Ebola virus (EBOV) infection blocks cellular production of alpha/beta interferon (IFN-␣Further, VP24 is found to specifically interact with karyopherin ␣1, the nuclear localization signal receptor for PY-STAT1, but not with karyopherin ␣2, ␣3, or ␣4. Overexpression of VP24 results in a loss of karyopherin ␣1-PY-STAT1 interaction, indicating that the VP24-karyopherin ␣1 interaction contributes to the block to IFN signaling. These data suggest that VP24 is likely to be an important virulence determinant that allows EBOV to evade the antiviral effects of IFNs.The filoviruses, Ebola virus (EBOV) and Marburg virus, cause periodic outbreaks of severe hemorrhagic fever in humans. In EBOV outbreaks consisting of more than 10 reported cases, mortality rates have ranged from 40 to 90% (41), and Marburg virus outbreaks have had reported case fatality rates ranging from 25 to 80% (13). This extreme virulence has made Ebola and Marburg viruses of concern both as naturally emerging pathogens and as potential bioweapons (41).The molecular mechanisms contributing to the severe pathogenesis of filovirus infection are poorly understood. Several potential mechanisms contributing to EBOV virulence have been reviewed (41). These include cytotoxicity of the viral glycoprotein, the production of proinflammatory cytokines, and the dysregulation of the coagulation cascade due to the production of tissue factor (14,20,21,62,64). Infection also appears to induce a general immune suppression (11, 53). Possible mechanisms contributing to this suppression include inhibition of dendritic cell activation and an induction of lymphocyte apoptosis (2,8,18,22,43). Each of these pathogenic processes likely occurs as a result of the active replication of the virus. Thus, the ability of the virus to counteract early antiviral responses, including those of the host's interferon system, likely plays an important role in EBOV virulence (41).EBOV encodes mechanisms to counteract the host interferon (IFN) response by blocking both production of IFN-␣/␤ and cellular responses to IFN-␣/␤ or -␥ treatment (6,24,26,27). We previously demonstrated that the EBOV VP35 protein suppresses IFN-␣/␤ production by inhibiting the activation of interferon regulatory factor 3 (IRF-3) (5, 7, 51), and subsequent studies confirm that VP35 exerts this function (8, 28). However, the manner in which EBOV blocks signaling from the IFN-␣/␤ or -␥ receptor has remained incompletely defined.IFN-␣/␤, a family of structurally related proteins, and IFN-␥ bind to two distinct receptors but activate similar signaling pathways (reviewed in reference 38). For both pathways, ligand binding activates receptor-associated Jak family tyrosine kinases. These undergo auto-and transphosphorylation and phosphorylate the cytoplasmic domains of the receptor subunits. The receptor-associated phosphotyrosine residues then serve as docking sites for the SH2 domains of STAT proteins. The receptor-associated STATs then undergo tyrosine-phosphorylation and form homo-or heterodimers via reciprocal SH2 domai...

Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era 1 . Recent work has recovered the gene sequences of this unusual strain 2 , so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes 3,4 . However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 hemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. In our study, each of 32 individuals tested that were born in or before 1915 exhibited seroreactivity with 1918 virus, nearly 90 years after the pandemic. Seven of 8 donor samples tested had circulating B cells that secreted antibodies that bound 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that exhibited potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but not with HAs of more contemporary human influenza viruses. The antibody genes exhibited an unusually Correspondence should be addressed to JEC (James.Crowe@vanderbilt.edu), CFB (Chris.Basler@mssm.edu) or ELA (Eric.Altschuler@umdnj.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Contributions XY and TT contributed equally to this work. XY, PAM, MDH and FSH made and cloned the mAbs, sequenced antibody genes, and performed IF experiments, CJK performed biosensor studies, TMT, CP, and LAP designed and performed in vivo studies, OM sequenced the HA genes of the H1N1 viruses used in this study and performed ELISA assays with these viruses. PVA assisted with HAI and neutralization assays and with cloning of recombinant HA molecules. JS and IAW provided recombinant HA; ELA led the clinical recruitment, ELA, CFB and JEC conceived of the experimental plan. CFB and JEC wrote the manuscript. All authors discussed the results and commented on the manuscript.Antibody nucleotide sequences are deposited in GenBank, accession numbers EU169674 through EU169679 and EU825947 through EU825950.Reprints and permissions information is available at npg.nature.com/reprints and permissions.The authors declare no competing financial interests. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 April 3. . We collected transformed cells from the wells corresponding to supernates exhibiting the highest levels of specific binding to the 1918 HA (derived from five donors) and fused them to the HMMA2.5 nonsecreting myeloma partner 7 using an electrofusion technique 8 . We isolated 17 unique hybridoma cell lines that secreted antibodies reactive with the 1918 HA from cell lines derived fro...

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.

Naturally occurring strains of Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently in clinical trials. Here, we have evaluated the possibility to enhance the cancer therapeutic potential of NDV by means of reverse genetics. Mice bearing s.c. implanted CT26 tumors were treated with intratumoral (i.t.) injections of a recombinant NDV modified to contain a highly fusogenic F protein. These treated mice exhibited significant reduction in tumor development compared with mice treated with the unmodified virus. Furthermore, mice in a CT26 metastatic tumor model treated with an i.v. injection of the genetically engineered NDV exhibited prolonged survival compared with wild-type control virus. In addition, we examined whether the oncolytic properties of NDV could be improved by expression of immunostimulatory molecules. In this regard, we engineered several NDVs to express granulocyte macrophage colony-stimulating factor, IFN-;, interleukin 2 (IL-2), or tumor necrosis factor A, and evaluated their therapeutic potential in an immunocompetent colon carcinoma tumor model. Mice bearing s.c. CT26 tumors treated with i.t. injections of recombinant NDV expressing IL-2 showed dramatic reductions in tumor growth, with a majority of the mice undergoing complete and long-lasting remission. Our data show the use of reverse genetics to develop enhanced recombinant NDV vectors as effective therapeutic agents for cancer treatment. [Cancer Res 2007;67(17):8285-92]

SUMMARYWe investigated cytokine profiles in interleukin (IL)-4 transgenic (Tg) mice with a skin inflammatory disease resembling human atopic dermatitis. cDNA microarray revealed that the mRNAs encoding IL-1 b , IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12p40, IL-13, tumour necrosis factor (TNF)-a , TNF-b and interferon (IFN)-g were up-regulated in the skin of late lesion Tg mice and to a lesser degree in nonlesion Tg mice when compared to those of non-Tg mice. Real time reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that the cDNA copy numbers of IL-1 b , IL-4, IL-6, IL-10, TNF-a and IFN-g from the skin of late, early and non-lesions increased significantly compared to nonTg mice. IL-2 and IL-12p40 cDNA copy numbers were increased significantly in early, but not late, lesions. Interestingly, IL-1 b , IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, TNF-a , and IFN-g cDNAs were increased significantly the skin of before-onset and/or non-lesion mice. Flow cytometry analyses demonstrated an increased percentage of keratinocytes producing IL-4 as the disease progressed. The percentage of IL-2, IL-4, IL-10 and IFN-g -producing T cells and IL-12-producing antigen-presenting cells in skin-draining lymph nodes and inflammatory skin also increased, particularly in mice with late lesion. These results suggest that disease induction is primarily triggered by Th2 cytokines and that Th1, Th2 and non-Th proinflammatory cytokines are all involved in the disease process.

We identified a novel cDNA (IG20) that is homologous to cDNAs encoding a protein differentially expressed in normal and neoplastic cells (DENN-SV) and human MADD (MAPK-activating death domain-containing protein). Furthermore, we show that the above variants most likely result from alternative splicing of a single gene. Functional analyses of these variants in permanently transfected HeLa cells revealed that IG20 and DENN-SV render them more susceptible or resistant to tumor necrosis factor ␣ (TNF-␣)-induced apoptosis, respectively. All variants tested could interact with TNF receptor 1 and activate ERK and nuclear factor B. However, relative to control cells, only cells expressing IG20 showed enhanced TNF-␣-induced activation of caspase-8 and -3, whereas cells expressing DENN-SV showed either reduced or no caspase activation. Transfection of these cells with a cDNA encoding CrmA maximally inhibited apoptosis in HeLa-IG20 cells. Our results show that IG20 can promote TNF-␣-induced apoptosis and activation of caspase-8 and -3 and suggest that it may play a novel role in the regulation of the pleiotropic effects of TNF-␣ through alternative splicing.

Antigen-presenting cells (APCs) are critical targets of Ebola virus (EBOV) infection in vivo.However, the susceptibility of monocytes to infection is controversial. Studies indicate productive monocyte infection, and yet monocytes are also reported to be resistant to EBOV GP-mediated entry. In contrast, monocyte-derived macrophages and dendritic cells are permissive for both EBOV entry and replication. Here, freshly isolated monocytes are demonstrated to indeed be refractory to EBOV entry. However, EBOV binds monocytes, and delayed entry occurs during monocyte differentiation. Cultured monocytes spontaneously downregulate the expression of viral entry restriction factors such as interferon-inducible transmembrane proteins, while upregulating the expression of critical EBOV entry factors cathepsin B and NPC1. Moreover, these processes are accelerated by EBOV infection. Finally, ectopic expression of NPC1 is sufficient to rescue entry into an undifferentiated, normally nonpermissive monocytic cell line. These results define the molecular basis for infection of APCs and suggest means to limit APC infection. Zaire Ebola virus (EBOV) is an emerging zoonotic pathogen that has caused outbreaks of viral hemorrhagic fever in humans with fatality rates approaching 90% (1). EBOV tropism toward antigen-presenting cells (APCs) is thought to play an important role in viral pathogenesis, contributing to the establishment of infection and to the development of hemorrhagic fever (2). EBOV productively infects APCs, including monocytes, macrophages, and dendritic cells (DCs), in vitro (3-9), and tissue sections from EBOV-infected humans and nonhuman primates contain APCs positive for EBOV antigen/nucleic acid, demonstrating APC infection in vivo (10-16). Although APCs serve as sites of virus amplification, their infection also deregulates APC function (2,17,18). This deregulation may contribute to the development of an ineffective antiviral immune response, as well as an intense and deregulated inflammatory response (4,19).Although monocytes, the most abundant blood-borne APCs, likely contribute to EBOV pathogenesis, an apparent discrepancy exists in our understanding of monocyte infection by EBOV (18). Specifically, although EBOV productively infects human bloodderived monocytes (4,20), several other studies demonstrate limited or restricted EBOV GP-mediated entry into monocytes (21-23).EBOV entry, which includes attachment and penetration into the target cell cytoplasm, is mediated by the surface glycoprotein (GP) (24). GP likely mediates viral attachment through the receptor-binding domain (RBD) located at its N terminus (25-28). Subsequent viral uptake likely occurs via macropinocytosis (29)(30)(31)(32). A number of cell surface molecules, including C-type lectins, have been identified as attachment or entry factors (33-38). However, none of these factors appears to function as an essential cell surface receptor. Additional host factors have also been implicated as regulating entry, including components of the homotypic fusio...