Picornaviruses are positive-stranded RNA viruses. Even though replication and translation of their genome take place in the cytoplasm, these viruses evolved different strategies to disturb nucleocytoplasmic trafficking of host proteins and RNA. The major targets of picornavirus are the phenylalanine-glycine (FG)-nucleoporins, which form a mesh in the central channel of the nuclear pore complex through which protein cargos and karyopherins are actively transported in both directions. Interestingly, while enteroviruses use the proteolytic activity of their 2A protein to degrade FG-nucleoporins, cardioviruses act by triggering phosphorylation of these proteins by cellular kinases. By targeting the nuclear pore complex, picornaviruses recruit nuclear proteins to the cytoplasm, where they increase viral genome translation and replication; they affect nuclear translocation of cytoplasmic proteins such as transcription factors that induce innate immune responses and retain host mRNA in the nucleus thereby preventing cell emergency responses and likely making the ribosomal machinery available for translation of viral RNAs.
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.
SummaryProteins from unrelated pathogens, including some RNA or DNA viruses and bacteria can recruit and activate cellular p90-ribosomal protein S6 kinases (RSKs) through a common linear motif. Data suggested a model where pathogens’ proteins act to dock the recruited RSKs toward specific substrates, which then act as effectors to the benefit of the pathogens. Using cardiovirus leader protein (L) as a paradigm, we show that pathogens’ proteins can modify the spectrum of RSK substrates in infected cells. L triggers nucleocytoplasmic trafficking perturbation and phenylalanine-glycine (FG)-nucleoporin hyperphosphorylation in an RSK-dependent fashion. Biotin ligase experiments identified FG-nucleoporins as common partners of L and RSK in infected cells. Using cells expressing an analog-sensitive RSK2 mutant, we show that L triggers direct phosphorylation of NUP98 and NUP214 by RSK2 in infected cells. Our data therefore demonstrate a novel virulence mechanism where pathogens’ proteins hijack and retarget cellular protein kinases of the RSK family.
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