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

Abstract

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.

Picornavirus security proteins promote the release of extracellular vesicle enclosed viruses via the modulation of host kinases

“…This was independent of upstream MAPKKK/MAPKK kinases [ 34 ], and may be caused by inhibition of protein phosphatases through EMCV L protein-binding domains. In line with our observation that the cardiovirus Leaders differ in their ability to activate P38, EMCV L was previously shown to employ both P38 and the ERK-RSK pathways to modulate host cell behavior, whereas TMEV L was found to rely predominantly on RSK [ 31 , 34 , 53 ]. P38 activation has also frequently been described during CVB3 infection [ 51 , 52 ], however the mechanism behind this activation remains elusive.…”

“…In contrast to MAVS, deletion of PKR, the antiviral sensor responsible for the formation of stress granules, partially restored the inability of EMCV lacking expression of its security protein EMCV L to induce EV and EV-enclosed virus release. In line with this finding, we observed that the induction of virus-induced EV subsets by EMCV L required the host factor RSK, a host kinase that was recently implicated in the inhibition of PKR by cardioviruses [ 31 , 53 ]. Together, these data suggest that PKR signaling acts as a negative regulator of EV-enclosed virus during infection.…”

“…The production of PKR and MAVS KO HeLa R19 cells by CRISPR-Cas9 gene editing has been described elsewhere [ 70 , 71 ]. HeLa M WT, RSK-1/2/3 triple KO, and RSK-2 reconstituted cells were a kind gift from dr. prof. T. Michiels (de Duve Institute, Université Catholique de Louvain, Belgium) and have been described in [ 53 ]. All cell lines were cultured in Dulbecco’s Modified Eagle Medium (high glucose, GlutaMAX, ThermoFischer Scientific, Waltham, MA, USA), supplemented with 10% fetal calf serum (FCS; GE Healthcare Bio-Sciences, Chicago, IL), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Paisley, United Kingdom) and kept in a humidified incubator at 37°C and 5% CO 2 .…”

Identification of RSK substrates using an analog-sensitive kinase approach

“…Therefore, unlike substrates of other kinases, substrates of the analog-sensitive kinase become thiophosphorylated. The gatekeeper residues, which restrict the kinase ATP pocket size, were identified as Leu141 for RSK1 and Leu152 for RSK4, based on the rule originally derived from c-SRC ( 26 ) and on the alignment with RSK2, which was recently successfully converted to analog-sensitive ( 18 ) ( Fig. 1 A ).…”

“…For RSK1-2, which are the most studied RSK isoforms, common substrates have been identified ( 17 ). In a previous work, we observed that pathogens such as cardioviruses could equally hijack any of the four RSK isoforms to disrupt nucleocytoplasmic trafficking in the cell ( 18 ) or to inhibit the antiviral kinase interferon-induced double-stranded RNA–activated kinase PKR ( 19 ). In line with these data, a recent study showed that all four RSK isoforms were phosphorylating very similar peptides in a peptide array type of screening ( 12 ).…”