PKR regulation by phosphorylation and antiviral activity of the PKR-ADAR1 axis
(2021)
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- Authors
- Supervisors
- Michiels, Thomas
- Abstract
- Protein kinase RNA-activated (PKR) and Double-stranded RNA-specific adenosine deaminase (ADAR1) are two double stranded RNA binding proteins, respectively involved in the antiviral response to viruses and in the metabolism of dsRNA molecules. PKR is a cellular protein kinase, that in response to dsRNA molecules generated during viral infections, gets activated and phosphorylates the translation initiation factor, eIF2a, leading to translational shutoff and apoptosis. As PKR thereby acts as a potent antiviral effector, many viruses evolved mechanisms to counteract its antiviral response. Previous studies showed that Theiler’s murine encephalomyelitis virus (TMEV), a cardiovirus belonging to the Picornaviridae family, can block PKR activation through the activity of its Leader (L) protein, an accessory protein of the virus known to block IFN gene transcription and perturb nucleocytoplasmic trafficking. In the first part of this thesis I contributed to a work showing that the L protein likely renders PKR insensitive to dsRNA molecules, possibly through the activation of cellular kinases. Next, we analyzed PKR phosphorylation modifications in the hope to identify potential phosphorylation sites responsible for PKR inhibition by TMEV L. We observed that the Ser6 residue located 3aa before the first double-stranded RNA binding motif (DRBM1) of PKR could be phosphorylated. A phospho-mimetic mutation of this site was inhibiting PKR activation after poly(I:C) transfection or viral infection, especially when combined to a phospho-mimetic mutation of the Ser97 residue, located 3aa before the second double- stranded RNA binding motif (DRBM2). We propose a model according to which phosphorylation occurring upstream of DRBMs would tighten the interaction of the DRBMs with the catalytic domain, blocking PKR in a closed conformation, and making it unable to be activated. ADAR1 is an editing enzyme, causing deamination of adenosines into inosines in dsRNA molecules, thus destabilizing dsRNA structures, either self or generated by viral infection. By disrupting dsRNA structures, ADAR1 limits PKR activation. Mutations in ADAR1 have been shown to cause Aicardi-Goutières syndrome, a disease where excessive interferon is generated in the absence of infection. Most of these mutations occur in the catalytic domain of the protein. One Aicardi-Goutières mutation (Pro193Ala) was however described in the Za domain of the protein. By reducing the editing activity and increasing the amount of dsRNA molecules, the mutations in the catalytic domain likely contribute to activate the various innate immunity pathways, which depend on the presence of dsRNA, thus enhancing the interferon response. Being able to modify dsRNA molecules, ADAR1 role during infection can be either antiviral or proviral, depending on the virus taken into consideration. We observed that one of the isoforms generated by ADAR1 gene, p150, has antiviral activity against TMEV, in spite of the fact that it inhibits PKR activation. We also observed that the Pro193Ala mutation decreased ADAR1 dsRNA binding ability, editing activity and slightly counteracted the antiviral role of p150 during TMEV infection. All together here we show that PKR can be regulated by phosphorylation events and that ADAR1 p150 isoform, despite being antagonist to PKR, has an antiviral role towards TMEV, likely by directly acting on its viral genome.
- Affiliations
Citations
Cesaro, T. (2021). PKR regulation by phosphorylation and antiviral activity of the PKR-ADAR1 axis. https://hdl.handle.net/2078.5/107619