk3+/+ and ripk3-/- MEFs infected with PR8 over a 24 hour time course revealed no evidence of either decreased infectivity or delayed kinetics of replication in cells lacking RIPK3 (see, Figure ID).

By 36 h.p.L, most (>80%) ripk3+/+ MEFs infected with IAV (hereafter PR8) were dead, while approximately half the population of infected ripk3-/- MEFs remained alive (see, Figure IE). Similarly infected ripk3-/- treated with zVAD or additionally lacking the apoptosis effectors caspase 8 or FADD remained mostly alive at this time point, indicating that the delayed death seen in ripk3-/- MEFs was mediated by a residual RIPK3 -independent FADD/caspase 8- dependent pathway of apoptosis (see, Figure IF). Neither TNF-a or TRAIL-mediated mechanisms, either alone or in combination, appeared to account for this pathway of RIPK3- independent apoptosis in IAV-infected MEFs (see, Figures 2B and 2C).

To extend these analyses to a physiologically relevant cell type, it was next assessed if RIPK3- driven cell death is triggered in lung type I alveolar epithelial cells (AECs) following infection with IAV. Employing the immortalized murine type I AEC cell line LET1, it was first confirmed that type I AECs express RIPK3 and its essential cell death regulators, RIPK1, FADD, MLKL and caspase 8, at levels comparable to those found in MEFs (see, Figure 3). Upon infection with PR8, LET1 cells showed clear evidence of CPE within 3 hours, and succumbed to infection within 12 h.p.i. (see, Figure 1G, left). CRISPR/Cas9-mediated ablation of RIPK3 expression protected LET1 cells from PR8 (see, Figure 1G, left), demonstrating that RIPK3-driven cell death mechanisms are conserved between MEFs and lung AECs. Notably, LET cells lacking RIPK3 produced significantly more progeny virions than control LET1 cells containing RIPK3, predictive of a prominent role for RIPK3 -driven cell death in clearance of virus from the infected lung (see, Figure 1G, right). IAV replication results in assembly of a RIPK3 complex containing RIPKl, FADD and MLKL. A molecular feature of RIPK3 dependent cell death is the stimulus-driven formation of a molecular complex called the necrosome, comprising as its core phosphorylated forms of RIPK3 and RIPKl, together with FADD and MLKL. From the necrosome, RIPK3 can activate both necroptosis and apoptosis, depending on target availability or RIPK3 activity. To test if IAV induced formation of a necrosome, RIPK3 was immunoprecipitated from lysates of PR8- infected MEFs, and immunoprecipitates were examined for the presence of slower-mobility forms of phosphorylated RIPKl. PR8 activated necrosome formation to a significant extent, as did the combination of PR8+zVAD (see, Figure 4 A, left and middle panels). Like TNF-a (see, Figure 4A, right panels), IAV infection also drove the recruitment of FADD and MLKL into the RIPK3-RIPK1 necrosome. As zVAD alone at this dose (25 μΜ) did not induce detectable necrosome formation, and as the combination of PR8+zVAD stabilized the necrosome to allow more reliable detection of IAV-activated RIPK3 -containing complexes over PR8 alone, this combination we used for subsequent dissection of the molecular determinants of IAV-triggered RIPK3 complex formation.

It was observed that PR8 induced a RIPK3-RIPK1 complex in MEFs beginning at -10 h.p.L, after the start of viral protein NS1 accumulation, which was detectable at -6-8 h.p.i (see, Figure 4B). Given that IAV-induced RIPK3 necrosome assembly was preceded by viral protein synthesis, it was next tested if active transcription, translation, or viral genome replication was required for IAV- induced RIPK3 activation. Pre-treatment of MEFs with the RNA polymerase II inhibitor Actinomycin D (ActD, which also blocks IAV replication: host RNA polymerase II is needed for export of IAV mRNAs from the nucleus to the cytoplasm) or with an inhibitor of mRNA translation, cycloheximide (CHX), effectively prevented both viral replication and RIPK3-RIPK1 complex formation (see, Figure 4C). Neither ActD nor CHX at these doses affected TNF-a-induced necrosome assembly (not shown). The IAV nucleoprotein inhibitor nucleozin also potently blocked RIPK3 -RIPKl association at doses that abrogated viral replication (see, Figure 4C). Together, these results demonstrate that IAV replication drives activation of RIPK3.

Next, kinase inhibitors specific for either RIPKl or RIPK3 were employed to identify roles for the kinase activity of each of these proteins in IAV-induced RIPK3 -RIPKl complex assembly. Downstream of TNFR1, the kinase activity of RIPKl, but not RIPK3, was necessary for necrosome formation (see, Figure 4D, left panels), although both classes of inhibitor were potent blockers of TNF-a-induced cell death (see, Figure 5 A). In contrast to how these kinases function in the TNF-a pathway, it was observed that the kinase activity of RIPK3 was required for IAV-induced necrosome formation, while that of RIPKl was largely dispensable (see, Figure 4D, right panels). Thus, while RIPK3 complexes activated by TNF-a and IAV both contain RIPKl, FADD, and MLKL, and are licensed by caspase inhibition, they display contrasting requirements for the kinase activities of RIPKl versus RIPK3.

Several innate-immune pathways are triggered by an acute RNA virus, many of which

(e.g., TNF-a, type I IFNs, TLR3) have been shown to activate RIPK3. To test if any of these pathways are responsible for necrosome formation and RIPK3-driven cell death in response to IAV infection, primary MEFs deficient in either PKR, IFNAR1, STAT1, RIG-I, MAVS, TNFR1 or MyD88/TRIF were infected and monitored for necrosome formation and RIPK3 -dependent cell death in these cells. IFNAR1, STAT1 and PKR are required for type I IFN-induced necroptosis in MEFs, while ablation of TNFR1, RIG-I/MAVS, or MyD88/TRIF was expected to abolish RIPK3 activation by, respectively, TNF-a, RLRs, and TLRs. In each case, RIPK3- dependent cell death (see, Figure 4E) and formation of the RIPK3-RIPK1 complex (see, Figure 6) were largely indistinguishable between wild-type MEFs and MEFs deficient in type I IFN, TNF-a, RLR or TLR signaling. These results indicate that IAV replication either induces

RIPK3- mediated cell death by mechanism(s) distinct from known RIPK3- activating pathways, or, alternatively, triggers redundant combinations of these pathways to activate RIPK3.

IAV activates parallel pathways of necroptosis and apoptosis downstream of RIPK3. Recent evidence shows that RIPK3 activates not only MLKL-driven necrotic cell death but, under certain conditions (e.g., when its kinase activity is inhibited or its conformation altered) caspase-dependent apoptosis as well. To distinguish between apoptotic and necrotic cell death mechanisms activated by IAV downstream of RIPK3, the effects of selective RIPKl and RIPK3 kinase inhibitors, as well as of the pan-caspase blocker zVAD, on viability of wild-type MEFs after infection with PR8 were examined. RIPKl kinase blockade with GSK'963, GSK'481 or Nec-1 did not have any discernible protective effect on viability of MEFs after infection with PR8 (see, Figure 5A), an observation in line with the inability of these agents to prevent IAV-driven necrosome formation (see, Figure. 4D). Surprisingly, RIPK3 kinase inhibition with GSK'872 or GSK'843 also failed to prevent PR8-induced cell death, although each of these inhibitors blocked PR8-induced necrosome assembly (see, Figure 4D) and was fully protective against TNF-a-induced necroptosis at the same doses (see, Figure 3A).

Similarly, zVAD by itself did not protect MEFs from PR8-induced cell deaths (see, Figure 5A) at a dose that prevented apoptosis induced by the combination of TRAIL and cycloheximide (not shown). But the combination of RIPK3 kinase inhibitors with zVAD conferred almost full protection against PR8-induced cell death in both MEFs (see, Figure 5 A) and LET1 AECs (see, Figure 5B). In similar studies on the necroptosis-competent human HT-29 cell line, it was observed that IAV- activated cell death was not singly prevented by either caspase or RIPK3 blockade, but was significantly rescued by the combined inhibition of caspases and RIPK3 kinase activity (see, Figure 5C). Thus, IAV-activated RIPK3 -dependent cell-death mechanisms appear conserved between humans and mice.

Notably, RIPK1 inhibitors deployed under similar conditions were incapable of protection against PR8 in the presence of zVAD (see, Figure 5A). Together with the results from the use of these inhibitors in the necrosome assembly experiments shown in Figure 4, these findings implicate a dominant role for the kinase activity of RIPK3, but not RIPK1, in PR8- driven necrotic death. They also indicate that parallel, redundant pathways of apoptosis and kinase-dependent necroptosis are activated by IAV infection downstream of RIPK3. In agreement with these observations, kinetic analysis of cell death in IAV-infected wild-type MEFs demonstrated that neither a caspase inhibitor nor an inhibitor of RIPK3 kinase function were singly able to protect against cell death, but the combined inhibition of caspases and RIPK3 kinase activity was significantly protective (see, Figure 5D).

IAV induced both MLKL phosphorylation and cleavage of caspase 8 in MEFs, only the first of which was inhibited by RIPK3 kinase blockade and only the second by zVAD (see, Figure 5E). Both MLKL phosphorylation and cleavage of caspase 8 were only seen in cells harboring actively-replicating IAV, and not in cells without detectable replicating virus, indicating that both arms of IAV-activated RIPK3 -dependent cell death are phenomena restricted to the infected cell, and not the result of bystander activity on surrounding, uninfected cells (see, Figure 7).

MLKL mediates necroptosis while FADD and caspase 8 drive apoptosis upon IAV infection. As MLKL has emerged as the primary effector of RIPK3- activated necroptosis, and as PR8-induced the robust phosphorylation of MLKL, the role of this pseudokinase in IAV- triggered cell death was next evaluated. Upon infection with PR8, primary mlkl-/- MEFs, unlike ripk3-l- MEFs, were not any more resistant to cell death than their wild-type counterparts, and succumbed to this virus with kinetics and magnitude indistinguishable from controls. The mlkl- /- MEFs were fully rescued from PR8-induced cell death by pretreatment with zVAD alone, without need for concurrent inhibition of the kinase activity of RIPK3 (see, Figure 8A).

Pretreatment of mlkl-/- MEFs with the caspase 8-specific inhibitor zIETD also provided significant protection against PR8, while RIPK3 kinase blockade by itself had no effect (see, Figure 8 A). As levels of key effector proteins, including RIPK1 and RIPK3, are comparable between mlkl+/+ and mlkl -/- MEFs (see, Figure 3), these results provide unambiguous evidence that IAV-activated death signaling bifurcates downstream of RIPK3 into MLKL-driven necroptosis (which requires the kinase activity of RIPK3) and caspase-mediated apoptosis (which does not). They also implicate caspase 8 as a dominant caspase in the RIPK3 -regulated cell death axis that results in apoptosis. Of note, zIETD consistently afforded less protection against PR8 than zVAD did, even when replenished at 12 hours, in agreement with findings that zIETD is weaker than zVAD at preventing caspase 8-driven apoptosis (see, Figure 9A).

Given the observation that the apoptosis adaptor protein FADD is recruited to the IAV- activated necrosome, it was next asked if FADD was involved in the IAV-activated apoptosis arm downstream of RIPK3. Accordingly, fadd-/- MEFs were infected with PR8 and the viability of these cells in the presence or absence of zVAD and/or RIPK3 inhibitors was monitored.

Fadd-/- MEFs were somewhat more resistant to IAV-induced cell death than wild-type MEFs. This resistance may be attributable, at least in part, to the reduced basal levels of MLKL seen in fadd-/- MEFs (see, Figure 6, see also Figure 8B). As fadd-/- MEFs display heightened basal necrosome activity, diminished MLKL levels may supply a survival advantage to these cells, allowing their continued propagation in culture. Nonetheless, the loss of viability oifadd-/- MEFs following PR8 infection was almost completely rescued by pretreatment of these cells with RIPK3 kinase inhibitors, but not with zVAD (see, Figure 8A).

These results indicate that MLKL and FADD each drive parallel, independent pathways of, respectively, necroptosis and apoptosis downstream of RIPK3. They also suggest that combined ablation of both MLKL and FADD will be needed to afford protection from IAV- induced cell death that is comparable to what is observed when RIPK3 is absent. To test this idea, mice homozygously null for both mlkl and fadd we regenerated. Ablating mlkl rescued the embryonic lethality of fadd-/- mice, and mlkl-/ -fadd-/- double knock-out mice are born at normal Mendelian frequency, exhibit no overt developmental defects, and survive into adulthood. When primary MEFs from mlkl-/-fadd-/- double knock-out mice were infected with IAV, they were observed to be remarkably (>95%) resistant to virus-induced cell death. Indeed, MEFs from these mice were even more resistant to IAV-triggered cell death than MEFs from npH-deficient mice, surviving for over 60 h.p.i. at an m.o.i. of 5 (see, Figure 9B), without any obvious defects in either IAV infectivity (not shown) or replication (see, Figure 9C). Thus, MLKL- and FADD- dependent cell death axes together almost completely account for IAV-triggered cell death, both downstream of RIPK3 and, indeed, independent of this kinase (see, Figures IF and 10).

In accord, mlkl-/- MEFs showed no defect in caspase 8 activation upon IAV infection, but displayed no MLKL phosphorylation, while fadd-/- MEFs were completely deficient in their capacity to activate caspase 8 after infection with IAV, but continue to support phosphorylation of MLKL (see, Figure 8B). Only mlkl-/-fadd-/- double knock-out MEFs, like ripk3-/- MEFs, were deficient in their ability to activate either pathway (see, Figure 8B). Both RIPK3 -dependent early apoptosis and RIPK3 -independent late apoptosis proceed normally on a background of MLKL deficiency, demonstrating the parallel nature of apoptosis and necroptosis pathways activated by IAV (see, Figure 9D).

The mlkl-/ -fadd-/- double knock-out MEFs displayed altered mobility of RIPKl and RIPK3 by SDS-PAGE, when compared to wild-type MEFs (see, Figure 3). Pre-incubation of mlkl-/- fadd-/- double knock-out MEFs with RIPKl or RIPK3 kinase inhibitors demonstrate that, at least in the case of RIPK3, the slower- migrating band represents an auto-phosphorylated version of this kinase (not shown), suggesting unanticipated roles for FADD and MLKL in homeostatic control of basal RIPK1/3 expression and activity.

Role of RIPKl in IAV-activated RIPK3 -dependent cell death pathways. Although RIPKl is robustly recruited to, and phosphorylated by, RIPK3 upon IAV infection, its role as a kinase in the execution of necroptosis following IAV infection appears unnecessary (see, Figure 5A). As RIPKl can function as an apoptosis adaptor downstream of RIPK3, the roles of this protein in RIPK3 -activated cell death upon IAV infection were investigated. To this end, ripkl -/- MEFs were infected with PR8 and the viability of these cells in the presence or absence of zVAD or the RIPK3 inhibitors GSK'843 and GSK'872 was examined. Ripkl-/- MEFs, unlike ripk3-/- MEFs, displayed no significant resistance to PR8, and succumbed to this virus with kinetics indistinguishable from wild-type MEFs (see, Figure 10A). Ripkl-/- MEFs, however, were almost- fully protected from PR8-induced cell death by pre-treatment with RIPK3 kinase inhibitors (but not with zVAD), demonstrating that, like FADD, RIPKl was essential for the apoptosis axis downstream of RIPK3. MEFs from two distinct RIPKl kinase-dead knock-in mice ripklk45a/k45a and ripkl dl38n/dl38n phenocopied wild-type MEFs in their cell death responses to IAV, demonstrating that the kinase activity of RIPKl was not required for activating apoptosis after infection (see, Figure 10A). The ripkl-/- MEFs, as well as MEFs from RIPKl kinase-dead knock-in mice, were resistant to TNF-a-induced necroptosis, where RIPKl functions as a kinase to activate RIPK3 and drive necrotic cell (see, Figure 10B).

In accordance with these findings, IAV-induced phosphorylation of MLKL was intact in ripkl-/- MEFs and in MEFs from RIPKl kinase-dead knock- in (ripkl dl38n/dl38n) mice, while caspase 8 activity was selectively lost in ripkl-/- MEFs, and not in MEFs expressing kinase-dead RIPKl (see, Figure IOC). Subsequent immunoprecipitation experiments revealed that PR8-induced association of FADD with RIPK3 was dependent on RIPKl (see, Figure 10D), placing RIPK1 upstream of FADD and functionally implicating RIPK1 as a kinase-independent adaptor protein that links RIPK3 to FADD and caspase 8 during IAV-activated apoptosis.

MLKL- and FADD-driven arms of RIPK3 -mediated cell death functionally overlap in protecting against lethal IAV infection in vivo. To examine the effect of RIPK3 deficiency on the host response to respiratory infection with IAV, cohorts of ripk3-/- mice, alongside their age- and sex-matched wild-type controls, were inoculated with PR8 and their survival over a time course of 18 days was monitored. It was observed that -25% of wild-type (ripk3+/+) controls succumbed to PR8 by 15 d.p.L; mice surviving past this time point recovered fully. Loss of ripk3 resulted in significantly increased lethality (p <0.05), with 60% of ripk3-/- mice succumbing to PR8 in the same time frame (see, Figure 11 A). When progeny virion production in infected mice was examined, no notable difference in virus output on day 3 p.i. was found, but ~ 10-fold more virus in the lungs of ripk3-/- mice than those of control animals on day 9 p.i. was observed (see, Figure 11C).

To identify the relative contributions of MLKL-mediated necroptosis versus FADD- induced apoptosis downstream of RIPK3 in control of IAV in vivo, wild-type (mlkl+/+ ), mlkl-/-, and mlkl-/ -f add-/- double knock-out mice were next infected with PR8 and their survival over 18 days was monitored. Fadd-/- mice are known to die in utero and were not tested. The mlkl ' ^' mice were not any more susceptible to IAV than their wild-type counterparts and the majority (-75%) of these mice survived this dose of PR8 to recover fully from infection (see, Figure 1 IB). In agreement with these findings, mlkl' ^' mice did not differ significantly from wild-type animals in lung progeny virion output either early (day 3) or late (day 9) post-infection (see, Figure 11C). Mice doubly-deficient in MLKL and FADD, however, were extremely susceptible to IAV-induced lethality, with none surviving past day 12 p.i (see, Figure 11B). These mice were also severely compromised in their capacity to limit IAV replication; although virus titers from infected mlkl-/-fadd-/- lungs were similar to those seen in wild-type animals early in infection, these titers did not drop during the resolution phase (day 9) of infection, remaining ~20-100-fold higher than levels observed in controls (see, Figure 11C).

To explore the basis for the elevated mortality of ripkJ-/- mice, histological analyses were performed on lungs 6 d.p.i. Lung sections were stained with anti-IAV antibody to determine the extent of infection and with hematoxylin/eosin to assess lesion severity.

Compared to ripk3+/+ littermate controls, ripk3-/- lungs exhibited a significant increase in virus spread within the infected lung (see, Figure 11D), consistent with our observations that ripk3-/- mice fail to effectively control IAV progeny virion production (see, Figure 6C).

Substantial pulmonary edema and an increase in alveolar fibrin deposition in ripk3-/- lungs was noted, indicative of severe damage to the capillaries and alveoli in these animals (see, Figure 1 IE). Deposition of fibrin around the alveoli of infected ripk3-/- lungs is likely indicative of a 'cytokine storm' -driven inflammatory response to increased virus spread within lung tissue.

As RIPK3 can also drive immune responses, we examined the effect of RIPK3 loss on the recruitment of T cells to the infected lung. Lungs from ripk3-/- displayed a markedly reduced infiltration of CD3+ cells, compared to ripk3+/+ controls, 6 d.p.i. (see, Figure 11F). To examine this effect in greater detail, the abundance of IAV- specific CD8+ T cells in

bronchio alveolar lavage (BAL) fluid were measured 9 d.p.i., and it was found that IAV-specific CD8+ T cell numbers were significantly diminished in ripk3-/- mice (see, Figure 11G, left). Moreover, responding ripk3-/- CD8+ T cells appeared less capable of mounting an efficient effector response, as evidenced by the lower frequencies of polyfunctional (IFN-y+TNF-a-i-) CD8+ T cells in BAL fluid of ripk3-/- mice, compared to ripk3+/+ controls (see, Figure 11G, right). Collectively, these findings denote that loss of RIPK3 results not only in increased IAV spread within the infected lung, but also in diminished antiviral CD8+ T cell responses to the infection.

Example 3: Determination of DAI sensitization of influenza A virus to activate RIPK3- dependent cell death: Materials and Methods

Mice and cells. Zbpl ' ^' , Ripk3 ^'A , ripkl ' ^' , and ripk3 ^' mice were housed in SPF facilities in house, and all in vivo experiments were conducted under protocols approved by a Committee on Use and Care of Animals. All primary mouse embryo fibroblasts (MEFs) were generated in- house from E14.5 embryos and used within five passages in experiments. For stable reconstitution studies, zbpl ^'1' MEFs immortalized by SV40 Large T antigen were infected with retroviruses expressing DAI mutants from the pQCXIH vector (Clontech). All cells were cultured in high-glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10- 15% heat- inactivated fetal bovine serum (Hyclone) and antibiotics.

Reagents. Biological and chemical reagents were from the following sources:

cycloheximide (MP Biomedicals), zVAD.fmk ( Bachem), interferon-β (PBL), and TNF-a (R&D systems). Antibodies to β-actin (Sigma), caspase 8 (Cell Signaling), cleaved caspase 8 (Cell Signaling), FADD (Millipore), IAV NS 1 (Santa Cruz), MLKL (Abgent or Millipore), p-MLKL (Abeam), RIG-I (Enzo), PKR (Santa Cruz), RIPl (BD Transduction lab), RIP3 (ProSci or Santa Cruz), STAT1 (BD Transduction labs), and p-STATl (Cell Signaling) were obtained from the indicated commercial sources. Secondary anti-mouse, -rabbit, and -rat IgG were purchased from Jackson ImmunoResearch. Viruses and virus infections. Influenza virus A/Puerto Rico/8/34 (PR8) was generated by reverse genetics. All IAV and influenza type B (IBV) strains were propagated by allantoic inoculation of embryonated hen's eggs with diluted (1 :106) seed virus. Virus titers were determined as 50% egg infectious dose (EID ₅₀ ). For in vivo studies, recombinant PR8-GFP virus was provided. Mice were anesthetized with isofluorane and infected i.n. with virus inoculum diluted in 30 xL of endotoxin-free phosphate-buffered saline (PBS). Mice were either monitored for survival and weight- loss over a period of 18 days or sacrificed at defined time points for analysis of histology and virus replication. Mice losing >35% body-weight were considered moribund and euthanized by CO ₂ asphyxiation. To determine progeny virus production in infected mice, lung homogenates were titered by plaque assay on Madin-Darby Canine Kidney cells.

Molecular modeling. The murine DAI Za2:zRNA complex was modeled based on the template of human ADAR Zed bound to zRNA (PDB code 2GXB, 33% identity, 53% similarity). DAI Za2 was aligned with the HHpred portion of the MPI bioinformatics Too t, and a model built using MODELLER. Two mouse DAI Za2 domains were superposed on the corresponding domains of the ADAR Za structure with RNA using the UCSF Chimera software and the zRNA co-ordinates were copied over to make the final model. Side chain rotamers were also considered using the SCWRL4 frame option to account for steric packing to RNA.

Purification and analysis of DAI-associated IAV RNA. HEK 293T cells seeded in 10 cm dishes and transfected with FLAG-tagged RIG-I or DAI constructs for 24 hours were infected with PR8 (MOI=2) for 12 hours and disrupted in 1 mL lysis buffer (50 mM HEPES, 150 mM KC1, 2 mM EDTA, 1 mM NaF, 0.5% NP40, 0.5 mM DTT, protease inhibitor cocktail, 25 units RNasin). An aliquot (5%) of lysate was saved for total RNA input control and immunoblot analysis. Lysates were then incubated with 30 μί^ιηρΐε anti-FLAG agarose bead slurry (Clone M2, Sigma- Aldrich) overnight with rotation at 4°C. Beads were collected by centrifugation, washed ten times with NT2 buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl ₂ , 0.05% NP40), resuspended in 250 μΐ. of DNase digestion buffer (40 mM Tris pH 8.0, 10 mM MgS0 ₄ , 1 mM CaCl ₂ ) and treated with 25U RNasin (Promega) and 2U DNAse I (NEB) at 37°C for 20 minutes. Beads were collected by centrifugation, washed with NT2, and resuspended in 100 μΐ. NT2 buffer. 10% of each sample was removed for immunoblot analysis. Samples were treated with 4 units proteinase K at 55°C for 30 minutes. 1 mL Tri-reagent (Sigma- Aldrich) was added to each sample, and RNA was harvested according to the manufacturer's instructions. For RNA Seq analyses, RNA was prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina). Briefly, total RNA was depleted of ribosomal RNA (rRNA-removal beads), fragmented, and reverse transcribed into cDNA using reverse transcriptase and random primers. Following second strand cDNA synthesis by DNA

Polymerase I in the presence of RNase H, an adenine was added to the 3 '-end and specific Illumina adapters were ligated to cDNAs. The ligation products were purified and enriched by PCR to create the final cDNA library, which was loaded on the MiSeq platform (Illumina). Bowtie2 was used to map reads to the PR8 genome. For PCR detection of DI particle genomes, RNA was reverse transcribed into cDNA using reverse transcriptase (Superscript ^® II RT, Thermo Fisher) with a universal genomic viral RNA (vRNA) primer (5 ' - AGCAAAAGCAGG- 3') (SEQ ID NO: 18), per manufacturer's instructions. PCR was then performed using the following primers PB2 : 5 '- ATGGAAAGAATAAAAGA

ACT A AG- 3' (SEQ ID NO: 19), and 5'-CTAATTGATGGCCATCCGAATTC-3' (SEQ ID NO:20).

Example 4: Determination of DAI sensitization of influenza A virus to activate RIPK3- dependent cell death: Experimental Results

DAI is required for IAV-induced cell death. In a focused screen for mediators of IAV- activated cell death, it was observed that MEFs from DAI-deficient {zbpl ^' ' ^' ) mice were extraordinarily resistant to death triggered by this virus. When evaluated over a period of 24 hours, near-confluent monolayers of primary, early-passage MEFs from two separately-housed zbpl ' ^' colonies uniformly displayed >90% viability when infected with IAV strain A/Puerto Rico/8/1934 (PR8, H1N1) while similarly-infected wild-type (C57BL/6) control MEFs manifested extensive cytopathic effect (CPE) and cell death by this time (see, Figures 12A and 12B). Notably, zbpl ' ^' MEFs were also resistant to cell death activated by seasonal H1N1 and H3N2 strains of IAV, as well as to IBV (see, Figure 16A), although these cells displayed levels of death effector proteins equivalent to controls (see, Figure 16B), and remained susceptible to necroptosis induced by TNF-a (see, Figure 12A). IAV entry, as measured by GFP-positivity 18 h.p.i. with recombinant PR8 expressing GFP (PR8-GFP), was equivalent between wild-type and zbpl ' ^' MEFs (see, Figure 12C). Virus proteins NP and NS1 were also produced at similar levels and with similar kinetics in PR8-infected wild-type and zbpl ' ^' MEFs (see, Figure 13D), arguing against a non-permissive cellular environment or diminished virus replication as responsible for survival of IAV-infected zbpl ' ^' MEFs.

To confirm a role for DAI in induction of cell death following IAV infection, immortalized zbpl ' ^' MEFs were reconstituted with either full-length DAI, or with a mutant of DAI (DAI mutRHIM), carrying a tetra-alanine substitution of the core RHIM sequence IQIG (aa 192-195), analogous to a human DAI mutant incapable of RHIM-based interactions. While immortalized zbpl ' ^' MEFs were resistant to IAV-induced cell death, reintroduction of full-length wild type DAI, but not DAI mutRHIM, into these cells fully restored susceptibility to IAV (see, Figure 12E). In a corollary experiment CRISPR/Cas9-based ablation of zbpl expression in wild- type MEFs rendered these cells resistant to IAV-induced cell death (see, Figure 12F). In neither case was susceptibility to TNF-a-induced necroptosis affected (see, Figures 16C and 16D).

To extend these findings to a cell type relevant to IAV replication in vivo, zbpl expression wad ablated in LETl cells before they were infected with IAV. The LETl cell line is derived from type I alveolar epithelium, a primary early target of IAV in the lung. LETl cells, unlike MEFs, support the complete IAV lifecycle, and produce progeny virions upon infection. It was previously observed that ablating ripk3 in these cells protects them from IAV induced cell death, allows unbridled virus replication, and increases progeny virion output. It has now been observed that ablating zbpl in LETl cells is similarly protective against cell death and supportive of increased productive virus replication: two distinct sgRNAs to murine zbpl both reduced IAV-triggered cell death by -70% in LET 1 cells (see, Figure 13G), comparable to the protection afforded by ablation of ripk3 itself. Notably, LETl cells in which zbpl was ablated, unlike cells lacking ripk3, were still susceptible to TNF-a-induced necroptosis (see, Figure 16E), underscoring the specific requirement for DAI in IAV, but not TNF-a, induced RIPK3 -driven cell death in this cell type as well. LETl cells lacking zbpl produced significantly more progeny IAV that LETl controls over a 30 hour timeframe, suggesting that these cells had become 'factories' for virus replication (see, Figure 12H).

DAI associates with RIPK3 and mediates both apoptosis and necroptosis in IAV- infected cells. IAV, uniquely among viruses studied thus far, activates both apoptosis and necroptosis downstream of RIPK3. It does so by nucleating a RIPK3 -containing necrosome complex that also comprises MLKL, which mediates necroptosis, as well as RIPK1 and FADD, which activate apoptosis. To test if DAI was required for necrosome assembly and consequent dual activation of apoptosis and necroptosis downstream of RIPK3, RIPK3 was

immunoprecipitated from IAV- infected wild-type and zbpl ' ^' MEFs, and precipitates were examined for the presence of DAI, RIPK1, and MLKL. As negative controls, ripk3 ^~ ' ^~ MEFs were included in these experiments. It was found that IAV infection induced the robust association of DAI with RIPK3 , and that DAI was essential for recruitment of both MLKL and RIPK1 to RIPK3 (see, Figure 13A). zbpl ' ^' MEFs were completely defective in both MLKL and caspase-8 activation upon IAV infection (see, Figure 13B), although necrosome assembly (not shown) and activation of MLKL (see, Figure 13B) in response to TNF-a occurred normally in the absence of DAI. LET1 cells in which expression of zbpl was ablated by CRISPR/Cas9 gene-targeting also failed to support either MLKL or caspase-8 activation flowing infection by IAV, although activation of MLKL by TNF-a was unaffected (see, Figure 17). Loss of RIPK3, expectedly, resulted in abolishment of MLKL activation in both MEFs and LET1 cells following either infection by IAV or exposure to TNF-a (see, Figures 14B and 17). Reintroduction of wild type DAI, but not DAI mutRHIM, into immortalized zbpl ' ^' MEFs restored both phosphorylation of MLKL and cleavage of caspase-8 in response to IAV (see, Figure 13C). In accordance with these findings, wild type DAI, but not DAI mutRHIM, robustly complexed with RIPK3 following IAV infection (see, Figure 13D). Together, these results demonstrate that DAI lies upstream of RIPK3 in the pathways leading to activation of MLKL and caspase-8, and requires its RHIM to associate with RIPK3 and activate these pathways.

It was previously shown that IAV activates a delayed RIPK3 -independent pathway of apoptosis that relies on FADD and caspase-8 and is activated between 24-36 h.p.i. Cells doubly- deficient in RIPK3 and FADD, RIPK3 and caspase-8, or MLKL and FADD, continue to survive past 36 hours, whereas >50% of ripk3 ^~ ' ^~ MEFs are dead by this time point. In fact, these double knockout MEFs (e.g., fadd' tnlkl' ^' MEFs) survive for up to 60 h.p.i without any obvious abatement of virus replication, while ripk3 ^~ ' ^~ MEFs are mostly dead by 48 h.p.i. It was observed that zbpl ' ^' were significantly more resistant to IAV than ripk3 ^~ ' ^~ MEFs, phenocopying in this regard the resistance of MEFs doubly-deficient in apoptosis and necroptosis pathways (see, Figure 13E) and suggesting that DAI may lie upstream of RIPK3-independent apoptosis as well.

It was hypothesized that, when RIPK3 is absent, DAI may instead employ RIPKl as a RHIM-containing adaptor to link replicating IAV to FADD and caspase-8; thus, co-ablation of RIPKl with RIPK3 will be needed to abrogate all major pathways of IAV-triggered programmed cell death. To test this idea, the kinetics of IAV- induced cell death were evaluated in ripkl ' ^' ripk3 ^~ ' ^~ double knockout MEFs, and it was found that these cells were as resistant to IAV as zbpl ^' ' ^' MEFs and fadd 'tnlkl ' ^' double knockout MEFs, each of which survived IAV beyond 36 h.p.i. (see, Figure 13E). Moreover, delayed caspase-8 activity seen in ripk3 ^~ ' ^~ MEFs and completely absent in zbpl ' ^' MEFs was largely abolished in ripkl ' ^~ ripk3 ^~ ' ^~ double knockout MEFs (see, Figure 13E). Thus, DAI also mediates IAV-activated RIPK3 -independent apoptosis, likely via recruitment of RIPKl and activation of a RIPKl-FADD-caspase-8 axis.

DAI senses IAV RNA. The results are believed to establish an absolute and specific role for DAI in RIPK3 -driven cell death responses to IAV, but do not clarify if DAI functions at the level of RIPK3 (as a co-factor needed for its activation) or if it operates upstream of RIPK3. In the latter scenario, DAI, a nucleic acid binding protein, may directly sense IAV nucleic acids produced during viral replication and link proliferating IAV to RIPK3. To distinguish between these alternatives, a mutagenesis approach aimed at identifying regions of DAI required for activation of RIPK3 and induction of cell death was undertaken. DAI possesses two tandem Za domains towards its N-terminus which, for convenience, were called Zal (a.a. 8-72) and Za2 (aa 84-147) (see, Figure 14A). Za2 is sometimes referred to as 'Ζβ' in previous studies, but Za2 is a more authentic descriptor of this domain that distinguishes it from the functionally-distinct Ζβ domain of ADAR1. These domains are followed by the RHIM (aa 184-200) and a C-terminal half containing a region (aa 314-411) shown to interact with TBK-l/IRF-3 in the pathway leading to production of type I IFNs (see, Figure 14A).

Deletion of the C-terminal TBK-l/IRF-3 binding region of DAI did not significantly impede the capacity of DAI to either trigger lysis of IAV infected cells or to activate RIPK3 , as measured by phosphorylation of MLKL, in these cells (see, Figure 14B). Similarly, singly deleting Zal had little effect on the magnitude of IAV-induced cell death (see, Figure 14B) or RIPK3 activation at 24 hours (see, Figure 14C), and only modestly affected the kinetics with which IAV killed the infected cell (see, Figure 18A). But deleting both the Zal and Za2 domains abolished cell death and RIPK3 activity.

Indeed, point mutations in two amino acids (N122 and Y126) in Za2 of DAI, previously shown to be essential for binding to Z-DNA and analogous to residues in ADAR1 Za known to contact Z-RNA, completely nullified the ability of DAI to induce cell death (see, Figure 14B) or stimulate RIPK3 (see, Figure 14C) upon IAV infection. Each of these mutants was expressed at levels comparable to wild type DAI (see, Figure 18B), and none of the mutations affected TNF- a-induced necroptosis signaling in these cells (see, Figures 14C and 17C). Caspase-8 activity downstream of RIPK3, like phosphorylation of MLKL, also required Za2 (not shown).

Together, these results implicate the second Za domain of DAI as critical for IAV-driven cell death responses, and suggest that this domain interacts with IAV RNA to initiate activation of DAI and subsequent signaling to RIPK3.

To determine if DAI bound RNA, a putative interaction between DAI Za2 and RNA was modeled using as template the published co-crystal structures of Za domains bound to Z- form DNA or RNA. The Za:Z-DNA and Za:Z-RNA structures bear remarkable similarity to each other (see, Figure 15D, left two panels). The conserved nucleic acid residues in these structures that correspond to N122 and Y126 in the nucleic acid recognition helix of mDAI Za2 are in identical positions, and very similar rotamers make contact with both Z-DNA and Z-RNA (see, Figure 14D, two left panels). Correspondingly, the model of mDAI Za2 complexed with Z-RNA strongly indicates that N122 and Y126 of mDAI Za2 are capable of making identical contacts to either Z-form nucleic acid (see, Figure 15D, third panel).

Indeed, when the mDAI Za2:RNA model was superimposed over to the known structure of hDAI Za2:DNA, Y126 is in an essentially identical rotamer conformation to the analogous tyrosine in hDAI Za2:DNA, while minor differences in the position N122 relative to the analogous asparagine in hDAI positions reflect a subtle change in the confirmation of the nucleic acid recognition helix in its DNA- versus RNA-bound forms (see, Figure 14D, fourth panel). The models are in general agreement with previous comparisons of Za domains bound to Z-DNA or Z-RNA, indicating that the DAI Za2 domain is similarly suited to bind either left- handed Z-form nucleic acid.

To test if DAI bound IAV RNA, and to identify these RNAs, FLAG-tagged DAI was transfected into 293T cells before these cells were infected with PR8. FLAG-affinity chromatography was then used to precipitate DAI from infected cells 12 h.p.L, eluted RNA co- precipitating with DAI, and eluates were examined for the presence of IAV RNA. In parallel, RNA co-precipitating with FLAG-RIG-I, a known sensor of IAV RNA, was also evaluated as a positive control. IAV-specific vRNA mapping to all eight IAV gene segments were readily detected in DAI immunoprecipitates (see, Figure 14E, top), but in a manner that was neither uniform between vRNA segments, nor equivalent across each segment. Instead, bound vRNAs mapped predominantly to the 5 ' and 3 ' ends of the three longer polymerase-encoding gene segments (PB2, PB1 , and PA). A significant number of reads also mapped to some of the shorter segments (e.g., NA and NP), this time more uniformly across the each individual segment, but not to others (e.g., HA). This pattern bears a resemblance to the spectrum of vRNAs co -precipitating with RIG-I (see, Figure 14E, bottom), which was previously shown to represent IAV subgenomic material packaged into defective interfering (DI) particles.

While significant RNA yields from IAV-infected cells expressing wild-type (or mutRHIM) DAI were obtained, the amount of eluted RNA from similarly- infected cells expressing Za2 mutants of DAI was far lower, and comparable to background yield from vector controls (see, Figure 18D), despite equivalent expression of each FLAG-tagged DAI construct (see, Figure 18C). Only wild-type DAI and DAI-mutRHIM, but not DAI Za2 mutants, associated with IAV vRNA (see, Figure 18E). Taken together, these results demonstrate that DAI binds IAV RNA in a manner requiring its Za2 domain, and likely senses IAV RNA structures found in DI particle sub-genomes that are also recognized by RIG-I.

DAI is required for protection against IAV in vivo. To examine the role of DAI in host defense to IAV, zbpl ' ^' mice were infected with PR8 at a dose (1000 EID ₅₀ ) that was not lethal to age-and sex-matched wild-type controls, and the survival of these mice was monitored over a time course of 18 days. All wild-type controls lost weight over the first week, but eventually recovered from infection by 15-18 dpi (see, Figures 15A and 15B). In contrast, -80% of zbpl ' ^' mice perished between 9 and 12 dpi (see, Figures 15A and 15B).

Next, virus replication was measured in lungs of mice infected with an even lower dose of PR8 (750 EID ₅₀ ) to delay some the lethality associated with zbpl loss. Progeny virion production was not notably different between lungs of wild type or zbpl ' ^' mice on day 6 p.L, but whereas virus was essentially cleared from wild-type lungs by 9 dpi, titers remained elevated in lungs from zbpl ' ^' animals (see, Figure 15C). In agreement with these findings, numerous virus- positive bronchiolar epithelial cells were observed in zbpl ' ^' lungs 9 dpi, by which time wild type lungs had largely cleared virus, and only cellular debris in the lumens of certain airways remained virus antigen-positive (see, Figure 15D). Moreover, hyaline membranes, indicative of diffuse severe alveolar damage in mice, were rarely seen in infected wild-type lungs 9 dpi, but were relatively common in zbpl ' ^' lungs (not shown). Without intending to be limited to any particular theory or mechanism of action, it is believed that these results signify that infected zbpl ' ^' bronchiolar epithelial cells have not undergone apoptosis or necroptosis and, thus, remain factories for virus production, greatly compromising lung function and resulting in mortality.

Example 5: Determination of DAI sensitization of influenza A virus to activate RIPK3- dependent cell death: Summary

Examples 3 and 4 provide evidence implicating DAI as the host sensor protein linking IA V replication to activation of RIPK3 and consequent induction of both necroptosis and apoptosis. A simple 'induced-proximity' model for how DAI senses IAV and stimulates RIPK3 is proposed (see, Figure 15E). In this model, DAI recognizes RNA intermediates produced during the IAV life cycle, which induces the multimerization of DAI. This model is based, in part, on the published co-crystal structure of DAI Za2 in complex with Z-DNA, which reveal dimers of Za2 associating with a single dsDNA double helix. Cystallographic studies have found that Za:Z-RNA complexes are conformationally very similar to Za:Z-DNA complexes, and the in silico models based on these structures suggest that Za2 domain of DAI associates with dsRNA as a dimer, bringing into proximity two or more molecules of DAI (see, Figure 14E). Once juxtaposed, RHIM-based associations between DAI monomers allows further oligomerization of DAI, and recruitment and subsequent oligomerization of RIPK3 (see, Figure 15E). Multimerization of DAI by IAV RNA may, at a minimum, suffice to recruit RIPK3, cluster this kinase, and initiate downstream death signaling (see, Figures 15A-15E). DAI also mediates RIPK3 -independent cell death during IAV infection, via a FADD/caspase 8 axis. This pathway is at least partially dependent on RIPK1 , suggesting that DAI may associate with RIPK1 to activate this alternative, delayed axis of RIPK3 -independent cell death (see, Figure 15E).

DAI appears to preferentially associate with shorter vRNAs, including internally- deleted variants of the polymerase gene segments previously identified as DI particle RNAs. Such subgenomic DI particle RNAs are produced when the IAV RNA dependent RNA polymerase (RdRp) falls off its antigenome template at the 5' end, but reattaches further downstream to continue transcription. These RNA segments are packaged in DI particles and retain the non-coding region, including the conserved 12 and 13 nucleotides at the 3' and 5' termini, respectively. The partial complementarity of these conserved nucleotides allows the formation of secondary 'corkscrew' structures that are recognized by the IAV RdRp. The profile of DAI-associated vRNA species bears similarity to vRNAs that associate with RIG-I during the course of IAV infection. It is believed that this suggests both DAI and RIG-I may recognize similar viral RNA species produced by IAV, with a propensity for subgenomic DI particle RNAs that may be improperly packaged and therefore more prone to detection by the host innate- immune machinery.

DAI also associates with some of the shorter IAV gene segments (most notably NA and NP) across their entire length, suggesting that dsRNA complexes of these genomic RNAs and their complementary antigenome templates serve as additional ligands for this sensor. The in silico models indicate that DAI Za2 dimers are unlikely to make contacts with A- form dsRNA as they do with Z-RNA, so whether IAV dsRNAs that function as DAI ligands adopt the Z conformation, perhaps as a result of torsional stress induced by negative supercoiling during viral replication.

ZBP1 (DAI) cDNA (SEQ ID NO:l):

agagctgcaa gaagcaccag gctcggccac ttcagaagcc ccagcctcga cctagcccaccctctcaggg ccacagtgca gaagcctgca cacctgccaa gtctctccga ctccttgcagctgctgtcag catggcccag gctcctgctg acccgggcag agaaggccac cttgaacaaa gaatcctgca ggtgctgaca gaggctggct ccccggtgaa acttgcccag ctggtgaagg aatgccaagc acccaagagg gagctcaacc aagtcctcta ccgaatgaaa aaggagttga aagtctccct cacatcccct gccacctggt gcttgggcgg gactgatcct gaaggcgagg gtcctgcaga gctggccttg tccagccctg ccgagaggcc ccagcaacat gcagctacaa ttccagagac ccctggccct cagttcagcc aacaacggga ggaagacatc tacaggtttc tcaaagacaa tggtccccag agggccctgg tcatcgccca agcactggga atgaggacag caaaagatgt gaaccgagac ttgtacagga tgaagagcag gcaccttctg gacatggatg agcagtccaa agcatggacg atttaccgcc cagaagattc tggaagaaga gcaaagtcag cctcaattat ttaccagcac aatccaatca acatgatctg ccagaatgga cccaacagct ggatttccat tgcaaactcc gaagccatcc agattggaca cgggaacatc attacaagac agacagtctc cagggaggac ggttccgccg gtccacgcca cctcccttca atggcaccag gtgattcctc aacttggggg accctagttg atccctgggg gccccaggac atccacatgg agcagtccat actgagacgg gtgcagctgg gacacagcaa tgagatgagg ctccacggcg tcccgtccga gggccctgcc cacatccccc ctggcagccc cccagtgttc cagtgcagtg ggaagcaggc agcagaggag agaggagctg aatcccagag gaagacactg acgtccagat cagccgggcg ggatcagtgg ggcagaccca ctgcgggatc agtggagcag aaagacgctt cccagacagc acaacagcac gagcggagtc tctgccactg ctgccggccc agaagcttcg tttgaagcaa gaattcccag tccaggaact caccctgagg gggaagccgc ccagagaatc cacatgaaat cgtgctttct cgaggacgcc accatcg

RIPK3 cDNA SEQ ID N0:2):

atgtcgtgcg tcaagttatg gcccagcggt gcccccgccc ccttggtgtc catcgaggaa ctggagaacc aggagctcgt cggcaaaggc gggttcggca cagtgttccg ggcgcaacat aggaagtggg gctacgatgt ggcggtcaag atcgtaaact caaaggcgat atccagggag gtcaaggcca tggcaagtct ggataacgaa ttcgtgctgc gcctagaagg ggttatcgag aaggtgaact gggaccaaga tcccaagccg gctctggtga ctaaattcat ggagaacggc tccttgtcgg ggctgctgca gtcccagtgc cctcggccct ggccgctcct ttgccgcctg ctgaaagaag tggtgcttgg gatgttttac ctgcacgacc agaacccggt gctcctgcac cgggacctca agccatccaa cgtcctgctg gacccagagc tgcacgtcaa gctggcagat tttggcctgt ccacatttca gggaggctca cagtcaggga cagggtccgg ggagccaggg ggcaccctgg gctacttggc cccagaactg tttgttaacg taaaccggaa ggcctccaca gccagtgacg tctacagctt cgggatccta atgtgggcag tgcttgctgg aagagaagtt gagttgccaa ccgaaccatc actcgtgtac gaagcagtgt gcaacaggca gaaccggcct tcattggctg agctgcccca agccgggcct gagactcccg gcttagaagg actgaaggag ctaatgcagc tctgctggag cagtgagccc aaggacagac cctccttcca ggaatgccta ccaaaaactg atgaagtctt ccagatggtg gagaacaata tgaatgctgc tgtctccacg gtaaaggatt tcctgtctca gctcaggagc agcaatagga gattttctat cccagagtca ggccaaggag ggacagaaat ggatggcttt aggagaacca tagaaaacca gcactctcgt aatgatgtca tggtttctga gtggctaaac aaactgaatc tagaggagcc tcccagctct gttcctaaaa aatgcccgag ccttaccaag aggagcaggg cacaagagga gcaggttcca caagcctgga cagcaggcac atcttcagat tcgatggccc aacctcccca gactccagag acctcaactt tcagaaacca gatgcccagc cctacctcaa ctggaacacc aagtcctgga ccccgaggga atcagggggc tgagagacaa ggcatgaact ggtcctgcag gaccccggag ccaaatccag taacagggcg accgctcgtt aacatataca actgctctgg ggtgcaagtt ggagacaaca actacttgac tatgcaacag acaactgcct tgcccacatg gggcttggca ccttcgggca aggggagggg cttgcagcac cccccaccag taggttcgca agaaggccct aaagatcctg aagcctggag caggccacag ggttggtata atcatagcgg gaaataa MLKL cDNA (SEQ ID NO:3):

atccagcaca gagaagcaaa caccagaagc ttctctccaa ctttcagttt ttctctaaac ttccagtcct tctcatactg tgtcctggtt ataatagttg ctgggacctt attctattct gaatccccag cccctaaaga aggcttggta ctgacgtgta cagcgtagtt acccagtgac tttggggaag gcaggaagag tgtggaaagg acaggaggaa ggaagggagg ggccgtggct ccgagctgcc tagaaagcgg ctccagggag ccagggcacc gtgagcctgg tggttggcag ctggagccac gtcggagggg gaagtgtcgc agcattctct gcaggcatca cagacctgag gcagtggcct ccggagggca ctggacagaa acagccatcc aagtggctga gtggagggac cctgctcaag tgcagctgca gtggccgggg tttccctcag gtagggatcg gggcgccttg tcgccgccag ccacgtgtgg cgtccggtac agtcagcaga gtgcagggtg cgggcaccag gaaagggggc gcaggggaac tcccgcgggc ctcgcgtttg caaacttctc gcctgggcag gaggcggtcg tgggaaagaa ggtggaagag cgagcttttt ggaactgtgc acgggacaga ttggacgcac acccctcggg aggcgcgaag gcatggaaaa tttgaagcat attatcaccc ttggccaggt catccacaaa cggtgtgaag agatgaaata ctgcaagaaa cagtgccggc gcctgggcca ccgcgtcctc ggcctgatca agcctctgga gatgctccag gaccaaggaa agaggagcgt gccctctgag aagttaacca cagccatgaa ccgcttcaag gctgccctgg aggaggctaa tggggagata gaaaagttca gcaatagatc caatatctgc aggtttctaa cagcaagcca ggacaaaata ctcttcaagg acgtgaacag gaagctgagt gatgtctgga aggagctctc gctgttactt caggttgagc aacgcatgcc tgtttcaccc ataagccaag gagcgtcctg ggcacaggaa gatcagcagg atgcagacga agacaggcga gctttccaga tgctaagaag agataatgaa aaaatagaag cttcactgag acgattagaa atcaacatga aagaaatcaa ggaaactttg aggcagtatt taccaccaaa atgcatgcag gagatcccgc aagagcaaat caaggagatc aagaaggagc agctttcagg atccccgtgg attctgctaa gggaaaatga agtcagcaca ctttataaag gagaatacca cagagctcca gtggccataa aagtattcaa aaaactccag gctggcagca ttgcaatagt gaggcagact ttcaataagg agatcaaaac catgaagaaa ttcgaatctc ccaacatcct gcgtatattt gggatttgca ttgatgaaac aggctacacc attcagaagc acctgaactc cacggaaaaa tcagaagctc aaacttcctg gtaactcaag gctaccaagt gaagatttgg gtacctggct gagtttgggg ctggcagcct tcagcacacc aatggtgtca atggcaggtg tgcatgggac aagaacatat gc

ZBP1 (DAI) RNA (SEQ ID N0:4):

agagcugcaa gaagcaccag gcucggccac uucagaagcc ccagccucga ccuagcccac ccucucaggg ccacagugca gaagccugca caccugccaa gucucuccga cuccuugcag cugcugucag cauggcccag gcuccugcug cccgggcag agaaggccac cuugaacaaa gaauccugca ggugcugaca gaggcuggcu ccccggugaa acuugcccag uggugaagg aaugccaagc acccaagagg gagcucaacc aaguccucua ccgaaugaaa aaggaguuga aagucucccu cacauccccu gccaccuggu gcuugggcgg gacugauccu gaaggcgagg guccugcaga gcuggccuug uccagcccug ccgagaggcc ccagcaacau gcagcuacaa uuccagagac cccuggcccu caguucagcc aacaacggga ggaagacauc uacagguuuc ucaaagacaa ugguccccag agggcccugg ucaucgccca agcacuggga augaggacag caaaagaugu gaaccgagac uuguacagga ugaagagcag gcaccuucug gacauggaug agcaguccaa agcauggacg auuuaccgcc cagaagauuc uggaagaaga gcaaagucag ccucaauuau uuaccagcac aauccaauca acaugaucug ccagaaugga cccaacagcu ggauuuccau ugcaaacucc gaagccaucc agauuggaca cgggaacauc auuacaagac agacagucuc cagggaggac gguuccgccg guccacgcca ccucccuuca auggcaccag gugauuccuc aacuuggggg acccuaguug aucccugggg gccccaggac auccacaugg agcaguccau acugagacgg gugcagcugg gacacagcaa ugagaugagg cuccacggcg ucccguccga gggcccugcc cacauccccc cuggcagccc cccaguguuc cagugcagug ggaagcaggc agcagaggag agaggagcug aaucccagag gaagacacug acguccagau cagccgggcg ggaucagugg ggcagaccca cugcgggauc aguggagcag aaagacgcuu cccagacagc acaacagcac gagcggaguc ucugccacug cugccggccc agaagcuucg uuugaagcaa gaauucccag uccaggaacu cacccugagg gggaagccgc ccagagaauc cacaugaaau cgugcuuucu cgaggacgcc accaucg

RIPK3 RNA (SEQ ID N0:5):

augucgugcg ucaaguuaug gcccagcggu gcccccgccc ccuugguguc caucgaggaa cuggagaacc aggagcucgu cggcaaaggc ggguucggca caguguuccg ggcgcaacau aggaaguggg gcuacgaugu ggcggucaag aucguaaacu caaaggcgau auccagggag gucaaggcca uggcaagucu ggauaacgaa uucgugcugc gccuagaagg gguuaucgag aaggugaacu gggaccaaga ucccaagccg gcucugguga cuaaauucau ggagaacggc uccuugucgg ggcugcugca gucccagugc ccucggcccu ggccgcuccu uugccgccug cugaaagaag uggugcuugg gauguuuuac cugcacgacc agaacccggu gcuccugcac cgggaccuca agccauccaa cguccugcug gacccagagc ugcacgucaa gcuggcagau uuuggccugu ccacauuuca gggaggcuca cagucaggga caggguccgg ggagccaggg ggcacccugg gcuacuuggc cccagaacug uuuguuaacg uaaaccggaa ggccuccaca gccagugacg ucuacagcuu cgggauccua augugggcag ugcuugcugg aagagaaguu gaguugccaa ccgaaccauc acucguguac gaagcagugu gcaacaggca gaaccggccu ucauuggcug agcugcccca agccgggccu gagacucccg gcuuagaagg acugaaggag cuaaugcagc ucugcuggag cagugagccc aaggacagac ccuccuucca ggaaugccua ccaaaaacug augaagucuu ccagauggug gagaacaaua ugaaugcugc ugucuccacg guaaaggauu uccugucuca gcucaggagc agcaauagga gauuuucuau cccagaguca ggccaaggag ggacagaaau ggauggcuuu aggagaacca uagaaaacca gcacucucgu aaugauguca ugguuucuga guggcuaaac aaacugaauc uagaggagcc ucccagcucu guuccuaaaa aaugcccgag ccuuaccaag aggagcaggg cacaagagga gcagguucca caagccugga cagcaggcac aucuucagau ucgauggccc aaccucccca gacucca accucaacuu ucagaaacca gaugcccagc ccuaccucaa cuggaacacc aaguccugga ccccgaggga aucagggi ugagagacaa ggcaugaacu gguccugcag gaccccggag ccaaauccag uaacagggcg accgcucguu aacauauaca acugcucugg ggugcaaguu ggagacaaca acuacuugac uaugcaacag acaacugccu ugcccacaug gggcuuggca ccuucgggca aggggagggg cuugcagcac cccccaccag uagguucgca agaaggcccu aaagauccug aagccuggag caggccacag gguugguaua aucauagcgg gaaauaa

MLKL RNA (SEQ ID NO: 6): auccagcaca gagaagcaaa caccagaagc uucucuccaa cuuucaguuu uucucuaaac uuccaguccu ucucauacug uguccugguu auaauaguug cugggaccuu auucuauucu gaauccccag ccccuaaaga aggcuuggua cugacgugua cagcguaguu acccagugac uuuggggaag gcaggaagag uguggaaagg acaggaggaa ggaagggagg ggccguggcu ccgagcugcc uagaaagcgg cuccagggag ccagggcacc gugagccugg ugguuggcag cuggagccac gucggagggg gaagugucgc agcauucucu gcaggcauca cagaccugag gcaguggccu ccggagggca cuggacagaa acagccaucc aaguggcuga guggagggac ccugcucaag ugcagcugca guggccgggg uuucccucag guagggaucg gggcgccuug ucgccgccag ccacgugugg cguccgguac agucagcaga gugcagggug cgggcaccag gaaagggggc gcaggggaac ucccgcgggc cucgcguuug caaacuucuc gccugggcag gaggcggucg ugggaaagaa gguggaagag cgagcuuuuu ggaacugugc acgggacaga uuggacgcac accccucggg aggcgcgaag gcauggaaaa uuugaagcau auuaucaccc uuggccaggu cauccacaaa cggugugaag agaugaaaua cugcaagaaa cagugccggc gccugggcca ccgcguccuc ggccugauca agccucugga gaugcuccag gaccaaggaa agaggagcgu gcccucugag aaguuaacca cagccaugaa ccgcuucaag gcugcccugg aggaggcuaa uggggagaua gaaaaguuca gcaauagauc caauaucugc agguuucuaa cagcaagcca ggacaaaaua cucuucaagg acgugaacag gaagcugagu gaugucugga aggagcucuc gcuguuacuu cagguugagc aacgcaugcc uguuucaccc auaagccaag gagcguccug ggcacaggaa gaucagcagg augcagacga agacaggcga gcuuuccaga ugcuaagaag agauaaugaa aaaauagaag cuucacugag acgauuagaa aucaacauga aagaaaucaa ggaaacuuug aggcaguauu uaccaccaaa augcaugcag gagaucccgc aagagcaaau caaggagauc aagaaggagc agcuuucagg auccccgugg auucugcuaa gggaaaauga agucagcaca cuuuauaaag gagaauacca cagagcucca guggccauaa aaguauucaa aaaacuccag gcuggcagca uugcaauagu gaggcagacu uucaauaagg agaucaaaac caugaagaaa uucgaaucuc ccaacauccu gcguauauuu gggauuugca uugaugaaac aggcuacacc auucagaagc accugaacuc cacggaaaaa ucagaagcuc aaacuuccug guaacucaag gcuaccaagu gaagauuugg guaccuggcu gaguuugggg cuggcagccu ucagcacacc aaugguguca auggcaggug ugcaugggac aagaacauau gc

ZBP1 cDNA complement (DAI) (SEQ ID NO:7):

tctcgacgtt cttcgtggtc cgagccggtg aagtcttcgg ggtcggagct ggatcgggtg ggagagtccc ggtgtcacgt cttcggacgt gtggacggtt cagagaggct gaggaacgtc gacgacagtc gtaccgggtc cgaggacgac tgggcccgtc tcttccggtg gaacttgttt cttaggacgt ccacgactgt ctccgaccga ggggccactt tgaacgggtc gaccacttcc ttacggttcg tgggttctcc ctcgagttgg ttcaggagat ggcttacttt ttcctcaact ttcagaggga gtgtagggga cggtggacca cgaacccgcc ctgactagga cttccgctcc caggacgtct cgaccggaac aggtcgggac ggctctccgg ggtcgttgta cgtcgatgtt aaggtctctg gggaccggga gtcaagtcgg ttgttgccct ccttctgtag atgtccaaag agtttctgtt accaggggtc tcccgggacc agtagcgggt tcgtgaccct tactcctgtc gttttctaca cttggctctg aacatgtcct acttctcgtc cgtggaagac ctgtacctac tcgtcaggtt tcgtacctgc taaatggcgg gtcttctaag accttcttct cgtttcagtc ggagttaata aatggtcgtg ttaggttagt tgtactagac ggtcttacct gggttgtcga cctaaaggta acgtttgagg cttcggtagg tctaacctgt gcccttgtag taatgttctg tctgtcagag gtccctcctg ccaaggcggc caggtgcggt ggagggaagt taccgtggtc cactaaggag ttgaaccccc tgggatcaac tagggacccc cggggtcctg taggtgtacc tcgtcaggta tgactctgcc cacgtcgacc ctgtgtcgtt actctactcc gaggtgccgc agggcaggct cccgggacgg gtgtaggggg gaccgtcggg gggtcacaag gtcacgtcac ccttcgtccg tcgtctcctc tctcctcgac ttagggtctc cttctgtgac tgcaggtcta gtcggcccgc cctagtcacc ccgtctgggt gacgccctag tcacctcgtc tttctgcgaa gggtctgtcg tgttgtcgtg ctcgcctcag agacggtgac gacggccggg tcttcgaagc aaacttcgtt cttaagggtc aggtccttga gtgggactcc cccttcggcg ggtctcttag gtgtacttta gcacgaaaga gctcctgcgg tggtagc RIPK3 cDNA complement (SEQ ID N0:8):

tacagcacgc agttcaatac cgggtcgcca cgggggcggg ggaaccacag gtagctcctt gacctcttgg tcctcgagca gccgtttccg cccaagccgt gtcacaaggc ccgcgttgta tccttcaccc cgatgctaca ccgccagttc tagcatttga gtttccgcta taggtccctc cagttccggt accgttcaga cctattgctt aagcacgacg cggatcttcc ccaatagctc ttccacttga ccctggttct agggttcggc cgagaccact gatttaagta cctcttgccg aggaacagcc ccgacgacgt cagggtcacg ggagccggga ccggcgagga aacggcggac gactttcttc accacgaacc ctacaaaatg gacgtgctgg tcttgggcca cgaggacgtg gccctggagt tcggtaggtt gcaggacgac ctgggtctcg acgtgcagtt cgaccgtcta aaaccggaca ggtgtaaagt ccctccgagt gtcagtccct gtcccaggcc cctcggtccc ccgtgggacc cgatgaaccg gggtcttgac aaacaattgc atttggcctt ccggaggtgt cggtcactgc agatgtcgaa gccctaggat tacacccgtc acgaacgacc ttctcttcaa ctcaacggtt ggcttggtag tgagcacatg cttcgtcaca cgttgtccgt cttggccgga agtaaccgac tcgacggggt tcggcccgga ctctgagggc cgaatcttcc tgacttcctc gattacgtcg agacgacctc gtcactcggg ttcctgtctg ggaggaaggt ccttacggat ggtttttgac tacttcagaa ggtctaccac ctcttgttat acttacgacg acagaggtgc catttcctaa aggacagagt cgagtcctcg tcgttatcct ctaaaagata gggtctcagt ccggttcctc cctgtcttta cctaccgaaa tcctcttggt atcttttggt cgtgagagca ttactacagt accaaagact caccgatttg tttgacttag atctcctcgg agggtcgaga caaggatttt ttacgggctc ggaatggttc tcctcgtccc gtgttctcct cgtccaaggt gttcggacct gtcgtccgtg tagaagtcta agctaccggg ttggaggggt ctgaggtctc tggagttgaa agtctttggt ctacgggtcg ggatggagtt gaccttgtgg ttcaggacct ggggctccct tagtcccccg actctctgtt ccgtacttga ccaggacgtc ctggggcctc ggtttaggtc attgtcccgc tggcgagcaa ttgtatatgt tgacgagacc ccacgttcaa cctctgttgt tgatgaactg atacgttgtc tgttgacgga acgggtgtac cccgaaccgt ggaagcccgt tcccctcccc gaacgtcgtg gggggtggtc atccaagcgt tcttccggga tttctaggac ttcggacctc gtccggtgtc ccaaccatat tagtatcgcc ctttatt

MLKL cDNA complement (SEQ ID N0:9):

taggtcgtgt ctcttcgttt gtggtcttcg aagagaggtt gaaagtcaaa aagagatttg aaggtcagga agagtatgac acaggaccaa tattatcaac gaccctggaa taagataaga cttaggggtc ggggatttct tccgaaccat gactgcacat gtcgcatcaa tgggtcactg aaaccccttc cgtccttctc acacctttcc tgtcctcctt ccttccctcc ccggcaccga ggctcgacgg atctttcgcc gaggtccctc ggtcccgtgg cactcggacc accaaccgtc gacctcggtg cagcctcccc cttcacagcg tcgtaagaga cgtccgtagt gtctggactc cgtcaccgga ggcctcccgt gacctgtctt tgtcggtagg ttcaccgact cacctccctg ggacgagttc acgtcgacgt caccggcccc aaagggagtc catccctagc cccgcggaac agcggcggtc ggtgcacacc gcaggccatg tcagtcgtct cacgtcccac gcccgtggtc ctttcccccg cgtccccttg agggcgcccg gagcgcaaac gtttgaagag cggacccgtc ctccgccagc accctttctt ccaccttctc gctcgaaaaa ccttgacacg tgccctgtct aacctgcgtg tggggagccc tccgcgcttc cgtacctttt aaacttcgta taatagtggg aaccggtcca gtaggtgttt gccacacttc tctactttat gacgttcttt gtcacggccg cggacccggt ggcgcaggag ccggactagt tcggagacct ctacgaggtc ctggttcctt tctcctcgca cgggagactc ttcaattggt gtcggtactt ggcgaagttc cgacgggacc tcctccgatt acccctctat cttttcaagt cgttatctag gttatagacg tccaaagatt gtcgttcggt cctgttttat gagaagttcc tgcacttgtc cttcgactca ctacagacct tcctcgagag cgacaatgaa gtccaactcg ttgcgtacgg acaaagtggg tattcggttc ctcgcaggac ccgtgtcctt ctagtcgtcc tacgtctgct tctgtccgct cgaaaggtct acgattcttc tctattactt ttttatcttc gaagtgactc tgctaatctt tagttgtact ttctttagtt cctttgaaac tccgtcataa atggtggttt tacgtacgtc ctctagggcg ttctcgttta gttcctctag ttcttcctcg tcgaaagtcc taggggcacc taagacgatt cccttttact tcagtcgtgt gaaatatttc ctcttatggt gtctcgaggt caccggtatt ttcataagtt ttttgaggtc cgaccgtcgt aacgttatca ctccgtctga aagttattcc tctagttttg gtacttcttt aagcttagag ggttgtagga cgcatataaa ccctaaacgt aactactttg tccgatgtgg taagtcttcg tggacttgag gtgccttttt agtcttcgag tttgaaggac cattgagttc cgatggttca cttctaaacc catggaccga ctcaaacccc gaccgtcgga agtcgtgtgg ttaccacagt taccgtccac acgtaccctg ttcttgtata eg

ZBP1 (DAI) RNA complement (SEQ ID NO:10):

ucucgacguu cuueguggue egagceggug aagucuuegg ggucggagcu ggaucgggug ggagaguccc ggugucacgu cuueggaegu guggaegguu cagagaggcu gaggaacguc gacgacaguc guacegggue cgaggacgac ugggcccguc ucuuccggug gaacuuguuu cuuaggacgu ccacgacugu cuccgaccga ggggccacuu ugaaegggue gaccacuucc uuacgguucg uggguueuce cucgaguugg uucaggagau ggcuuacuuu uuccucaacu uucagaggga guguagggga egguggacca cgaacccgcc cugacuagga cuuccgcucc caggacgucu cgaccggaac agguegggae ggcucuccgg ggucguugua cgucgauguu aaggucucug gggaccggga gucaaguegg uuguugcecu ecuueuguag auguccaaag aguuucuguu accagggguc ucccgggacc aguagegggu ucgugacccu uacuccuguc guuuucuaca cuuggcucug aacauguccu acuucucguc cguggaagac cuguaccuac ucgucagguu ucguaccugc uaaauggegg gucuueuaag accuucuucu cguuucaguc ggaguuaaua aauggucgug uuagguuagu uguacuagac ggucuuaccu ggguugucga ccuaaaggua acguuugagg cuuegguagg ucuaaccugu geccuuguag uaauguucug ucugucagag gucccuccug ccaaggcggc caggugeggu ggagggaagu uaccgugguc cacuaaggag uugaaccccc ugggaucaac uagggacccc egggguccug uagguguacc ucgucaggua ugacucugcc cacgucgacc cugugucguu acucuacucc gaggugcege agggcaggcu cccgggacgg guguaggggg gaecgueggg gggucacaag gucacgucac ccuucguccg ucgucuccuc ucuccucgac uuagggucuc cuucugugac ugcaggucua gucggcccgc ccuagucacc ccgucugggu gacgcccuag ucaccucguc uuucugcgaa gggucugucg uguugucgug cucgccucag agacggugac gacggccggg ucuucgaagc aaacuucguu cuuaaggguc agguccuuga gugggacucc cccuucggcg ggucucuuag guguacuuua gcacgaaaga gcuccugcgg ugguagc

RIPK3 RNA complement (SEQ ID NO: 11):

uacagcacgc aguucaauac cgggucgcca cgggggcggg ggaaccacag guagcuccuu gaccucuugg uccucgagca gccguuuccg cccaagccgu gucacaaggc ccgcguugua uccuucaccc cgaugcuaca ccgccaguuc uagcauuuga guuuccgcua uaggucccuc caguuccggu accguucaga ccuauugcuu aagcacgacg cggaucuucc ccaauagcuc uuccacuuga cccugguucu aggguucggc cgagaccacu gauuuaagua ccucuugccg aggaacagcc ccgacgacgu cagggucacg ggagccggga ccggcgagga aacggcggac gacuuucuuc accacgaacc cuacaaaaug gacgugcugg ucuugggcca cgaggacgug gcccuggagu ucgguagguu gcaggacgac cugggucucg acgugcaguu cgaccgucua aaaccggaca gguguaaagu cccuccgagu gucagucccu gucccaggcc ccucgguccc ccgugggacc cgaugaaccg gggucuugac aaacaauugc auuuggccuu ccggaggugu cggucacugc agaugucgaa gcccuaggau uacacccguc acgaacgacc uucucuucaa cucaacgguu ggcuugguag ugagcacaug cuucgucaca cguuguccgu cuuggccgga aguaaccgac ucgacggggu ucggcccgga cucugagggc cgaaucuucc ugacuuccuc gauuacgucg agacgaccuc gucacucggg uuccugucug ggaggaaggu ccuuacggau gguuuuugac uacuucagaa ggucuaccac cucuuguuau acuuacgacg acagaggugc cauuuccuaa aggacagagu cgaguccucg ucguuauccu cuaaaagaua gggucucagu ccgguuccuc ccugucuuua ccuaccgaaa uccucuuggu aucuuuuggu cgugagagca uuacuacagu accaaagacu caccgauuug uuugacuuag aucuccucgg agggucgaga caaggauuuu uuacgggcuc ggaaugguuc uccucguccc guguucuccu cguccaaggu guucggaccu gucguccgug uagaagucua agcuaccggg uuggaggggu cugaggucuc uggaguugaa agucuuuggu cuacgggucg ggauggaguu gaccuugugg uucaggaccu ggggcucccu uagucccccg acucucuguu ccguacuuga ccaggacguc cuggggccuc gguuuagguc auugucccgc uggcgagcaa uuguauaugu ugacgagacc ccacguucaa ccucuguugu ugaugaacug auacguuguc uguugacgga acggguguac cccgaaccgu ggaagcccgu uccccucccc gaacgucgug gggggugguc auccaagcgu ucuuccggga uuucuaggac uucggaccuc guccgguguc ccaaccauau uaguaucgcc cuuuauu

MLKL RNA complement (SEQ ID NO:12):

uaggucgugu cucuucguuu guggucuucg aagagagguu gaaagucaaa aagagauuug aaggucagga agaguaugac acaggaccaa uauuaucaac gacccuggaa uaagauaaga cuuagggguc ggggauuucu uccgaaccau gacugcacau gucgcaucaa ugggucacug aaaccccuuc cguccuucuc acaccuuucc uguccuccuu ccuucccucc ccggcaccga ggcucgacgg aucuuucgcc gaggucccuc ggucccgugg cacucggacc accaaccguc gaccucggug cagccucccc cuucacagcg ucguaagaga cguccguagu gucuggacuc cgucaccgga ggccucccgu gaccugucuu ugucgguagg uucaccgacu caccucccug ggacgaguuc acgucgacgu caccggcccc aaagggaguc caucccuagc cccgcggaac agcggcgguc ggugcacacc gcaggccaug ucagucgucu cacgucccac gcccgugguc cuuucccccg cguccccuug agggcgcccg gagcgcaaac guuugaagag cggacccguc cuccgccagc acccuuucuu ccaccuucuc gcucgaaaaa ccuugacacg ugcccugucu aaccugcgug uggggagccc uccgcgcuuc cguaccuuuu aaacuucgua uaauaguggg aaccggucca guagguguuu gccacacuuc ucuacuuuau gacguucuuu gucacggccg cggacccggu ggcgcaggag ccggacuagu ucggagaccu cuacgagguc cugguuccuu ucuccucgca cgggagacuc uucaauuggu gucgguacuu ggcgaaguuc cgacgggacc uccuccgauu accccucuau cuuuucaagu cguuaucuag guuauagacg uccaaagauu gucguucggu ccuguuuuau gagaaguucc ugcacuuguc cuucgacuca cuacagaccu uccucgagag cgacaaugaa guccaacucg uugcguacgg acaaaguggg uauucgguuc cucgcaggac ccguguccuu cuagucgucc uacgucugcu ucuguccgcu cgaaaggucu acgauucuuc ucuauuacuu uuuuaucuuc gaagugacuc ugcuaaucuu uaguuguacu uucuuuaguu ccuuugaaac uccgucauaa auggugguuu uacguacguc cucuagggcg uucucguuua guuccucuag uucuuccucg ucgaaagucc uaggggcacc uaagacgauu cccuuuuacu ucagucgugu gaaauauuuc cucuuauggu gucucgaggu caccgguauu uucauaaguu uuuugagguc cgaccgucgu aacguuauca cuccgucuga aaguuauucc ucuaguuuug guacuucuuu aagcuuagag gguuguagga cgcauauaaa cccuaaacgu aacuacuuug uccgaugugg uaagucuucg uggacuugag gugccuuuuu agucuucgag uuugaaggac cauugaguuc cgaugguuca cuucuaaacc cauggaccga cucaaacccc gaccgucgga agucgugugg uuaccacagu uaccguccac acguacccug uucuuguaua eg

ZBP1 (DAI) Protein (SEQ ID NO: 13):

MAQAPADPGR EGHLEQRILQ VLTEAGSPVK LAQLVKECQA PKRELNQVLY RMKKELKVSL TSPATWCLGG TDPEGEGPAE LALSSPAKRP QQHAATIPET PGPQFSQQRE EDIYRFLKDN GPQRALVIAQ ALGMRTAKDV NRDLYRMKSR HLLDMDEQSK AWTIYRPEDS GRRAKSASII YQHNPINMIC QNGPNSWISI ANSEAIQIGH GNIITRQTVS REDGSAGPRH LPSMAPGDSS TWGTLVDPWG PQDIHMERSI LRRVQLGHSN EMRLHGVPSE GPAHIPPGSP PVSATAAGPE ASFEARIPSP GTHPEGEAAQ RIHMKSCFLE DATIGNSNKM SISPGVAGPG GVAGSGEGEP GEDAGRRPAD TQSRSHFPRD IGQPITPSHS KLTPKLETMT LGNRSHKAAE GSHYVDEASH EGSWWGGGI RIPK3 Protein (SEQ ID NO: 14):

MSCVKLWPSG APAPLVSIEE LENQELVGKG GFGTVFRAQH RKWGYDVAVK IVNSKAISRE VKAM AS LDNE FVLRLEGVIE KVNWDQDPKP ALVTKFMENG SLSGLLQSQC PRPWPLLCRL LKEVVLGMFY LHDQNPVLLH RDLKPSNVLL DPELHVKLAD FGLSTFQGGS QSGTGSGEPG GTLGYLAPEL FVNVNRKAST ASDVYSFGIL MWAVLAGREV ELPTEPSLVY EAVCNRQNRP SLAELPQAGP ETPGLEGLKE LMQLCWSSEP KDRPSFQECL PKTDEVFQMV ENNMNAAVST VKDFLSQLRS SNRRFSIPES GQGGTEMDGF RRTIENQHSR NDVMVSEWLN KLNLEEPPSS VPKKCPSLTK RSRAQEEQVP QAWTAGTSSD SMAQPPQTPE TSTFRNQMPS PTSTGTPSPG PRGNQGAERQ GMNWSCRTPE PNPVTGRPLV NIYNCSGVQV GDNNYLTMQQ TTALPTWGLA PSGKGRGLQH PPPVGSQEGP KDPEAWSRPQ GWYNHSGK

MLKL Protein (SEQ ID NO:15):

MENLKHIITL GQ VIHKRCEE MKYCKKQCRR LGHRVLGLIK PLEMLQDQGK RSVPSEKLTT AMNRFKAALE EANGEIEKFS NRSNICRFLT ASQDKILFKD VNRKLSDVWK ELSLLLQVEQ RMPVSPISQG ASWAQEDQQD ADEDRRAFQM LRRDNEKIEA SLRRLEINMK EIKETLRQYL PPKCMQEIPQ EQIKEIKKEQ LSGSPWILLR ENEVSTLYKG EYHRAPVAIK VFKKLQAGSI AIVRQTFNKE IKTMKKFESP NILRIFGICI DETVTPPQFS IVMEYCELGT LRELLDREKD LTLGKRMVLV LGAARGLYRL HHSEAPELHG KIRSSNFLVT QGYQVKLAGF ELRKTQTSMS LGTTREKTDR VKSTAYLSPQ ELEDVFYQYD VKSEIYSFGI VLWEIATGDI PFQGCNSEKI RKLVAVKRQQ EPLGEDCPSE LREIIDECRA HDPSVRPSVD EILKKLSTFS K CRISPR 1 (SEQ ID NO: 16): tctggagtcacacaagagtcccct

CRISPR 2 (SEQ ID NO: 17): gctcagtacatctacatggacaagtccttg

Universal genomic viral RNA (vRNA) primer (SEQ ID NO:18): agcaaaagcagg

Primer 1 (SEQ ID NO: 19): atggaaagaataaaagaactaag

Primer 2 (SEQ ID NO:20): ctaattgatggccatccgaattc

Targeted sequence (SEQ ID NO:21): tgagaacgttctgctcctgc

The present disclosure is not limited to the embodiments described and exemplified above, but is capable of variation and modification within the scope of the appended claims.