Michaela Gack and colleagues have found that disturbance of the cell cytoskeleton is a necessary priming step for activation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. They show that remodelling of the actin cytoskeleton, which commonly occurs during virus infection, drives relocation of a PPP1R12C phosphatase complex to RIG-I and MDA5, where it dephosphorylates the RLRs to enable their activation.

RLRs are intracellular pattern-recognition receptors that sense RNA and upregulate antiviral responses via the MAVS signalling pathway. Dephosphorylation of caspase activation and recruitment domains (CARDs) in the amino-terminal of RLRs is mediated by the PP1α and PP1γ phosphatases and is necessary for RLR multimerization, MAVS binding and downstream signalling. However, PP1α/γ have many other cellular substrates, and it is not known how these phosphatases are specifically recruited to RLRs.

Credit: Simon Bradbrook/Springer Nature Limited

Previous work from the Gack laboratory had identified eight putative PP1 regulatory proteins; in the current study Acharya et al. used siRNA-mediated knockdown to determine if these proteins directly regulate RLRs. They found that silencing of PPP1R12C, but not the other candidate proteins, enhanced phosphorylation of RIG-I and MDA5 to a similar extent as PP1α/γ deletion, suggesting PPP1R12C supports the dephosphorylation of RLRs. In agreement with this, depletion of PPP1R12C impaired IFNβ induction in cells stimulated with synthetic dsRNA or infected with SARS-CoV-2, Zika virus or vesicular stomatitis virus (VSV). Similar findings of impaired IFNβ induction and antiviral gene expression were seen in PPP1R12C knockout cells, and in response to VSV infection, Ppp1r12c-deficient mice showed impaired innate immune responses, enhanced viral replication and higher mortality.

Further experiments showed that infection with various RNA viruses causes PPP1R12C binding to RIG-I and MDA5. PPP1R12C also showed increased PP1 binding following virus infection, and the authors found that it recruits PP1 phosphatases to the RLRs through the formation of PP1–PPP1R12C–RLR complexes.

PPP1R12C regulates cytoskeleton dynamics as part of the myosin phosphatase complex; therefore, the authors hypothesized that actin cytoskeleton disturbance may displace PPP1R12C from F-actin to promote PP1–PPP1R12C–RLR complex formation. They confirmed this idea using both viral and non-infectious triggers of cytoskeleton disturbance. Notably, the authors found that full activation of RLRs requires both RNA binding and actin cytoskeleton disturbance. They showed that inducible expression of immunostimulatory RNA in cells only led to antiviral gene expression if cells were also treated with agents that disturb the cytoskeleton and cause relocalization of PPP1R12C.

“full activation of RLRs requires both RNA binding and actin cytoskeleton disturbance”

These findings challenge the current view that the presence of immunostimulatory RNA is sufficient for RLR activation. Instead, the authors propose that full RLR activation requires two key trigger steps: first, actin cytoskeleton disturbance to prime RLRs via PP1–PPP1R12C–RLR complex formation, and second the detection of immunostimulatory RNA.