PKR and TLR3 trigger distinct signals that coordinate the induction of antiviral apoptosis

Abstract

RIG-I-like receptors (RLRs), protein kinase R (PKR), and endosomal Toll-like receptor 3 (TLR3) sense viral non-self RNA and are involved in cell fate determination. However, the mechanisms by which intracellular RNA induces apoptosis, particularly the role of each RNA sensor, remain unclear. We performed cytoplasmic injections of different types of RNA and elucidated the molecular mechanisms underlying viral dsRNA-induced apoptosis. The results obtained revealed that short 5′-triphosphate dsRNA, the sole ligand of RIG-I, induced slow apoptosis in a fraction of cells depending on IRF-3 transcriptional activity and IFN-I production. However, intracellular long dsRNA was sensed by PKR and TLR3, which activate distinct signals, and synergistically induced rapid apoptosis. PKR essentially induced translational arrest, resulting in reduced levels of cellular FLICE-like inhibitory protein and functioned in the TLR3/TRIF-dependent activation of caspase 8. The present results demonstrated that PKR and TLR3 were both essential for inducing the viral RNA-mediated apoptosis of infected cells and the arrest of viral production.

Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses

“…Notably, this pathway gene-set is functionally related to other gene-sets in the apoptosis and PRR/inflammatory/pathogen response sub-clusters (manually isolated from these two sub-clusters in Cluster-1, Figure 10 ), exhibiting gene overlap with 15/19 of these gene-sets, with eight of these resulting in similarity coefficients >0.02 and thus displayed in Figure 10 . This reflects the substantial crosstalk that exists between programmed cell death and PRR signalling in response to infection [ 67 , 112 ].…”

“…The circumstances under which this occurs are quite diverse. For example, PKR-mediated translational inhibition leads to apoptosis [ 115 , 116 ] via depletion of cFLIP protein [ 112 ] which acts as an important inhibitor of caspase-8 ( Figure 11 b) [ 132 , 133 ]. PKR phosphorylation by PACT (in response to stress) can also lead to translational inhibition leading to caspase-8 dependent apoptosis [ 134 ], as can overexpression of PKR [ 135 , 136 , 137 ].…”

“…The eif2ak2 gene encoding PKR was also among the top 250 most significant DEGs in this study ( Table S4 ) and identified as an important hub gene in functional network in Figure 9 , being ranked 3rd overall ( Table S7 ). Given the importance of this ISG in terms of antiviral defence [ 112 , 115 , 145 ], particularly regarding programmed cell death, we hypothesized that the knock-out (KO) of the eif2ak2 gene may impact CyHV-3 clearance in zebrafish larvae.…”

“…If programmed cell death also features heavily in the response to CyHV-3 infection in these mutant zebrafish strains, as earlier observations in the WT strain suggested ( Figure 8 , Figure 11 and Video S1 ), these processes would need to be mediated via other mechanisms. Notably, in addition to IFN-stimulated PKR/PKZ-mediated programmed cell death [ 112 , 113 , 129 ], these processes can be stimulated by other cytokines such as FAS, TNFα, and TRAIL [ 154 , 155 , 156 , 157 ] (the zebrafish orthologs for these proteins are encoded by the faslg , tnfa , and tnfsf10 genes, respectively). Like IFN, these cytokines also operate by binding to their respective cell membrane receptors and downstream interactions between these and various other proteins are required to initiate apoptosis and/or necroptosis.…”

“…The list of zebrafish orthologs/paralogs corresponding to each node in these pathways can be accessed in the KEGG database using the corresponding gene-set references (Herpes simplex virus 1 infection (DRE05168), Apoptosis (DRE04210) and Necroptosis (DRE04217)), which can then be cross-referenced with data in Table S5 (using NCBI/Entrez/GenBank Gene IDs or Gene Symbols). Key genes involved in IFN-stimulated PKR-mediated programmed cell death, i.e., translational inhibition [ 114 , 116 , 128 ] leading to apoptosis [ 112 ] (blue), IFN-stimulated PKR-mediated apoptosis [ 129 , 130 ] (pink), and IFN-stimulated PKR-mediated necroptosis [ 113 ] (yellow) are highlighted. Genes with dashed line borders indicate instances where downregulation, translational inhibition or post-translational inactivation of protein products promote the processes in question (see main text and references provided within this caption for details).…”