PACT prevents aberrant activation of PKR by endogenous dsRNA without sequestration

The innate immune sensor PKR for double-stranded RNA (dsRNA) is critical for antiviral defense, but its aberrant activation by cellular dsRNA is linked to various diseases. The dsRNA-binding protein PACT plays a critical yet controversial role in the PKR pathway. We demonstrate that PACT is a direct and specific suppressor of PKR against endogenous dsRNA ligands like inverted-repeat Alu RNAs, which robustly activate PKR in the absence of PACT. PACT-mediated inhibition does not involve competition for dsRNA binding. Instead, PACT impairs PKR’s ability to scan along dsRNA––a process necessary for PKR molecules to encounter and autophoshorylate each other for activation. By scanning along dsRNA and directly interacting with PKR, PACT restricts PKR’s movement on dsRNA, reducing the likelihood of PKR molecular collisions and subsequent autophosphorylation, effectively inhibiting PKR without sequestering dsRNA. Consequently, PKR inhibition is more robust with longer and less abundant dsRNA, and minimal with abundant or short dsRNA. Thus, PACT functions to adjust the PKR activation threshold for long endogenous dsRNA without altering its inherent activity, revealing new mechanisms for establishing self-tolerance.