Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: The SIV-induced IL-1β production requires receptor-interacting protein kinase 1 (RIPK1) kinase activity. ( A ) PAMs were transfected with a negative control siRNA (siControl) or RIPK1 -targeting siRNA (siRIPK1) for 24 h and infected with Sk02 at an MOI of 1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β was analyzed by Western blotting. RIPK1 knockdown efficiency was determined by real-time PCR. ( B ) PAMs were infected with Sk02 at an MOI of 1 for 20 h in the presence of a vehicle (DMSO) or increasing concentrations (80 and 160 μM) of RIPK1 kinase inhibitor, Nec-1. Porcine IL-1β levels from supernatants were measured by ELISA. Expression of pro-IL-1β and viral NS1 was analyzed by Western blotting. ( C ) Amino acid sequences of the N-terminal ends of the kinase domains in porcine, murine, and human RIPK1 (based on the GenBank accession numbers XM_005665536, NM_009068, and NM_003804, respectively) were aligned using Clustal Omega. Amino acid positions are based on the human RIPK1 and asterisks indicate conserved residues among the three species. K41/K42 in porcine RIPK1, K45/K46 in murine RIPK1, and K45 in human RIPK1 are underscored. ( D ) For the NLRP3 inflammasome reconstitution assay, HEK293T cells were co-transfected with plasmids expressing porcine NLRP3 inflammasome components (NLRP3, ASC, and procaspase-1) and pro-IL-1β along with Myc-vector or Myc-tagged WT/mutant human RIPK1 for 16 h. Porcine IL-1β levels from supernatants were measured by ELISA. Protein expression was analyzed by Western blotting. ( E ) HEK293T cells were transfected as in ( D ) along with the Myc-vector or Myc-tagged porcine RIPK1 WT/mutant for 16 h. ELISA and Western blotting were done as in ( D ). Statistical analysis was done with one-way ANOVA except for the knockdown efficiency data done with an unpaired t -test. * p < 0.05; ** p < 0.01; *** p < 0.001. Results are representative of three independent experiments.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Activity Assay, Transfection, Negative Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Knockdown, Real-time Polymerase Chain Reaction, Reconstitution Assay, Plasmid Preparation, Mutagenesis

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: RIPK1 interacts with DRP1. HEK293T cells were co-transfected with the Flag-vector or Flag-RIPK1 construct along with the DRP1-Myc construct for 24 h. Cell lysates were subjected to co-IP with Flag antibody, and the expression of Flag-RIPK1 and DRP1-Myc in input and IP samples was analyzed by Western blotting. Results are representative of three independent experiments.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Transfection, Plasmid Preparation, Construct, Co-Immunoprecipitation Assay, Expressing, Western Blot

Journal: Viruses

Article Title: Swine Influenza Virus Induces RIPK1/DRP1-Mediated Interleukin-1 Beta Production

doi: 10.3390/v10080419

Figure Lengend Snippet: A proposed model of RIPK1/DRP1-mediated IL-1β production in SIV-infected PAMs. Recognition of IAV RNA by endosomal TLRs or RIG-I is known to induce pro-IL-1β synthesis. SIV infection turns on the RIPK1/DRP1 signaling for the NLRP3 inflammasome activation in PAMs. SIV seems to utilize the RIPK1 function, while how RIPK1 is activated by SIV or whether an upstream sensor plays a role in its activation is unclear. By interacting with DRP1 and through its kinase activity, RIPK1 induces the phosphorylation of porcine DRP1 at S579. Upon DRP1 translocation to mitochondria, mitochondrial fission occurs and ROS is generated. This promotes the activation of NLRP3 that is required for the NLRP3 inflammasome assembly and caspase-1 activation. Active caspase-1 converts pro-IL-1β into mature IL-1β, which is critical for SIV-induced lung inflammation. Sensing LPS by TLR4 is known to induce the pro-IL-1β expression, and this also induces the DRP1 phosphorylation leading to the NLRP3 inflammasome activation in PAMs, although it is unclear whether LPS activates any upstream regulator of RIPK1.

Article Snippet: Myc-tagged human RIPK1 (#44159, Addgene, Cambridge, MA, USA) was a gift from Xin Lin [ ].

Techniques: Infection, Activation Assay, Activity Assay, Phospho-proteomics, Translocation Assay, Generated, Expressing