The Zaire ebolavirus protein VP24 was previously demonstrated to inhibit alpha/beta interferon (IFN-␣/␤)-and IFN-␥-induced nuclear accumulation of tyrosine-phosphorylated STAT1 (PY-STAT1) and to inhibit IFN-␣/␤-and IFN-␥-induced gene expression. These properties correlated with the ability of VP24 to interact with the nuclear localization signal receptor for PY-STAT1, karyopherin ␣1. Here, VP24 is demonstrated to interact not only with overexpressed but also with endogenous karyopherin ␣1. Mutational analysis demonstrated that VP24 binds within the PY-STAT1 binding region located in the C terminus of karyopherin ␣1. In addition, VP24 was found to inhibit PY-STAT1 binding to both overexpressed and endogenous karyopherin ␣1. We assessed the binding of both PY-STAT1 and the VP24 proteins from Zaire, mouse-adapted Zaire, and Reston Ebola viruses for interaction with all six members of the human karyopherin ␣ family. We found, in contrast to previous studies, that PY-STAT1 can interact not only with karyopherin ␣1 but also with karyopherins ␣5 and ␣6, which together comprise the NPI-1 subfamily of karyopherin ␣s. Similarly, all three VP24s bound and inhibited PY-STAT1 interaction with karyopherins ␣1, ␣5, and ␣6. Consistent with their ability to inhibit the karyopherin-PY-STAT1 interaction, Zaire, mouse-adapted Zaire, and Reston Ebola virus VP24s displayed similar capacities to inhibit IFN-␤-induced gene expression in human and mouse cells. These findings suggest that VP24 inhibits interaction of PY-STAT1 with karyopherins ␣1, ␣5, or ␣6 by binding within the PY-STAT1 binding region of the karyopherins and that this function is conserved among the VP24 proteins of different Ebola virus species.

Previous studies have demonstrated that Marburg viruses (MARV) and Ebola viruses (EBOV) inhibit interferon (IFN)-α/β signaling but utilize different mechanisms. EBOV inhibits IFN signaling via its VP24 protein which blocks the nuclear accumulation of tyrosine phosphorylated STAT1. In contrast, MARV infection inhibits IFNα/β induced tyrosine phosphorylation of STAT1 and STAT2. MARV infection is now demonstrated to inhibit not only IFNα/β but also IFNγ-induced STAT phosphorylation and to inhibit the IFNα/β and IFNγ-induced tyrosine phosphorylation of upstream Janus (Jak) family kinases. Surprisingly, the MARV matrix protein VP40, not the MARV VP24 protein, has been identified to antagonize Jak and STAT tyrosine phosphorylation, to inhibit IFNα/β or IFNγ-induced gene expression and to inhibit the induction of an antiviral state by IFNα/β. Global loss of STAT and Jak tyrosine phosphorylation in response to both IFNα/β and IFNγ is reminiscent of the phenotype seen in Jak1-null cells. Consistent with this model, MARV infection and MARV VP40 expression also inhibit the Jak1-dependent, IL-6-induced tyrosine phosphorylation of STAT1 and STAT3. Finally, expression of MARV VP40 is able to prevent the tyrosine phosphorylation of Jak1, STAT1, STAT2 or STAT3 which occurs following over-expression of the Jak1 kinase. In contrast, MARV VP40 does not detectably inhibit the tyrosine phosphorylation of STAT2 or Tyk2 when Tyk2 is over-expressed. Mutation of the VP40 late domain, essential for efficient VP40 budding, has no detectable impact on inhibition of IFN signaling. This study shows that MARV inhibits IFN signaling by a mechanism different from that employed by the related EBOV. It identifies a novel function for the MARV VP40 protein and suggests that MARV may globally inhibit Jak1-dependent cytokine signaling.

Dendritic cells (DCs), important early targets of Ebola virus (EBOV) infection in vivo, are activated by Ebola virus-like particles (VLPs). To better understand this phenomenon, we have systematically assessed the response of DCs to VLPs of different compositions. VLPs containing the viral matrix protein (VP40) and the viral glycoprotein (GP), were found to induce a proinflammatory response highly similar to a prototypical DC activator, LPS. This response included the production of several proinflammatory cytokines, activation of numerous transcription factors including NF-kappaB, the functional importance of which was demonstrated by employing inhibitors of NF-kappaB activation, and activation of ERK1/2 MAP kinase. In contrast, VLPs constituted with a mutant GP lacking the heavily glycosylated mucin domain showed impaired NF-kappaB and Erk activation and induced less DC cytokine production. We conclude that the GP mucin domain is required for VLPs to stimulate human dendritic cells through NF-kappaB and MAPK signaling pathways.

Marburgviruses are zoonotic pathogens that cause lethal hemorrhagic fever in humans and nonhuman primates. However, they do not cause lethal disease in immunocompetent mice unless they are adapted to this species. The adaptation process can therefore provide insight into the specific virus-host interactions that determine virulence. In primate cells, the Lake Victoria marburgvirus Musoke strain ( Filoviruses, which include the genera Marburgvirus and Ebolavirus (EBOV), are causative agents of severe hemorrhagic fevers in humans and nonhuman primates. Human case fatality rates have reached up to 90% in some outbreaks. The genus Marburgvirus includes a single species, Lake Victoria marburgvirus that is comprised of two lineages; one is represented by a 1987 isolate from Kenya (Marburg Ravn virus [RAVV]), and the other is comprised of multiple strains, including Ozolin, Popp, Musoke (abbreviated as MARV here), and Angola (10,14,33).Filoviruses counteract the innate immune responses of primate hosts, preventing the production of and cellular responses to alpha/beta interferon (IFN-␣/␤) (1,28,29,40). IFN-␣/␤ and IFN-␥ are cytokines that induce in cells an antiviral state and are of particular importance to innate antiviral immunity (3, 36). Binding of IFN-␣/␤ to its receptor results in the tyrosine phosphorylation of the Janus kinases Jak1 and Tyk2, which subsequently phosphorylate the transcription factors STAT1 and STAT2. STAT1 and STAT2 form a heterodimer and associate with the IFN regulatory factor 9 (IRF9), translocate into the nucleus, and activate a set of genes involved in antiviral response (25). The absence of Jak1 leads to lack of STAT activation (11,21,30,44).Filovirus proteins that antagonize IFN responses have been identified. EBOV VP35 inhibits the production of IFN-␣/␤ by antagonizing the RIG-I signaling pathway (1,6,12,17). EBOV VP24 inhibits cellular responses to IFN-␣/␤ and IFN-␥ by interacting with the NPI-1 subfamily of karyopherin ␣ nuclear transport proteins, preventing the nuclear accumulation of tyrosine phosphorylated STAT1 (20,28,29). Surprisingly, given the similar genomic organization of EBOVs and Marburg viruses, MARV was demonstrated to block cellular responses to IFNs by a unique mechanism (40). Specifically, while the MARV VP24 protein did not exhibit binding to karyopherin ␣ proteins and did not block IFN signaling, the MARV major matrix protein VP40, which drives viral budding (8,16,34,39), inhibits Jak1-dependent signaling pathways, including the IFN-␣/␤, IFN-␥, and interleukin-6 pathways (40). For the IFN-␣/␤ pathway, VP40 inhibits activation of Jak1, resulting in the absence of IFN-␣/␤-induced Jak1, Tyk2, STAT1, and STAT2 tyrosine phosphorylation (40).The assembly and budding functions of filovirus VP40 proteins are related to their ability to bud from cells, and expression of VP40 proteins results in the release of virus-like particles (VLPs) from the cell surface (13,15,16,23,32,39). Implicated in the budding process are short amino acid motifs known as late domains. EBOV VP40 ...

Stochastic processes are intrinsic phenomena that perturb developmental processes. However, the canalization process restricts the magnitude of perturbation and hence the magnitude of morphological variation during development. Heat-shock protein 90 (Hsp90) chaperones are a class of proteins stabilizing a network of 'client' proteins that are involved in diverse signal transduction pathways affecting development. Here it is reported that a reduction of Hsp90 gene dose creates canalization perturbations that affect many aspects of Arabidopsis development and results in a plethora of morphological alterations. Hence, Hsp90 restricts stochastic phenomena by minimizing perturbations, thereby canalizing development. It is also shown that morphogenesis is determined by three mutually inter-related parameters: genotype, environment, and time. Hsp90 is involved in the interaction of these three parameters which ultimately affect developmental processes. The amount of phenotypic variation upon the reduction of Hsp90 function could be perceived as adaptive and could have an impact on the evolutionary process.