Ebola Virus VP24 Proteins Inhibit the Interaction of NPI-1 Subfamily Karyopherin α Proteins with Activated STAT1

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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-foldlower 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. IMPORTANCEThe 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.

supporting

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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VP24-Karyopherin Alpha Binding Affinities Differ between Ebolavirus Species, Influencing Interferon Inhibition and VP24 Stability

Schwarz

Edwards

Diederichs

et al. 2017

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“…The other EBOV protein that antagonizes the innate immune response, VP24, acts by mechanisms distinct from those of VP35. VP24 interacts with karyopherin ␣ nuclear transporters to prevent translocation of STAT1 to the nucleus (29)(30)(31)(32) and also binds to heterogeneous ribonuclear protein complex C1/C2 (hnRNP C1/ C2), which normally interacts with karyopherin ␣1, and partially alters the nuclear transport of hnRNP C1/C2 (33). The interaction of VP24 with karyopherin ␣1 is completely reversed by the mutation K142A (29).…”

mentioning

confidence: 99%

Different Temporal Effects of Ebola Virus VP35 and VP24 Proteins on Global Gene Expression in Human Dendritic Cells

Ilinykh

Lubaki

Widen

et al. 2015

Ebola virus (EBOV) causes Filoviruses cause a severe hemorrhagic fever in humans and nonhuman primates with a mortality in humans of up to 90% (1). Filoviruses include five species, Zaire ebolavirus, Bundibugyo ebolavirus, Sudan ebolavirus, Taï Forest ebolavirus, and Reston ebolavirus, belonging to the genus Ebolavirus, and a single species, Marburg marburgvirus, belonging to the genus Marburgvirus (2). Filovirus outbreaks occur in Central Africa regularly; in 2012, four filovirus outbreaks occurred in Uganda and the Democratic Republic of the Congo (3), and a new outbreak with a previously unknown clade of Ebola virus (EBOV), which belongs to the species Zaire ebolavirus, started in Guinea in the winter of 2013-2014 (4) and spread to Liberia, Sierra Leone, Nigeria, Senegal, Mali, Spain, the United States, and the United Kingdom (5). Because of the extremely high mortality of the disease caused by filoviruses and the lack of approved vaccine and treatments, work with these viruses is performed under the biosafety level 4 (BSL-4) biocontainment.EBOV causes a severe immunosuppression in both humans and nonhuman primate models, characterized by a deficient T cell response, lymphopenia, and T cell apoptosis, despite the lack of infection of T cells (6)(7)(8)(9)(10)(11)(12). On the other hand, dendritic cells (DC), which are the most effective antigen-presenting cells and are infected by EBOV, do not undergo normal maturation despite the infection (13-15). Because DC play a key role in initiation of the adaptive immune response by processing viral antigens and presenting them to naive lymphocytes, the deficient T cell response may be linked to the deficient and/or aberrant stimulation of T cells by the infected DC.EBOV has two proteins, VP35 and VP24, which antagonize the

“…A dysregulation of the host immune response is characteristic of primate MARV infections (10). In EBOV, VP24 antagonizes signal transduction from the interferon receptor by interacting with karyopherin ␣ (11)(12)(13)(14). In MARV, instead of VP24, the VP40 protein antagonizes signal transduction by inhibiting the phos-phorylation of JAK1, TYK1, STAT1, and STAT2 under the stimuli of both type I and type II IFNs (15).…”

mentioning

confidence: 99%

Crystal Structure of Marburg Virus VP40 Reveals a Broad, Basic Patch for Matrix Assembly and a Requirement of the N-Terminal Domain for Immunosuppression

Oda

Noda

Wijesinghe

et al. 2016

Marburg virus (MARV), a member of the filovirus family, causes severe hemorrhagic fever with up to 90% lethality. MARV matrix protein VP40 is essential for assembly and release of newly copied viruses and also suppresses immune signaling in the infected cell. Here we report the crystal structure of MARV VP40. We found that MARV VP40 forms a dimer in solution, mediated by N-terminal domains, and that formation of this dimer is essential for budding of virus-like particles. We also found the N-terminal domain to be necessary and sufficient for immune antagonism. The C-terminal domains of MARV VP40 are dispensable for immunosuppression but are required for virus assembly. The C-terminal domains are only 16% identical to those of Ebola virus, differ in structure from those of Ebola virus, and form a distinct broad and flat cationic surface that likely interacts with the cell membrane during virus assembly. IMPORTANCEMarburg virus, a cousin of Ebola virus, causes severe hemorrhagic fever, with up to 90% lethality seen in recent outbreaks. Molecular structures and visual images of the proteins of Marburg virus are essential for the development of antiviral drugs. One key protein in the Marburg virus life cycle is VP40, which both assembles the virus and suppresses the immune system. Here we provide the molecular structure of Marburg virus VP40, illustrate differences from VP40 of Ebola virus, and reveal surfaces by which Marburg VP40 assembles progeny and suppresses immune function. Marburg virus (MARV) and the related Ebola virus (EBOV) both belong to the Filoviridae family. These are enveloped viruses with a nonsegmented single-stranded and negative-sense RNA genome. Genomes of Marburg viruses differ from those of ebolaviruses by more than 50% at the nucleotide level (1). MARV causes severe and rapidly progressing hemorrhagic fever in humans and nonhuman primates, with lethality ranging from 25% to over 90% depending on the geographic location and viral strain (2). The 19-kb MARV genome encodes seven structural proteins. Each of these proteins is of critical importance, and most are known to perform multiple functions during the viral life cycle.VP40 is the Marburg virus matrix protein, which builds the protein shell underneath the viral envelope and confers the hallmark filamentous morphology to the Marburg virion. VP40 alone is able to induce the assembly and budding of filamentous viruslike particles (VLPs), which resemble authentic virions (3, 4). The intracellular distribution of filovirus VP40 varies during the progression of the viral life cycle (5, 6), as it orchestrates the distribution of the other viral components and viral assembly (6, 7). The majority of it, however, traffics to and associates with the cellular membrane (5,7,8). Within the virion, VP40 interacts with both the lipid envelope and the core nucleocapsid complex, which contains the NP, VP35, L, VP30, and VP24 proteins (9). VP40 also interacts with the GP viral surface protein. Indeed, coexpression of VP40 and GP alters the distributi...