Modulation Of PKR Activity During HIV Infection And Cellular Stress By PACT And TRBP

A crucial component of the cellular response to stress is the attenuation of protein synthesis to allow the cell to dedicate resources for the restoration of homeostasis or towards the induction of apoptotic cell death in case the stressors overwhelm the cell. This process is itself regulated by one of the four eIF2α kinases of which PKR (Protein Kinase R) is responsible for inhibiting general translation during viral infection, oxidative stress, ER stress, heat shock or serum withdrawal. During viral infection, PKR is transcriptionally induced by interferon but remains latent until it interacts with dsRNA. This interaction induces a conformational change that activates PKR’s catalytic activity, resulting in the phosphorylation of the eukaryotic initiation factor eIF2α and the cessation of both general and viral protein synthesis. This inhibition of viral protein synthesis is however short-lived, as several viral and host cellular factors are coopted by viruses to neutralize PKR’s catalytic activity against viral replication. One such cellular factor is PACT (Protein Activator of PKR), PKR’s protein activator during non-viral stress, which interacts strongly with PKR during HIV infection but does not activate its catalytic activity. We investigated the mechanisms behind PACT’s inability to activate PKR robustly during HIV infection. Our results show that PACT acts synergistically with the HIV trans-activator, Tat, dsRNA-containing mRNAs, as well as the adenosine deaminase, ADAR1 to form a PKR inhibitory complex to facilitate the translation of viral mRNAs during HIV infection. Most importantly, these results elucidate a pathway that could be a target of antiviral therapy to promote PKR activation and reduce viral load in infected cells. During non-viral stress, PKR’s activity is regulated negatively by the TAR RNA Binding Protein, TRBP. TRBP regulates PKR activity by interacting with PKR as well as PACT. Stress-induced phosphorylation of PACT at Serine 287 weakens its interaction with TRBP, while increasing PACT’s homomeric interactions and heteromeric interactions with PKR. The role, if any, of similar stress-induced post-translational modifications on TRBP’s ability to form homomeric interactions and heteromeric interactions with PKR as well as to inhibit PKR have remained unclear. In this light, we investigated whether TRBP is subject to stress-induced phosphorylation and how that might alter TRBP-TRBP and TRBP-PKR interactions as well as TRBP’s ability to inhibit PKR. Our results demonstrate that TRBP is phosphorylated by the Mitogen Activated Protein Kinases JNK and ERK in response to oxidative stress, and consequently forms strong homomeric interactions with PKR, resulting in increased inhibition of PKR and better cell recovery during oxidative stress. PKR, PACT, and TRBP ‘s homomeric and heteromeric interactions are primarily mediated by the evolutionarily conserved dsRNA binding motif (dsRBM) present in all three proteins as well as in several other dsRNA binding proteins. We investigated the contributions of the two copies of dsRBMs in PACT to PACT’s interactions with dsRNA, PACT, TRBP, and PKR. Our results establish that each motif contributes to a varying extent towards PACT’s interaction with its known binding partners, and highlight the importance of PACT homodimerization for PKR activation