Rotavirus Structural Proteins and dsRNA Are Required for the Human Primary Plasmacytoid Dendritic Cell IFNα Response

Rotaviruses are the leading cause of severe dehydrating diarrhea in children worldwide. Rotavirus-induced immune responses, especially the T and B cell responses, have been extensively characterized; however, little is known about innate immune mechanisms involved in the control of rotavirus infection. Although increased levels of systemic type I interferon (IFNα and β) correlate with accelerated resolution of rotavirus disease, multiple rotavirus strains, including rhesus rotavirus (RRV), have been demonstrated to antagonize type I IFN production in a variety of epithelial and fibroblast cell types through several mechanisms, including degradation of multiple interferon regulatory factors by a viral nonstructural protein. This report demonstrates that stimulation of highly purified primary human peripheral plasmacytoid dendritic cells (pDCs) with either live or inactivated RRV induces substantial IFNα production by a subset of pDCs in which RRV does not replicate. Characterization of pDC responses to viral stimulus by flow cytometry and Luminex revealed that RRV replicates in a small subset of human primary pDCs and, in this RRV-permissive small subset, IFNα production is diminished. pDC activation and maturation were observed independently of viral replication and were enhanced in cells in which virus replicates. Production of IFNα by pDCs following RRV exposure required viral dsRNA and surface proteins, but neither viral replication nor activation by trypsin cleavage of VP4. These results demonstrate that a minor subset of purified primary human peripheral pDCs are permissive to RRV infection, and that pDCs retain functionality following RRV stimulus. Additionally, this study demonstrates trypsin-independent infection of primary peripheral cells by rotavirus, which may allow for the establishment of extraintestinal viremia and antigenemia. Importantly, these data provide the first evidence of IFNα induction in primary human pDCs by a dsRNA virus, while simultaneously demonstrating impaired IFNα production in primary human cells in which RRV replicates. Rotavirus infection of primary human pDCs provides a powerful experimental system for the study of mechanisms underlying pDC-mediated innate immunity to viral infection and reveals a potentially novel dsRNA-dependent pathway of IFNα induction.     

Viral Infection of Human Lung Macrophages Increases PDL1 Expression via IFNβ

Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.     

Oenothein B,a Cyclic Dimeric Ellagitannin Isolated from Epilobium angustifolium,Enhances IFNγ Production by Lymphocytes

Oenothein B is a polyphenol isolated from Epilobium angustifolium and other plant sources, which has been reported to exhibit immunomodulatory properties. Oenothein B is known to activate myeloid cells and induce the production of IL-1 and other cytokines. However, its effects on lymphocytes are unknown. In this report, we show that oenothein B stimulated innate lymphocytes, including bovine and human γδ T cells and NK cells, resulting in either increased CD25 and/or CD69 expression. We also demonstrate that oenothein B enhanced the production of interferon-γ (IFNγ) by bovine and human NK cells alone and in combination with interleukin-18 (IL-18), a response not observed with other commonly studied polyphenols. Furthermore, we demonstrate that oenothein B enhanced the production of IFNγ by human T cells. Since IFNγ contributes to antitumor, antibacterial, and antiviral cell responses, these data suggest an additional mechanism that could account, at least in part, for the immune enhancing properties of oenothein B.     

Tonsilar NK Cells Restrict B Cell Transformation by the Epstein-Barr Virus via IFN-��

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56^brightCD16⁻ NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-γ (IFN-γ) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-γ concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.     

Activation Mechanisms of Natural Killer Cells during Influenza Virus Infection

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.     

Healthy Neonates Possess a CD56-Negative NK Cell Population with Reduced Anti-Viral Activity

Background

Neonatal Natural Killer (NK) cells show functional impairment and expansion of a CD56 negative population of uncertain significance.

Methods

NK cells were isolated from cord blood and from adult donors. NK subpopulations were identified as positive or negative for the expression of CD56 and characterized for expression of granzyme B and surface markers by multi-parameter flow cytometry. Cell function was assessed by viral suppression and cytokine production using autologous lymphocytes infected with HIV. Activating (NKp30, NKp46) and inhibitory (Siglec-7) markers in healthy infants and adults were compared with viremic HIV-infected adults.

Results

Cord blood contained increased frequencies of CD56 negative (CD56neg) NK cells with reduced expression of granzyme B and reduced production of IFNγ and the CC-class chemokines RANTES, MIP1α and MIP1β upon stimulation. Both CD56pos and CD56neg NK subpopulations showed impaired viral suppression in cord blood, with impairment most marked in the CD56neg subset. CD56neg NK cells from cord blood and HIV-infected adults shared decreased inhibitory and activating receptor expression when compared with CD56pos cells.

Conclusions

CD56neg NK cells are increased in number in normal infants and these effectors show reduced anti-viral activity. Like the expanded CD56neg population described in HIV-infected adults, these NK cells demonstrate functional impairments which may reflect inadequate development or activation.     

Chronic HCV Infection Affects the NK Cell Phenotype in the Blood More than in the Liver

Although epidemiological and functional studies have implicated NK cells in protection and early clearance of HCV, the mechanism by which they may contribute to viral control is poorly understood, particularly at the site of infection, the liver. We hypothesized that a unique immunophenotypic/functional NK cell signature exists in the liver that may provide insights into the contribution of NK cells to viral control. Intrahepatic and blood NK cells were profiled from chronically infected HCV-positive and HCV-negative individuals. Baseline expression of activating and inhibitory receptors was assessed, as well as functional responses following stimulation through classic NK cell pathways. Independent of HCV infection, the liver was enriched for the immunoregulatory CD56^bright NK cell population, which produced less IFNγ and CD107a but comparable levels of MIP1β, and was immunophenotypically distinct from their blood counterparts. This profile was mostly unaltered in chronic HCV infection, though different expression levels of NKp46 and NKG2D were associated with different grades of fibrosis. In contrast to the liver, chronic HCV infection associated with an enrichment of CD161^lowperforin^high NK cells in the blood correlated with increased AST and 2B4 expression. However, the association of relatively discrete changes in the NK cell phenotype in the liver with the fibrosis stage nevertheless suggests an important role for the NK response. Overall these data suggest that tissue localization has a more pervasive effect on NK cells than the presence of chronic viral infection, during which these cells might be mostly attuned to limiting immunopathology. It will be important to characterize NK cells during early HCV infection, when they should have a critical role in limiting infection.     

NK-, NKT- and CD8-Derived IFNγ Drives Myeloid Cell Activation and Erythrophagocytosis,Resulting in Trypanosomosis-Associated Acute Anemia

African trypanosomes are the causative agents of Human African Trypanosomosis (HAT/Sleeping Sickness) and Animal African Trypanosomosis (AAT/Nagana). A common hallmark of African trypanosome infections is inflammation. In murine trypanosomosis, the onset of inflammation occurs rapidly after infection and is manifested by an influx of myeloid cells in both liver and spleen, accompanied by a burst of serum pro-inflammatory cytokines. Within 48 hours after reaching peak parasitemia, acute anemia develops and the percentage of red blood cells drops by 50%. Using a newly developed in vivo erythrophagocytosis assay, we recently demonstrated that activated cells of the myeloid phagocytic system display enhanced erythrophagocytosis causing acute anemia. Here, we aimed to elucidate the mechanism and immune pathway behind this phenomenon in a murine model for trypanosomosis. Results indicate that IFNγ plays a crucial role in the recruitment and activation of erythrophagocytic myeloid cells, as mice lacking the IFNγ receptor were partially protected against trypanosomosis-associated inflammation and acute anemia. NK and NKT cells were the earliest source of IFNγ during T. b. brucei infection. Later in infection, CD8+ and to a lesser extent CD4+ T cells become the main IFNγ producers. Cell depletion and transfer experiments indicated that during infection the absence of NK, NKT and CD8+ T cells, but not CD4+ T cells, resulted in a reduced anemic phenotype similar to trypanosome infected IFNγR-/- mice. Collectively, this study shows that NK, NKT and CD8+ T cell-derived IFNγ is a critical mediator in trypanosomosis-associated pathology, driving enhanced erythrophagocytosis by myeloid phagocytic cells and the induction of acute inflammation-associated anemia.     

Type I Interferon Upregulates Bak and Contributes to T Cell Loss during Human Immunodeficiency Virus (HIV) Infection

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression.     

Loss of Cytotoxicity and Gain of Cytokine Production in Murine Tumor-Activated NK Cells

NK cells are able to form a functional memory suggesting that some NK cells are surviving the activation process. We hypothesized that NK cell activation causes the development of a distinct NK cell subset and studied the fate of murine post-activation NK cells. Activation was achieved by in vivo and in vitro exposures to the melanoma tumor cell line B16 that was followed by differentiation in IL-2. When compared with control NK cells, post-activation CD25⁺ NK cells expressed little granzyme B or perforin and had low lysis activity. Post-activation NK cells expressed CD27, CD90, CD127, and were low for CD11b suggesting that tumor-induced activation is restricted to an early NK cell subset. Activation of NK cells led to decreases of CD16, CD11c and increases of CD62L and the IL-18 receptor. In vivo activated but not control NK cells expressed a variety of cytokines that included IFNγ, TNFα, GM-CSF and IL-10. These data suggest that the exposure of a subset of peripheral NK cells to the B16 tumor environment caused an exhaustion of their cytolytic capacity but also a gain in their ability to produce cytokines.     

NK Cell–Like Behavior of Vα14i NK T Cells during MCMV Infection

Immunity to the murine cytomegalovirus (MCMV) is critically dependent on the innate response for initial containment of viral replication, resolution of active infection, and proper induction of the adaptive phase of the anti-viral response. In contrast to NK cells, the Vα14 invariant natural killer T cell response to MCMV has not been examined. We found that Vα14i NK T cells become activated and produce significant levels of IFN-γ, but do not proliferate or produce IL-4 following MCMV infection. In vivo treatment with an anti-CD1d mAb and adoptive transfer of Vα14i NK T cells into MCMV-infected CD1d^−/− mice demonstrate that CD1d is dispensable for Vα14i NK T cell activation. In contrast, both IFN-α/β and IL-12 are required for optimal activation. Vα14i NK T cell–derived IFN-γ is partially dependent on IFN-α/β but highly dependent on IL-12. Vα14i NK T cells contribute to the immune response to MCMV and amplify NK cell–derived IFN-γ. Importantly, mortality is increased in CD1d^−/− mice in response to high dose MCMV infection when compared to heterozygote littermate controls. Collectively, these findings illustrate the plasticity of Vα14i NK T cells that act as effector T cells during bacterial infection, but have NK cell–like behavior during the innate immune response to MCMV infection.     

Epstein-Barr Virus Interferes with the Amplification of IFNα Secretion by Activating Suppressor of Cytokine Signaling 3 in Primary Human Monocytes

Background

Epstein-Barr virus is recognized to cause lymphoproliferative disorders and is also associated with cancer. Evidence suggests that monocytes are likely to be involved in EBV pathogenesis, especially due to a number of cellular functions altered in EBV-infected monocytes, a process that may affect efficient host defense. Because type I interferons (IFNs) are crucial mediators of host defense against viruses, we investigated the effect of EBV infection on the IFNα pathway in primary human monocytes.

Methodology/Principal Findings

Infection of monocytes with EBV induced IFNα secretion but inhibited the positive feedback loop for the amplification of IFNα. We showed that EBV infection induced the expression of suppressor of cytokine signaling 3 (SOCS3) and, to a lesser extent, SOCS1, two proteins known to interfere with the amplification of IFNα secretion mediated by the JAK/STAT signal transduction pathway. EBV infection correlated with a blockage in the activation of JAK/STAT pathway members and affected the level of phosphorylated IFN regulatory factor 7 (IRF7). Depletion of SOCS3, but not SOCS1, by small interfering RNA (siRNA) abrogated the inhibitory effect of EBV on JAK/STAT pathway activation and significantly restored IFNα secretion. Finally, transfection of monocytes with the viral protein Zta caused the upregulation of SOCS3, an event that could not be recapitulated with mutated Zta.

Conclusions/Significance

We propose that EBV protein Zta activates SOCS3 protein as an immune escape mechanism that both suppresses optimal IFNα secretion by human monocytes and favors a state of type I IFN irresponsiveness in these cells. This immunomodulatory effect is important to better understand the aspects of the immune response to EBV.     

Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Tonsilar NK cells restrict B cell transformation by the Epstein-Barr virus via IFN-gamma

Cells of the innate immune system act in synergy to provide a first line of defense against pathogens. Here we describe that dendritic cells (DCs), matured with viral products or mimics thereof, including Epstein-Barr virus (EBV), activated natural killer (NK) cells more efficiently than other mature DC preparations. CD56(bright)CD16(-) NK cells, which are enriched in human secondary lymphoid tissues, responded primarily to this DC activation. DCs elicited 50-fold stronger interferon-gamma (IFN-gamma) secretion from tonsilar NK cells than from peripheral blood NK cells, reaching levels that inhibited B cell transformation by EBV. In fact, 100- to 1,000-fold less tonsilar than peripheral blood NK cells were required to achieve the same protection in vitro, indicating that innate immune control of EBV by NK cells is most efficient at this primary site of EBV infection. The high IFN-gamma concentrations, produced by tonsilar NK cells, delayed latent EBV antigen expression, resulting in decreased B cell proliferation during the first week after EBV infection in vitro. These results suggest that NK cell activation by DCs can limit primary EBV infection in tonsils until adaptive immunity establishes immune control of this persistent and oncogenic human pathogen.     

A Fluorescence Reporter Model Defines “Tip-DCs” as the Cellular Source of Interferon β in Murine Listeriosis

Production of type I interferons, consisting mainly of multiple IFNα subtypes and IFNβ, represents an essential part of the innate immune defense against invading pathogens. While in most situations, namely viral infections, this class of cytokines is indispensable for host survival they mediate a detrimental effect during infection with L. monocytogenes by rendering macrophages insensitive towards IFNγ signalling which leads to a lethal bacterial pathology in mice. Due to a lack of suitable analytic tools the precise identity of the cell population responsible for type I IFN production remains ill-defined and so far these cells have been described to be macrophages. As in general IFNβ is the first type I interferon to be produced, we took advantage of an IFNβ fluorescence reporter-knockin mouse model in which YFP is expressed from a bicistronic mRNA linked by an IRES to the endogenous ifnb mRNA to assess the IFNβ production on a single cell level in situ. Our results showed highest frequencies and absolute numbers of IFNβ⁺ cells in the spleen 24 h after infection with L. monocytogenes where they were located predominately in the white pulp within the foci of infection. Detailed FACS surface marker analyses, intracellular cytokine stainings and T cell proliferation assays revealed that the IFNβ⁺ cells were a phenotypically and functionally further specialized subpopulation of TNF and iNOS producing DCs (Tip-DCs) which are known to be essential for the early containment of L. monocytogenes infection. We proved that the IFNβ⁺ cells exhibited the hallmark characteristics of Tip-DCs as they produced iNOS and TNF and possessed T cell priming abilities. These results point to a yet unappreciated ambiguous role for a multi-effector, IFNβ producing subpopulation of Tip-DCs in controlling the balance between containment of L. monocytogenes infection and effects detrimental to the host driven by IFNβ.     

Marburg Virus Evades Interferon Responses by a Mechanism Distinct from Ebola Virus

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.