Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: Identification of CoV-RHIMs in Nsp13 and Nsp14 of SARS-CoV-2 and bats as hosts in viral RHIM evolution in RNA viruses (A) Schematic representation showing the genomic organization of SARS-CoV-2 and CoV-RHIMs in Nsp13 and Nsp14 proteins encoded by ORF1b. (B) Amino acid residues and their relative conservation in RHIMs of human proteins (RIPK1, RIPK3, and ZBP1), M45 of murine cytomegalovirus (MCMV), ICP6 and ICP10 of herpes simplex virus (HSV), ORF20 of varicella-zoster virus (VZV) and Nsp13 and Nsp14 of CoVs (SARS-CoV-2, MERS-CoV and CoV-NL63). Human ZBP1 consists of two RHIM sequences (ZBP1-R1 and ZBP1-R2). (C) Phylogenetic tree representing the relatedness of different CoV genera based on protein sequences encoded by ORF1ab. (D–F) The phylogenetic tree and the relative conservation of CoV-RHIM-1 in Nsp13 (D), CoV-RHIM-2 (E), and CoV-RHIM-3 (F) in Nsp14 across CoVs. (G) Bat species from Yangochiroptera and Yinpterochiroptera suborders considered for analyzing bat-originated RIPK3 and ZBP1 protein sequences. (H) Protein sequence alignment and phylogenetic trees showing relative conservation of human and bat RHIM-sequences within RIPK3 and ZBP1 proteins. (I) The potential viral RHIM signatures in lyssavirus rabies, picornavirus, and hantaviruses that originate from bats. The core tetrad residues and the proximal conserved residues of RHIMs are highlighted in bold; conserved or identical residue positions in RHIM sequences are highlighted in red; less conserved but chemically similar residue positions in RHIM sequences are highlighted in blue. Asterisk (∗) symbols indicate Wuhan SARS-CoV-2 isolates.

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: Virus, Sequencing, Residue

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: Nsp13 promotes RNA-binding channel-dependent bat cell death, and CoV-RHIM-1 function is less critical (A) Microscopic analysis of Tb1 Lu cells infected with lentiviruses expressing SARS-CoV-2 Nsp13-WT, Nsp13-Tet-mut, Nsp13-Δ1B, Nsp13- Δ1B-Rec1A-Rec2A and EGFP followed by puromycin treatment. Scale bar, 50μm. (B) Immunoblot analysis of lysates from Tb1 Lu cells showing expression of Nsp13-WT and its mutants after lentivirus transduction. (C) Representative Caspase-3/7 and Sytox green staining images of Tb1 Lu cells infected with lentiviruses expressing SARS-CoV-2 Nsp13-WT, Nsp13-Tet-mut, Nsp13-Δ1B and Nsp13- Δ1B-Rec1A-Rec2A followed by puromycin treatment acquired by Incucyte imaging analysis system. Scale bar, 200μm. (D) Real-time cell death measurement by Caspase-3/7 and Sytox green staining of Tb1 Lu cells infected as in panel-C. ∗∗∗∗ p < 0.0001, ∗∗∗ p = 0.0003, ∗∗ p = 0.0046 (Sytox green staining), ∗∗ p = 0.0086 (Caspase-3/7 staining), ns, not significant (two-way ANOVA, n = 3). Data shown are mean ± SEM. (E and F) Immunoblot analysis of RIPK1, RIPK3, ZBP1 and MLKL in lysates from `Tb1Lu cells after mock treatment, Nsp13 or Poly(I:C) transfection. The antibodies used for detecting these proteins were specific to human and mouse proteins. (G and H) Real-time cell death measurement by Sytox green staining of Tb1 Lu (G) and HT-29 cells (H), after treatment of TNF, zVAD and SMACmimetic (SMACmim). ∗∗∗∗ p < 0.0001 (two-way ANOVA, n = 3), ns – not significant (two-way ANOVA). (I and J) Real-time cell death measurement by Sytox green staining of Tb1 Lu cells transfected with Poly(I:C) (I) or treated with Curaxin (J) ∗∗∗∗ p < 0.0001 (two-way ANOVA, n = 3). Data shown are mean ± SEM. (K) Immunoblot analysis of phosphorylated MLKL (pMLKL) in lysates from Tb1Lu cells after mock or Nsp13 transfection.

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: RNA Binding Assay, Infection, Expressing, Western Blot, Transduction, Staining, Imaging, Transfection

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 Nsp13 shows RHIM-dependent interaction with host ZBP1 and RIPK3 proteins (A and B) Immunoblot analysis of anti-Strep-Tag (Nsp13), anti-RIPK1, anti-HA-tag (RIPK3), and anti-ZBP1 immunoprecipitates (A) and whole cell lysates (inputs) (B) from HEK-293T cell lysates expressing Nsp13 alone or co-expressing Nsp13, RIPK3 and ZBP1. (C and D) Immunoblot analysis of anti-Strep-Tag (Nsp13) and anti-HA-tag (ZBP1) immunoprecipitates (C) and inputs (D) from HEK-293T cell lysates expressing Nsp13 WT, Nsp13-Tet-Mut, Δ1B, ZBP1-WT and ZBP1-RHIM-Mut alone or in indicated combinations of Nsp13 and ZBP1 constructs. (E) Confocal microscopy imaging of HEK-293T cells expressing Nsp13-EGFP or ZBP1-dTomoto alone or co-expressing both the constructs. Scale bar, 10μm. (F) Confocal microscopy imaging of HEK-293Tcells expressing Nsp13-EGFP and in combination with ZBP1-HA or RIPK3-HA. Scale bar, 10μm. (G) Immunoblot analysis of anti-Strep-Tag (Nsp13) and anti-HA-tag (RIPK3) immunoprecipitates from insoluble fractions of HEK-293T cell lysates expressing Nsp13-WT, Nsp13-Tet-Mut and RIPK3 individually or in indicated combinations of Nsp13 and RIPK3 constructs. L.E. – long exposure; S.E. – short exposure. (H) Inputs for insoluble and soluble fractions of cell lysates for Panel-G.

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: Western Blot, Strep-tag, Expressing, Construct, Confocal Microscopy, Imaging

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 Nsp13 triggers host-RHIM protein oligomerization and large complex formation (A) Immunoblot analysis of crosslinked lysates of HEK-293T cells expressing Nsp13-WT, RIPK3 and ZBP1 individually or co-expressing Nsp13-WT+ZBP1, Nsp13-WT+RIPK3, Nsp13-WT+ZBP1+RIPK3 or ZBP1+RIPK3, in non-reduced (without BME) and reduced (with BME) conditions. O- oligomer complexes; M-Monomer. (B and C) Visualization of SARS-CoV-2 Nsp13 and ZBP1 (B) or RIPK3 (C) in HEK-293T cells using DNA-PAINT imaging. Scale bars, 5 μm (complete cell image); 200nm (magnified images).

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: Western Blot, Expressing, Imaging

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 Nsp13 promotes ZBP1-RIPK3 signaling-dependent cell death and is regulated by intracellular RNA ligands (A) Real-time cell death measurement by Sytox green staining of WT and Zbp1 −/− L929 cells ectopically expressing Nsp13-WT and Nsp13-Tet-mut after IAV, IAV+zVAD and mock infection. ∗∗∗ p = 0.0005, ∗ p = 0.0307, ns, not significant (two-way ANOVA). (B) Real-time cell death measurement by Sytox green staining of ZBP1 expressing HT-29 cells after Nsp13 transfection and IAV+zVAD infection. (C and D) Cell death measurement by Sytox green staining of WT and Zbp1 −/− L929 cells expressing Nsp13-WT after LMB+IFN-β (C) and Curaxin (D) treatment. Data shown are mean ± SEM. (E) Microscopic images of cell death in L929 treated as indicated and acquired by Incucyte imaging analysis for the cells. Scale bar, 200μm. (F) Real-time analysis of cell death of mock or Nsp13 transfected L929 cells treated/infected with IFN-β alone, IAV alone, IAV + IFN-β, LMB alone or LMB + IFN-β. ∗∗∗∗ p < 0.0001, ∗∗ p = 0.0022, ∗ p = 0.0310, ns, not significant (two-way ANOVA, n = 3). Data shown are mean ± SEM.

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: Staining, Expressing, Infection, Transfection, Imaging

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 encode Z-RNA forming genomic segments that enhance Nsp13-mediated cell death (A) Specific SARS-CoV-2 genome segments with high Z-RNA forming dsRNA conformations. The orange box outlines represent alternate purine-pyrimidine repeats with Z-RNA forming and Zα-domain binding potential. (B) SDS-PAGE gel picture representing purified human ZBP1-Zα1α2 domains. (C and D) Octet binding of SARS-CoV-2 Z-RNAs (SC2-zRNA), control Z-RNA (alternate purine-pyrimidine repeats favoring Z-RNA conformation) and poly(I:C) at 100nM (D) and 200nM (E) concentration to human ZBP1-Zα1α2 domains. (E) Electrophoretic mobility shift assay of control Z-RNA and SARS-CoV-2 Z-RNAs with human ZBP1-Zα1α2 domains to monitor their interaction. (F) Real-time cell death measurement by Sytox green staining of Nsp13 expressing L929 cells after transient transfection with control zRNA, SARS-CoV-2 Z-RNAs (SC2-zRNA) or Poly(I:C). ∗∗∗∗ p < 0.0001 (two-way ANOVA, n = 3). Data shown are mean ± SEM.

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: Binding Assay, SDS Page, Purification, Control, Concentration Assay, Electrophoretic Mobility Shift Assay, Staining, Expressing, Transfection

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet:

Article Snippet: pCMV-Human-ZBP1 cDNA , Sino Biological , HG19385-UT.

Techniques: Virus, Recombinant, Transfection, Staining, Western Blot, Electron Microscopy, Plasmid Preparation, Software, Microscopy

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: Identification of CoV-RHIMs in Nsp13 and Nsp14 of SARS-CoV-2 and bats as hosts in viral RHIM evolution in RNA viruses (A) Schematic representation showing the genomic organization of SARS-CoV-2 and CoV-RHIMs in Nsp13 and Nsp14 proteins encoded by ORF1b. (B) Amino acid residues and their relative conservation in RHIMs of human proteins (RIPK1, RIPK3, and ZBP1), M45 of murine cytomegalovirus (MCMV), ICP6 and ICP10 of herpes simplex virus (HSV), ORF20 of varicella-zoster virus (VZV) and Nsp13 and Nsp14 of CoVs (SARS-CoV-2, MERS-CoV and CoV-NL63). Human ZBP1 consists of two RHIM sequences (ZBP1-R1 and ZBP1-R2). (C) Phylogenetic tree representing the relatedness of different CoV genera based on protein sequences encoded by ORF1ab. (D–F) The phylogenetic tree and the relative conservation of CoV-RHIM-1 in Nsp13 (D), CoV-RHIM-2 (E), and CoV-RHIM-3 (F) in Nsp14 across CoVs. (G) Bat species from Yangochiroptera and Yinpterochiroptera suborders considered for analyzing bat-originated RIPK3 and ZBP1 protein sequences. (H) Protein sequence alignment and phylogenetic trees showing relative conservation of human and bat RHIM-sequences within RIPK3 and ZBP1 proteins. (I) The potential viral RHIM signatures in lyssavirus rabies, picornavirus, and hantaviruses that originate from bats. The core tetrad residues and the proximal conserved residues of RHIMs are highlighted in bold; conserved or identical residue positions in RHIM sequences are highlighted in red; less conserved but chemically similar residue positions in RHIM sequences are highlighted in blue. Asterisk (∗) symbols indicate Wuhan SARS-CoV-2 isolates.

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: Virus, Sequencing, Residue

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: Nsp13 promotes RNA-binding channel-dependent bat cell death, and CoV-RHIM-1 function is less critical (A) Microscopic analysis of Tb1 Lu cells infected with lentiviruses expressing SARS-CoV-2 Nsp13-WT, Nsp13-Tet-mut, Nsp13-Δ1B, Nsp13- Δ1B-Rec1A-Rec2A and EGFP followed by puromycin treatment. Scale bar, 50μm. (B) Immunoblot analysis of lysates from Tb1 Lu cells showing expression of Nsp13-WT and its mutants after lentivirus transduction. (C) Representative Caspase-3/7 and Sytox green staining images of Tb1 Lu cells infected with lentiviruses expressing SARS-CoV-2 Nsp13-WT, Nsp13-Tet-mut, Nsp13-Δ1B and Nsp13- Δ1B-Rec1A-Rec2A followed by puromycin treatment acquired by Incucyte imaging analysis system. Scale bar, 200μm. (D) Real-time cell death measurement by Caspase-3/7 and Sytox green staining of Tb1 Lu cells infected as in panel-C. ∗∗∗∗ p < 0.0001, ∗∗∗ p = 0.0003, ∗∗ p = 0.0046 (Sytox green staining), ∗∗ p = 0.0086 (Caspase-3/7 staining), ns, not significant (two-way ANOVA, n = 3). Data shown are mean ± SEM. (E and F) Immunoblot analysis of RIPK1, RIPK3, ZBP1 and MLKL in lysates from `Tb1Lu cells after mock treatment, Nsp13 or Poly(I:C) transfection. The antibodies used for detecting these proteins were specific to human and mouse proteins. (G and H) Real-time cell death measurement by Sytox green staining of Tb1 Lu (G) and HT-29 cells (H), after treatment of TNF, zVAD and SMACmimetic (SMACmim). ∗∗∗∗ p < 0.0001 (two-way ANOVA, n = 3), ns – not significant (two-way ANOVA). (I and J) Real-time cell death measurement by Sytox green staining of Tb1 Lu cells transfected with Poly(I:C) (I) or treated with Curaxin (J) ∗∗∗∗ p < 0.0001 (two-way ANOVA, n = 3). Data shown are mean ± SEM. (K) Immunoblot analysis of phosphorylated MLKL (pMLKL) in lysates from Tb1Lu cells after mock or Nsp13 transfection.

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: RNA Binding Assay, Infection, Expressing, Western Blot, Transduction, Staining, Imaging, Transfection

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 Nsp13 shows RHIM-dependent interaction with host ZBP1 and RIPK3 proteins (A and B) Immunoblot analysis of anti-Strep-Tag (Nsp13), anti-RIPK1, anti-HA-tag (RIPK3), and anti-ZBP1 immunoprecipitates (A) and whole cell lysates (inputs) (B) from HEK-293T cell lysates expressing Nsp13 alone or co-expressing Nsp13, RIPK3 and ZBP1. (C and D) Immunoblot analysis of anti-Strep-Tag (Nsp13) and anti-HA-tag (ZBP1) immunoprecipitates (C) and inputs (D) from HEK-293T cell lysates expressing Nsp13 WT, Nsp13-Tet-Mut, Δ1B, ZBP1-WT and ZBP1-RHIM-Mut alone or in indicated combinations of Nsp13 and ZBP1 constructs. (E) Confocal microscopy imaging of HEK-293T cells expressing Nsp13-EGFP or ZBP1-dTomoto alone or co-expressing both the constructs. Scale bar, 10μm. (F) Confocal microscopy imaging of HEK-293Tcells expressing Nsp13-EGFP and in combination with ZBP1-HA or RIPK3-HA. Scale bar, 10μm. (G) Immunoblot analysis of anti-Strep-Tag (Nsp13) and anti-HA-tag (RIPK3) immunoprecipitates from insoluble fractions of HEK-293T cell lysates expressing Nsp13-WT, Nsp13-Tet-Mut and RIPK3 individually or in indicated combinations of Nsp13 and RIPK3 constructs. L.E. – long exposure; S.E. – short exposure. (H) Inputs for insoluble and soluble fractions of cell lysates for Panel-G.

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: Western Blot, Strep-tag, Expressing, Construct, Confocal Microscopy, Imaging

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 Nsp13 triggers host-RHIM protein oligomerization and large complex formation (A) Immunoblot analysis of crosslinked lysates of HEK-293T cells expressing Nsp13-WT, RIPK3 and ZBP1 individually or co-expressing Nsp13-WT+ZBP1, Nsp13-WT+RIPK3, Nsp13-WT+ZBP1+RIPK3 or ZBP1+RIPK3, in non-reduced (without BME) and reduced (with BME) conditions. O- oligomer complexes; M-Monomer. (B and C) Visualization of SARS-CoV-2 Nsp13 and ZBP1 (B) or RIPK3 (C) in HEK-293T cells using DNA-PAINT imaging. Scale bars, 5 μm (complete cell image); 200nm (magnified images).

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: Western Blot, Expressing, Imaging

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 Nsp13 promotes ZBP1-RIPK3 signaling-dependent cell death and is regulated by intracellular RNA ligands (A) Real-time cell death measurement by Sytox green staining of WT and Zbp1 −/− L929 cells ectopically expressing Nsp13-WT and Nsp13-Tet-mut after IAV, IAV+zVAD and mock infection. ∗∗∗ p = 0.0005, ∗ p = 0.0307, ns, not significant (two-way ANOVA). (B) Real-time cell death measurement by Sytox green staining of ZBP1 expressing HT-29 cells after Nsp13 transfection and IAV+zVAD infection. (C and D) Cell death measurement by Sytox green staining of WT and Zbp1 −/− L929 cells expressing Nsp13-WT after LMB+IFN-β (C) and Curaxin (D) treatment. Data shown are mean ± SEM. (E) Microscopic images of cell death in L929 treated as indicated and acquired by Incucyte imaging analysis for the cells. Scale bar, 200μm. (F) Real-time analysis of cell death of mock or Nsp13 transfected L929 cells treated/infected with IFN-β alone, IAV alone, IAV + IFN-β, LMB alone or LMB + IFN-β. ∗∗∗∗ p < 0.0001, ∗∗ p = 0.0022, ∗ p = 0.0310, ns, not significant (two-way ANOVA, n = 3). Data shown are mean ± SEM.

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: Staining, Expressing, Infection, Transfection, Imaging

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet: SARS-CoV-2 encode Z-RNA forming genomic segments that enhance Nsp13-mediated cell death (A) Specific SARS-CoV-2 genome segments with high Z-RNA forming dsRNA conformations. The orange box outlines represent alternate purine-pyrimidine repeats with Z-RNA forming and Zα-domain binding potential. (B) SDS-PAGE gel picture representing purified human ZBP1-Zα1α2 domains. (C and D) Octet binding of SARS-CoV-2 Z-RNAs (SC2-zRNA), control Z-RNA (alternate purine-pyrimidine repeats favoring Z-RNA conformation) and poly(I:C) at 100nM (D) and 200nM (E) concentration to human ZBP1-Zα1α2 domains. (E) Electrophoretic mobility shift assay of control Z-RNA and SARS-CoV-2 Z-RNAs with human ZBP1-Zα1α2 domains to monitor their interaction. (F) Real-time cell death measurement by Sytox green staining of Nsp13 expressing L929 cells after transient transfection with control zRNA, SARS-CoV-2 Z-RNAs (SC2-zRNA) or Poly(I:C). ∗∗∗∗ p < 0.0001 (two-way ANOVA, n = 3). Data shown are mean ± SEM.

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: Binding Assay, SDS Page, Purification, Control, Concentration Assay, Electrophoretic Mobility Shift Assay, Staining, Expressing, Transfection

Journal: iScience

Article Title: Bat RNA viruses employ viral RHIMs orchestrating species-specific cell death programs linked to Z-RNA sensing and ZBP1-RIPK3 signaling

doi: 10.1016/j.isci.2024.111444

Figure Lengend Snippet:

Article Snippet: The coding sequence of human ZBP1 (hZBP1) was amplified from pCMV-Human-ZBP1 cDNA clone expression plasmid (Sino Biological Inc.) and subcloned into pLVX-EF1alpha-2xStrep-IRES-Puro backbone using EcoRI and BamHI restriction sites.

Techniques: Virus, Recombinant, Transfection, Staining, Western Blot, Electron Microscopy, Plasmid Preparation, Software, Microscopy