Cardiovirus leader proteins retarget RSK kinases toward alternative substrates to perturb nucleocytoplasmic traffic

Proteins from some unrelated pathogens, including small RNA viruses of the family Picornaviridae, large DNA viruses such as Kaposi sarcoma-associated herpesvirus and even bacteria of the genus Yersinia can recruit cellular p90-ribosomal protein S6 kinases (RSKs) through a common linear motif and maintain the kinases in an active state. On the one hand, pathogens' proteins might hijack RSKs to promote their own phosphorylation (direct target model). On the other hand, some data suggested that pathogens' proteins might dock the hijacked RSKs toward a third interacting partner, thus redirecting the kinase toward a specific substrate. We explored the second hypothesis using the Cardiovirus leader protein (L) as a paradigm. The L protein is known to trigger nucleocytoplasmic trafficking perturbation, which correlates with hyperphosphorylation of phenylalanine-glycine (FG)-nucleoporins (FG-NUPs) such as NUP98. Using a biotin ligase fused to either RSK or L, we identified FG-NUPs as primary partners of the L-RSK complex in infected cells. An L protein mutated in the central RSK-interaction motif was readily targeted to the nuclear envelope whereas an L protein mutated in the C-terminal domain still interacted with RSK but failed to interact with the nuclear envelope. Thus, L uses distinct motifs to recruit RSK and to dock the L-RSK complex toward the FG-NUPs. Using an analog-sensitive RSK2 mutant kinase, we show that, in infected cells, L can trigger RSK to use NUP98 and NUP214 as direct substrates. Our data therefore illustrate a novel virulence mechanism where pathogens' proteins hijack and retarget cellular protein kinases toward specific substrates, to promote their replication or to escape immunity. Author summary Nuclear pore complexes (NPCs) are multiprotein complexes forming gates in the nuclear envelope that allow the passage of specific proteins and RNAs between the nuclear and cytoplasmic compartments. Within NPCs, phenylalanine and glycine-rich nucleoporins (FG-NUPs) form a mesh needed to sort specific proteins and RNAs that move in and out of the nucleus. Our previous work showed that some viral and bacterial proteins were able to bind cellular kinases called RSKs and to maintain those kinases in an active state. Here we show that a short protein encoded by small RNA viruses of the genus Cardiovirus can act as an adapter molecule, which recruits and directs activated RSK kinases toward the NPCs where they phosphorylate the FG-NUPs. As a consequence, phosphorylated FG-NUPs disrupt the mesh and trigger the uncontrolled diffusion of molecules through the nuclear pore. Perturbation of nucleocytoplasmic trafficking can benefit cardioviruses by preventing the initiation of innate immune responses or by providing nuclear cell components to the cytoplasmic virus replication complexes. Our data support the concept that proteins expressed by pathogens are able to retarget host cell enzymes toward alternative substrates, to the benefit of the pathogen.

Automated summary

It has been discovered that certain pathogen proteins can hijack cellular protein kinases to promote their own replication or evade immunity by redirecting them to specific substrates.