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Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1.

ABSTRACT: During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C terminus in complex with eVP24. In the complex, eVP24 recognizes a unique nonclassical nuclear localization signal (NLS) binding site on KPNA5 that is necessary for efficient PY-STAT1 nuclear transport. eVP24 binds KPNA5 with very high affinity to effectively compete with and inhibit PY-STAT1 nuclear transport. In contrast, eVP24 binding does not affect the transport of classical NLS cargo. Thus, eVP24 counters cell-intrinsic innate immunity by selectively targeting PY-STAT1 nuclear import while leaving the transport of other cargo that may be required for viral replication unaffected.

SUBMITTER: Xu W

PROVIDER: S-EPMC4188415 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1.

Xu Wei W Edwards Megan R MR Borek Dominika M DM Feagins Alicia R AR Mittal Anuradha A Alinger Joshua B JB Berry Kayla N KN Yen Benjamin B Hamilton Jennifer J Brett Tom J TJ Pappu Rohit V RV Leung Daisy W DW Basler Christopher F CF Amarasinghe Gaya K GK

Cell host & microbe 20140801 2

During antiviral defense, interferon (IFN) signaling triggers nuclear transport of tyrosine-phosphorylated STAT1 (PY-STAT1), which occurs via a subset of karyopherin alpha (KPNA) nuclear transporters. Many viruses, including Ebola virus, actively antagonize STAT1 signaling to counteract the antiviral effects of IFN. Ebola virus VP24 protein (eVP24) binds KPNA to inhibit PY-STAT1 nuclear transport and render cells refractory to IFNs. We describe the structure of human KPNA5 C terminus in complex ...[more]

PMID: 25121748

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Project description:Zaire ebolavirus (EBOV), Bundibugyo ebolavirus (BDBV), and Reston ebolavirus (RESTV) belong to the same genus but exhibit different virulence properties. VP24 protein, a structural protein present in all family members, blocks interferon (IFN) signaling and likely contributes to virulence. Inhibition of IFN signaling by EBOV VP24 (eVP24) involves its interaction with the NPI-1 subfamily of karyopherin alpha (KPNA) nuclear transporters. Here, we evaluated eVP24, BDBV VP24 (bVP24), and RESTV VP24 (rVP24) interactions with three NPI-1 subfamily KPNAs (KPNA1, KPNA5, and KPNA6). Using purified proteins, we demonstrated that each VP24 binds to each of the three NPI-1 KPNAs. bVP24, however, exhibited approximately 10-fold-lower KPNA binding affinity than either eVP24 or rVP24. Cell-based assays also indicate that bVP24 exhibits decreased KPNA interaction, decreased suppression of IFN induced gene expression, and a decreased half-life in transfected cells compared to eVP24 or rVP24. Amino acid sequence alignments between bVP24 and eVP24 also identified residues within and surrounding the previously defined eVP24-KPNA5 binding interface that decrease eVP24-KPNA affinity or bVP24-KPNA affinity. VP24 mutations that lead to reduced KPNA binding affinity also decrease IFN inhibition and shorten VP24 half-lives. These data identify novel functional differences in VP24-KPNA interaction and reveal a novel impact of the VP24-KPNA interaction on VP24 stability. The interaction of Ebola virus (EBOV) VP24 protein with host karyopherin alpha (KPNA) proteins blocks type I interferon (IFN) signaling, which is a central component of the host innate immune response to viral infection. Here, we quantitatively compared the interactions of VP24 proteins from EBOV and two members of the Ebolavirus genus, Bundibugyo virus (BDBV) and Reston virus (RESTV). The data reveal lower binding affinity of the BDBV VP24 (bVP24) for KPNAs and demonstrate that the interaction with KPNA modulates inhibition of IFN signaling and VP24 stability. The effect of KPNA interaction on VP24 stability is a novel functional consequence of this virus-host interaction, and the differences identified between viral species may contribute to differences in pathogenesis.

Project description:Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.

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Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1.

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Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively compete with nuclear import of phosphorylated STAT1.

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