Mice deficient in STAT1 but not STAT2 or IRF9 develop a lethal CD4+ T-cell-mediated disease following infection with lymphocytic choriomeningitis virus

Interferon (IFN) signaling is crucial for antiviral immunity. While type I IFN signaling is mediated by STAT1, STAT2, and IRF9, type II IFN signaling requires only STAT1. Here, we studied the roles of these signaling factors in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild-type (WT) mice and mice lacking either STAT2 or IRF9, LCMV infection was nonlethal, and the virus either was cleared (WT) or established persistence (STAT2 knockout [KO] and IRF9 KO). However, in the case of STAT1 KO mice, LCMV infection was lethal and accompanied by severe multiorgan immune pathology, elevated expression of various cytokine genes in tissues, and cytokines in the serum. This lethal phenotype was unaltered by the coabsence of the gamma interferon (IFN-γ) receptor and hence was not dependent on IFN-γ. Equally, the disease was not due to a combined defect in type I and type II IFN signaling, as IRF9 KO mice lacking the IFN-γ receptor survived infection with LCMV. Clearance of LCMV is mediated normally by CD8(+) T cells. However, the depletion of these cells in LCMV-infected STAT1 KO mice was delayed, but did not prevent, lethality. In contrast, depletion of CD4(+) T cells prevented lethality in LCMV-infected STAT1 KO mice and was associated with a reduction in tissue immune pathology. These studies highlight a fundamental difference in the role of STAT1 versus STAT2 and IRF9. While all three factors are required to limit viral replication and spread, only STAT1 has the unique function of preventing the emergence of a lethal antiviral CD4(+) T-cell response.     

Inhibitor of κB kinase epsilon (IKK(epsilon)), STAT1, and IFIT2 proteins define novel innate immune effector pathway against West Nile virus infection

West Nile virus is an emerging virus whose virulence is dependent upon viral evasion of IFN and innate immune defenses. The actions of IFN-stimulated genes (ISGs) impart control of virus infection, but the specific ISGs and regulatory pathways that restrict West Nile virus (WNV) are not defined. Here we show that inhibitor of κB kinase ε (IKKε) phosphorylation of STAT1 at serine 708 (Ser-708) drives IFIT2 expression to mediate anti-WNV effector function of IFN. WNV infection was enhanced in cells from IKKε(-/-) or IFIT2(-/-) mice. In IKKε(-/-) cells, the loss of IFN-induced IFIT2 expression was linked to lack of STAT1 phosphorylation on Ser-708 but not Tyr-701 nor Ser-727. STAT1 Ser-708 phosphorylation occurs independently of IRF-3 but requires signaling through the IFN-α/β receptor as a late event in the IFN-induced innate immune response that coincides with IKKε-responsive ISGs expression. Biochemical analyses show that STAT1 tyrosine dephosphorylation and CRM1-mediated STAT1 nuclear-cytoplasmic shuttling are required for STAT1 Ser-708 phosphorylation. When compared with WT mice, WNV-infected IKKε(-/-) mice exhibit enhanced kinetics of virus dissemination and increased pathogenesis concomitant with loss of STAT1 Ser-708 phosphorylation and IFIT2 expression. Our results define an IFN-induced IKKε signaling pathway of specific STAT1 phosphorylation and IFIT2 expression that imparts innate antiviral immunity to restrict WNV infection and control viral pathogenesis.     

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

The Bacteriostatic Protein Lipocalin 2 Is Induced in the Central Nervous System of Mice with West Nile Virus Encephalitis

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.     

Innate STAT1-dependent genomic response of neurons to the antiviral cytokine alpha interferon

Alpha/beta interferons (IFNs-alpha/beta) are cytokines that play an essential role in the host defense against viral infection. Our previous studies have shown that the key IFN signaling molecule STAT1 is highly elevated and activated in central nervous system neurons during viral infection and in transgenic mice with astrocyte production of IFN-alpha (glial fibrillary acidic protein [GFAP]-IFN-alpha), suggesting that neurons are a very responsive target cell population for IFNs. To elucidate the genomic response of neurons to IFN-alpha, we undertook studies both in vitro and in vivo. Gene chip analysis was applied to RNA from IFN-alpha-treated or untreated primary cortical neuronal cultures derived from embryonic day 15 fetal wild-type or STAT1 knockout (KO) mice. The expression of 51 known and 5 unknown genes was increased significantly by more than twofold after exposure of wild-type but not STAT1 KO neurons to IFN-alpha. Some more highly expressed genes included IFN-induced 15-kDa protein, ubiquitin-specific protease 18, glucocorticoid attenuated response genes, IFN-induced GTPases, and the chemokine CXCL10. For several of these genes, the gene chip findings were confirmed by RNase protection assays. In addition, examination of the expression of some of these selected genes revealed that they were increased in neurons in the brain of either GFAP-IFN-alpha mice or mice infected with lymphocytic choriomeningitis virus. In conclusion, our study revealed a robust STAT1-dependent genomic response of neurons to IFN-alpha, highlighting an innate potential of these cells to defend against viral infection in the brain.     

Interferon-Induced Ifit2/ISG54 Protects Mice from Lethal VSV Neuropathogenesis

Interferon protects mice from vesicular stomatitis virus (VSV) infection and pathogenesis; however, it is not known which of the numerous interferon-stimulated genes (ISG) mediate the antiviral effect. A prominent family of ISGs is the interferon-induced with tetratricopeptide repeats (Ifit) genes comprising three members in mice, Ifit1/ISG56, Ifit2/ISG54 and Ifit3/ISG49. Intranasal infection with a low dose of VSV is not lethal to wild-type mice and all three Ifit genes are induced in the central nervous system of the infected mice. We tested their potential contributions to the observed protection of wild-type mice from VSV pathogenesis, by taking advantage of the newly generated knockout mice lacking either Ifit2 or Ifit1. We observed that in Ifit2 knockout (Ifit2 ^−/−) mice, intranasal VSV infection was uniformly lethal and death was preceded by neurological signs, such as ataxia and hind limb paralysis. In contrast, wild-type and Ifit1 ^−/− mice were highly protected and survived without developing such disease. However, when VSV was injected intracranially, virus replication and survival were not significantly different between wild-type and Ifit2^−/− mice. When administered intranasally, VSV entered the central nervous system through the olfactory bulbs, where it replicated equivalently in wild-type and Ifit2 ^−/− mice and induced interferon-β. However, as the infection spread to other regions of the brain, VSV titers rose several hundred folds higher in Ifit2 ^−/− mice as compared to wild-type mice. This was not caused by a broadened cell tropism in the brains of Ifit2 ^−/− mice, where VSV still replicated selectively in neurons. Surprisingly, this advantage for VSV replication in the brains of Ifit2^−/− mice was not observed in other organs, such as lung and liver. Pathogenesis by another neurotropic RNA virus, encephalomyocarditis virus, was not enhanced in the brains of Ifit2 ^−/− mice. Our study provides a clear demonstration of tissue-, virus- and ISG-specific antiviral action of interferon.     

CD40-CD40 ligand interactions promote trafficking of CD8+ T cells into the brain and protection against West Nile virus encephalitis

Recent studies have established a protective role for T cells during primary West Nile virus (WNV) infection. Binding of CD40 by CD40 ligand (CD40L) on activated CD4+ T cells provides an important costimulatory signal for immunoglobulin class switching, antibody affinity maturation, and priming of CD8+ T-cell responses. We examined here the function of CD40-dependent interactions in limiting primary WNV infection. Compared to congenic wild-type mice, CD40(-/-) mice uniformly succumbed to WNV infection. Although CD40(-/-) mice produced low levels of WNV-specific immunoglobulin M (IgM) and IgG, viral clearance from the spleen and serum was not altered, and CD8+ T-cell priming in peripheral lymphoid tissues was normal. Unexpectedly, CD8+ T-cell trafficking to the central nervous system (CNS) was markedly impaired in CD40(-/-) mice, and this correlated with elevated WNV titers in the CNS and death. In the brains of CD40(-/-) mice, T cells were retained in the perivascular space and did not migrate into the parenchyma, the predominant site of WNV infection. In contrast, in wild-type mice, T cells trafficked to the site of infection in neurons. Beside its role in maturation of antibody responses, our experiments suggest a novel function of CD40-CD40L interactions: to facilitate T-cell migration across the blood-brain barrier to control WNV infection.     

The interferon-inducible gene viperin restricts West Nile virus pathogenesis

Type I interferon (IFN) signaling coordinates an early antiviral program in infected and uninfected cells by inducing IFN-stimulated genes (ISGs) that modulate viral entry, replication, and assembly. However, the specific antiviral functions in vivo of most ISGs remain unknown. Here, we examined the contribution of the ISG viperin to the control of West Nile virus (WNV) in genetically deficient cells and mice. While modest increases in levels of WNV replication were observed for primary viperin(-/-) macrophages and dendritic cells, no appreciable differences were detected in deficient embryonic cortical neurons or fibroblasts. In comparison, viperin(-/-) adult mice infected with WNV via the subcutaneous or intracranial route showed increased lethality and/or enhanced viral replication in central nervous system (CNS) tissues. In the CNS, viperin expression was induced in both WNV-infected and adjacent uninfected cells, including activated leukocytes at the site of infection. Our experiments suggest that viperin restricts the infection of WNV in a tissue- and cell-type-specific manner and may be an important ISG for controlling viral infections that cause CNS disease.     

Antibody prophylaxis and therapy against West Nile virus infection in wild-type and immunodeficient mice

West Nile virus (WNV) is a mosquito-borne Flavivirus that causes encephalitis in a subset of susceptible humans. Current treatment for WNV infections is supportive, and no specific therapy or vaccine is available. In this study, we directly tested the prophylactic and therapeutic efficacy of polyclonal antibodies against WNV. Passive administration of human gamma globulin or mouse serum prior to WNV infection protected congenic wild-type, B-cell-deficient ( micro MT), and T- and B-cell-deficient (RAG1) C57BL/6J mice. Notably, no increased mortality due to immune enhancement was observed. Although immune antibody completely prevented morbidity and mortality in wild-type mice, its effect was not durable in immunocompromised mice: many micro MT and RAG1 mice eventually succumbed to infection. Thus, antibody by itself did not completely eliminate viral reservoirs in host tissues, consistent with an intact cellular immune response being required for viral clearance. In therapeutic postexposure studies, human gamma globulin partially protected against WNV-induced mortality. In micro MT mice, therapy had to be initiated within 2 days of infection to gain a survival benefit, whereas in the wild-type mice, therapy even 5 days after infection reduced mortality. This time point is significant because between days 4 and 5, WNV was detected in the brains of infected mice. Thus, passive transfer of immune antibody improves clinical outcome even after WNV has disseminated into the central nervous system.     

Expression of interferon-induced antiviral genes is delayed in a STAT1 knockout mouse model of Crimean-Congo hemorrhagic fever

ABSTRACT: Background Crimean Congo hemorrhagic fever (CCHF) is a tick-borne hemorrhagic zoonosis associated with high mortality. Pathogenesis studies and the development of vaccines and antivirals against CCHF have been severely hampered by the lack of suitable animal model. We recently developed and characterized a mature mouse model for CCHF using mice carrying STAT1 knockout (KO). Findings Given the importance of interferons in controlling viral infections, we investigated the expression of interferon pathway-associated genes in KO and wild-type (WT) mice challenged with CCHF virus. We expected that the absence of the STAT1 protein would result in minimal expression of IFN-related genes. Surprisingly, the KO mice showed high levels of IFN-stimulated gene expression, beginning on day 2 post-infection, while in WT mice challenged with virus the same genes were expressed at similar levels on day 1. Conclusions We conclude that CCHF virus induces similar type I IFN responses in STAT1 KO and WT mice, but the delayed and dysregulated response in the KO mice permits rapid viral dissemination and fatal illness.     

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18(-/-) mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18(-/-) mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages from Usp18(-/-) mice showed restricted LCMV replication. Moreover, MEF from Usp18(-/-) mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.     

Identification of Five Interferon-Induced Cellular Proteins That Inhibit West Nile Virus and Dengue Virus Infections

Interferons (IFNs) are key mediators of the host innate antiviral immune response. To identify IFN-stimulated genes (ISGs) that instigate an antiviral state against two medically important flaviviruses, West Nile virus (WNV) and dengue virus (DENV), we tested 36 ISGs that are commonly induced by IFN-α for antiviral activity against the two viruses. We discovered that five ISGs efficiently suppressed WNV and/or DENV infection when they were individually expressed in HEK293 cells. Mechanistic analyses revealed that two structurally related cell plasma membrane proteins, IFITM2 and IFITM3, disrupted early steps (entry and/or uncoating) of the viral infection. In contrast, three IFN-induced cellular enzymes, viperin, ISG20, and double-stranded-RNA-activated protein kinase, inhibited steps in viral proteins and/or RNA biosynthesis. Our results thus imply that the antiviral activity of IFN-α is collectively mediated by a panel of ISGs that disrupt multiple steps of the DENV and WNV life cycles.West Nile virus (WNV) and dengue virus (DENV) are mosquito-borne flaviviruses that cause invasive neurological diseases and lethal hemorrhagic fever in humans, respectively (6, 32). Since its first incursion into New York City in 1999, WNV has rapidly spread throughout the continental United States and has recently reached South America (29, 34). In most cases, WNV infection of people resolves as an asymptomatic or a mild febrile illness. However, approximately 1% of infections result in severe neurological disorders, such as encephalitis and meningitis (27). Unlike WNV, for which people are only accidental hosts, DENV has fully adapted to humans (32). It has apparently lost the need for an enzootic cycle and causes a range of diseases in people, from acute febrile illness to life-threatening dengue hemorrhagic fever/dengue shock syndrome (6). Four distinct serotypes of DENV have spread throughout the tropical and subtropical parts of the world, with an estimated 50 to 100 million human cases annually and about 2.5 billion people worldwide being at risk of infection (32). Effective antiviral therapies and vaccines to treat or prevent WNV and DENV infections in humans are not yet available.Type I interferons (IFNs), represented by IFN-α and IFN-β, have been demonstrated to play an essential role in defending against WNV and DENV infections. For example, mice with deficiencies in the induction of type I IFNs and the receptor or JAK-STAT signal transduction pathway of the cytokines are vulnerable to WNV and DENV infections (7, 38, 42, 49-51). In addition, a strain of WNV that fails to block the type I IFN signal transduction pathway is phenotypically attenuated in mice (23, 50). Clinically, during acute DENV infection, innate immune responses play a key role in determining disease outcome (35), and resolution of WNV infection requires effective IFN-mediated innate host responses (23, 43, 53). Therefore, understanding how the IFN-mediated innate immune response functions is one of the critical frontiers in the molecular biology of WNV and DENV pathogenesis (1, 44).IFNs inhibit virus infection by induction of IFN-stimulated genes (ISGs) that disrupt distinct steps of the viral replication cycle (47). However, although IFN treatment of cells induces the expression of hundreds of cellular genes (9), only approximately a dozen ISGs have been experimentally demonstrated to instigate an antiviral state against selected viruses (41). As mentioned above, although there is ample evidence suggesting that IFN-mediated innate immunity plays a critical role in defending against WNV and DENV infections, the underlying antiviral mechanism of the cytokines remains to be understood (6, 16, 31). With WNV, previous studies suggested that mice lacking double-stranded-RNA-activated protein kinase (PKR) and RNase L were more susceptible to the virus infection and had increased viral loads in multiple peripheral organs and neuronal tissues, in comparison with congenic wild-type mice (43). In addition, genetic studies showed that a nonsense mutation in the gene encoding the 2′,5′-oligoadenylate synthetase 1b (OAS1b) isoform was associated with WNV susceptibility in mice, and expression of wild-type OAS1b in mouse fibroblasts efficiently inhibited WNV infection (22, 33, 37, 45). For DENV, it was reported recently that viperin was among the highly induced ISGs in DENV-infected cells and overexpression of viperin in A549 cells significantly reduced DENV replication (13).In principle, to understand how IFNs inhibit DENV and WNV infections, it is essential to know the repertoire of ISGs that are directly implicated in antiviral action and understand how these antiviral ISGs work individually and coordinately to limit virus replication. To achieve this goal, we set out to systematically identify the ISGs that are able to inhibit infection with the two viruses and elucidate their antiviral mechanisms.     

IL-13 regulates Th17 secretion of IL-17A in an IL-10-dependent manner

IL-13 is a central mediator of airway hyperresponsiveness and mucus expression, both hallmarks of asthma. IL-13 is found in the sputum of patients with asthma; therefore, IL-13 is an attractive drug target for treating asthma. We have shown previously that IL-13 inhibits Th17 cell production of IL-17A and IL-21 in vitro. Th17 cells are associated with autoimmune diseases, host immune responses, and severe asthma. In this study, we extend our in vitro findings and determine that IL-13 increases IL-10 production from Th17-polarized cells and that IL-13-induced IL-10 production negatively regulates the secretion of IL-17A and IL-21. To determine if IL-13 negatively regulates lung IL-17A expression via an IL-10-dependent mechanism in vivo, we used a model of respiratory syncytial virus (RSV) strain A2 infection in STAT1 knockout (KO) mice that increases lung IL-17A and IL-13 expression, cytokines not produced during RSV infection in wild-type mice. To test the hypothesis that IL-13 negatively regulates lung IL-17A expression, we created STAT1/IL-13 double KO (DKO) mice. We found that RSV-infected STAT1/IL-13 DKO mice had significantly greater lung IL-17A expression compared with that of STAT1 KO mice and that increased IL-17A expression was abrogated by anti-IL-10 Ab treatment. RSV-infected STAT1/IL-13 DKO mice also had increased neutrophil infiltration compared with that of RSV-infected STAT1 KO mice. Neutralizing IL-10 increased the infiltration of inflammatory cells into the lungs of STAT1 KO mice but not STAT1/IL-13 DKO mice. These findings are vital to understanding the potential side effects of therapeutics targeting IL-13. Inhibiting IL-13 may decrease IL-10 production and increase IL-17A production, thus potentiating IL-17A-associated diseases.     

Promotion of alpha/beta interferon induction during in vivo viral infection through alpha/beta interferon receptor/STAT1 system-dependent and -independent pathways

Viruses and viral components can be potent inducers of alpha/beta interferons (IFN-alpha/beta). In culture, IFN-alpha/beta prime for their own expression, in response to viruses, through interferon regulatory factor 7 (IRF-7) induction. The studies presented here evaluated the requirements for functional IFN receptors and the IFN signaling molecule STAT1 in IFN-alpha/beta induction during infections of mice with lymphocytic choriomeningitis virus (LCMV). At 24 h after infection, levels of induced IFN-alpha/beta in serum were reduced 90 to 95% in IFN-alpha/beta receptor-deficient (IFN-alpha/betaR(-/-)) and STAT1(-/-) mice compared to those in wild-type mice. However, at 48 h, these mice showed elevated expression in the serum whereas IFN-alpha/beta levels were still reduced >75% in IFN-alpha/betagammaR(-/-) mice even though the viral burden was heavy. Levels of IFN-beta, IFN-alpha4, and non-IFN-alpha4 subtype mRNA expression correlated with IFN-alpha/beta bioactivity, and all IFN-alpha/beta subtypes were coincidentally detectable. IRF-7 mRNA was induced under conditions of IFN-alpha/beta production, including late production in IFN-alpha/betaR(-/-) mice. These data demonstrate that the presence of the virus alone is not sufficient to induce IFN-alpha/beta during LCMV infection in vivo. Instead, autocrine amplification through the IFN-alpha/betaR is necessary for optimal induction. In the absence of a functional IFN-alpha/betaR, however, alternative mechanisms, independent of STAT1 but requiring a functional IFN-gammaR, take over.     

Whole-genome expression profiling reveals that inhibition of host innate immune response pathways by Ebola virus can be reversed by a single amino acid change in the VP35 protein

Ebola hemorrhagic fever is a rapidly progressing acute febrile illness characterized by high virus replication, severe immunosuppression, and case fatalities of ca. 80%. Inhibition of phosphorylation of interferon regulatory factor 3 (IRF-3) by the Ebola VP35 protein may block the host innate immune response and play an important role in the severity of disease. We used two precisely defined reverse genetics-generated Ebola viruses to investigate global host cell responses resulting from the inhibition of IRF-3 phosphorylation. The two viruses encoded either wild-type (WT) VP35 protein (recEbo-VP35/WT) or VP35 with an arginine (R)-to-alanine (A) amino acid substitution at position 312 (recEbo-VP35/R312A) within a previously defined IRF-3 inhibitory domain. When sucrose-gradient purified virus was used for infection, host cell whole-genome expression profiling revealed striking differences in human liver cell responses to these viruses differing by a single amino acid. The inhibition of host innate immune responses by WT Ebola virus was so potent that little difference in interferon and antiviral gene expression could be discerned between cells infected with purified WT, inactivated virus, or mock-infected cells. However, infection with recEbo-VP35/R312A virus resulted in a strong innate immune response including increased expression of MDA-5, RIG-I, RANTES, MCP-1, ISG-15, ISG-54, ISG-56, ISG-60, STAT1, IRF-9, OAS, and Mx1. The clear gene expression differences were obscured if unpurified virus stocks were used to initiate infection, presumably due to soluble factors present in virus-infected cell supernatant preparations. Ebola virus VP35 protein clearly plays a pivotal role in the potent inhibition of the host innate immune responses, and the present study indicates that VP35 has a wider effect on host cell responses than previously shown. The ability to eliminate this inhibitory effect with a single amino acid change in VP35 demonstrates the critical role this protein must play in the severe aspects this highly fatal disease.