Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion

Adaptive cellular immunity is required to clear HSV-1 infection in the periphery. Myeloid dendritic cells (DCs) are the first professional Ag-presenting cell to encounter the virus after primary and secondary infection and thus the consequences of their infection are important in understanding the pathogenesis of the disease and the response to the virus. Following HSV-1 infection, both uninfected and infected human DCs acquire a more mature phenotype. In this study, we demonstrate that type I IFN secreted from myeloid DC mediates bystander activation of the uninfected DCs. Furthermore, we confirm that this IFN primes DCs for elevated IL-12 p40 and p70 secretion. However, secretion of IFN is not responsible for the acquisition of a mature phenotype by HSV-1-infected DC. Rather, virus binding to a receptor on the cell surface induces DC maturation directly, through activation of the NF-kappaB and p38 MAPK pathways. The binding of HSV glycoprotein D is critical to the acquisition of a mature phenotype and type I IFN secretion. The data therefore demonstrate that DCs can respond to HSV exposure directly through recognition of viral envelope structures. In the context of natural HSV infection, the coupling of viral entry to the activation of DC signaling pathways is likely to be counterbalanced by viral disruption of DC maturation. However, the parallel release of type I IFN may result in paracrine activation so that the DCs are nonetheless able to mount an adaptive immune response.     

TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses

TLR signaling leads to dendritic cell (DC) maturation and immunity to diverse pathogens. The stimulation of TLRs by conserved viral structures is the only described mechanism leading to DC maturation after a virus infection. In this report, we demonstrate that mouse myeloid DCs mature normally after in vivo and in vitro infection with Sendai virus (SeV) in the absence of TLR3, 7, 8, or 9 signaling. DC maturation by SeV requires virus replication not necessary for TLR-mediated triggering. Moreover, DCs deficient in TLR signaling efficiently prime for Th1 immunity after infection with influenza or SeV, generating IFN-gamma-producing T cells, CTLs and antiviral Abs. We have previously demonstrated that SeV induces DC maturation independently of the presence of type I IFN, which has been reported to mature DCs in a TLR-independent manner. The data presented here provide evidence for the existence of a novel intracellular pathway independent of TLR-mediated signaling responsible for live virus triggering of DC maturation and demonstrate its critical role in the onset of antiviral immunity. The revelation of this pathway should stimulate invigorating research into the mechanism for virus-induced DC maturation and immunity.     

IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

Th2-inducing pathological conditions such as parasitic diseases increase susceptibility to viral infections through yet unclear mechanisms. We have previously reported that IL-4, a pivotal Th2 cytokine, suppresses the response of murine bone-marrow-derived conventional dendritic cells (cDCs) and splenic DCs to Type I interferons (IFNs). Here, we analyzed cDC responses to TLR7 and TLR9 ligands, R848 and CpGs, respectively. We found that IL-4 suppressed the gene expression of IFNβ and IFN-responsive genes (IRGs) upon TLR7 and TLR9 stimulation. IL-4 also inhibited IFN-dependent MHC Class I expression and amplification of IFN signaling pathways triggered upon TLR stimulation, as indicated by the suppression of IRF7 and STAT2. Moreover, IL-4 suppressed TLR7- and TLR9-induced cDC production of pro-inflammatory cytokines such as TNFα, IL-12p70 and IL-6 by inhibiting IFN-dependent and NFκB-dependent responses. IL-4 similarly suppressed TLR responses in splenic DCs. IL-4 inhibition of IRGs and pro-inflammatory cytokine production upon TLR7 and TLR9 stimulation was STAT6-dependent, since DCs from STAT6-KO mice were resistant to the IL-4 suppression. Analysis of SOCS molecules (SOCS1, −2 and −3) showed that IL-4 induces SOCS1 and SOCS2 in a STAT6 dependent manner and suggest that IL-4 suppression could be mediated by SOCS molecules, in particular SOCS2. IL-4 also decreased the IFN response and increased permissiveness to viral infection of cDCs exposed to a HIV-based lentivirus. Our results indicate that IL-4 modulates and counteracts pro-inflammatory stimulation induced by TLR7 and TLR9 and it may negatively affect responses against viruses and intracellular parasites.     

Herpes simplex virus virion host shutoff (vhs) activity alters periocular disease in mice

During lytic infection, the virion host shutoff (vhs) protein of herpes simplex virus (HSV) mediates the rapid degradation of RNA and shutoff of host protein synthesis. In mice, HSV type 1 (HSV-1) mutants lacking vhs activity are profoundly attenuated. HSV-2 has significantly higher vhs activity than HSV-1, eliciting a faster and more complete shutoff. To examine further the role of vhs activity in pathogenesis, we generated an intertypic recombinant virus (KOSV2) in which the vhs open reading frame of HSV-1 strain KOS was replaced with that of HSV-2 strain 333. KOSV2 and a marker-rescued virus, KOSV2R, were characterized in cell culture and tested in an in vivo mouse eye model of latency and pathogenesis. The RNA degradation kinetics of KOSV2 was identical to that of HSV-2 333, and both showed vhs activity significantly higher than that of KOS. This demonstrated that the fast vhs-mediated degradation phenotype of 333 had been conferred upon KOS. The growth of KOSV2 was comparable to that of KOS, 333, and KOSV2R in cell culture, murine corneas, and trigeminal ganglia and had a reactivation frequency similar to those of KOS and KOSV2R from explanted latently infected trigeminal ganglia. There was, however, significantly reduced blepharitis and viral replication within the periocular skin of KOSV2-infected mice compared to mice infected with either KOS or KOSV2R. Taken together, these data demonstrate that heightened vhs activity, in the context of HSV-1 infection, leads to increased viral clearance from the skin of mice and that the replication of virus in the skin is a determining factor for blepharitis. These data also suggest a role for vhs in modulating host responses to HSV infection.     

Herpes simplex virus ICP27 is required for virus-induced stabilization of the ARE-containing IEX-1 mRNA encoded by the human IER3 gene

Herpes simplex virus (HSV) stifles cellular gene expression during productive infection of permissive cells, thereby diminishing host responses to infection. Host shutoff is achieved largely through the complementary actions of two viral proteins, ICP27 and virion host shutoff (vhs), that inhibit cellular mRNA biogenesis and trigger global mRNA decay, respectively. Although most cellular mRNAs are thus depleted, some instead increase in abundance after infection; perhaps surprisingly, some of these contain AU-rich instability elements (AREs) in their 3'-untranslated regions. ARE-containing mRNAs normally undergo rapid decay; however, their stability can increase in response to signals such as cytokines and virus infection that activate the p38/MK2 mitogen-activated protein kinase (MAPK) pathway. We and others have shown that HSV infection stabilizes the ARE mRNA encoding the stress-inducible IEX-1 mRNA, and a previous report from another laboratory has suggested vhs is responsible for this effect. However, we now report that ICP27 is essential for IEX-1 mRNA stabilization whereas vhs plays little if any role. A recent report has documented that ICP27 activates the p38 MAPK pathway, and we detected a strong correlation between this activity and stabilization of IEX-1 mRNA by using a panel of HSV type 1 (HSV-1) isolates bearing an array of previously characterized ICP27 mutations. Furthermore, IEX-1 mRNA stabilization was abrogated by the p38 inhibitor SB203580. Taken together, these data indicate that the HSV-1 immediate-early protein ICP27 alters turnover of the ARE-containing message IEX-1 by activating p38. As many ARE mRNAs encode proinflammatory cytokines or other immediate-early response proteins, some of which may limit viral replication, it will be of great interest to determine if ICP27 mediates stabilization of many or all ARE-containing mRNAs.     

Deletion of the virion host shutoff protein (vhs) from herpes simplex virus (HSV) relieves the viral block to dendritic cell activation: potential of vhs- HSV vectors for dendritic cell-mediated immunotherapy

Herpes simplex virus (HSV) infects dendritic cells (DC) efficiently but with minimal replication. HSV, therefore, appears to have evolved the ability to enter DC even though they are nonpermissive for virus growth. This provides a potential utility for HSV in delivering genes to DC for vaccination purposes and also suggests that the life cycle of HSV usually includes the infection of DC. However, DC infected with HSV usually lose the ability to become activated following infection (M. Salio, M. Cella, M. Suter, and A. Lanzavecchia, Eur. J. Immunol. 29:3245-3253, 1999; M. Kruse, O. Rosorius, F. Kratzer, G. Stelz, C. Kuhnt, G. Schuler, J. Hauber, and A. Steinkasserer, J. Virol. 74:7127-7136, 2000). We report that for DC to retain the ability to become activated following HSV infection, the virion host shutoff protein (vhs) must be deleted. vhs usually functions to destabilize mRNA in favor of the production of HSV proteins in permissive cells. We have found that it also plays a key role in the inactivation of DC and is therefore likely to be important for immune evasion by the virus. Here, vhs would be anticipated to prevent DC activation in the early stages of infection of an individual with HSV, reducing the induction of cellular immune responses and thus preventing virus clearance during repeated cycles of virus latency and reactivation. Based on this information, replication-incompetent HSV vectors with vhs deleted which allow activation of DC and the induction of specific T-cell responses to delivered antigens have been constructed. These responses are greater than if DC are loaded with antigen by incubation with recombinant protein.     

Induction of potent protection against acute and latent herpes simplex virus infection in mice vaccinated with dendritic cells

Background aimsDendritic cells (DCs) are the most potent antigen presenting cells of the immune system and have been under intense study with regard to their use in immunotherapy against cancer and infectious disease agents. In the present study, DCs were employed to assess their value in protection against live virus challenge in an experimental model using lethal and latent herpes simplex virus (HSV) infection in Balb/c mice.MethodsDCs obtained ex vivo in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 were loaded with HSV-1 proteins (DC/HSV-1 vaccine). Groups of mice were vaccinated twice, 7 days apart, via subcutaneous, intraperitoneal or intramuscular routes with DC/HSV-1 and with mock (DC without virus protein) and positive (alum adjuvanted HSV-1 proteins [HSV-1/ALH]) control vaccines. After measuring anti-HSV-1 antibody levels in blood samples, mice were given live HSV-1 intraperitoneally or via ear pinna to assess the protection level of the vaccines with respect to lethal or latent infection challenge.ResultsIntramuscular, but not subcutaneous or intraperitoneal, administration of DC/HSV-1 vaccine provided complete protection against lethal challenge and establishment of latent infection as assessed by death and virus recovery from the trigeminal ganglia. It was also shown that the immunity was not associated with antibody production because DC/HSV-1 vaccine, as opposed to HSV-1/ALH vaccine, produced very little, if any, HSV-1-specific antibody.ConclusionsOverall, our results may have some impact on the design of vaccines against genital HSV as well as chronic viral infections such as hepatitis B virus, hepatitis C virus and human immunodeficiency virus.     

Varicella-zoster virus (VZV) ORF17 protein induces RNA cleavage and is critical for replication of VZV at 37 degrees C but not 33 degrees C

Varicella-zoster virus (VZV) open reading frame 17 (ORF17) is homologous to herpes simplex virus (HSV) UL41, which encodes the viral host shutoff protein (vhs). HSV vhs induces degradation of mRNA and rapid shutoff of host protein synthesis. An antibody to ORF17 protein detected a 46-kDa protein in VZV-infected cells. While HSV vhs is located in virions, VZV ORF17 protein was not detectable in virions. ORF17 protein induced RNA cleavage, but to a substantially lesser extent than HSV-1 vhs. Expression of ORF17 protein did not inhibit expression from a beta-galactosidase reporter plasmid, while HSV type 1 vhs abolished reporter expression. Two VZV ORF17 deletion mutants were constructed to examine the role of ORF17 in virus replication. While the ORF17 VZV mutants grew to peak titers that were similar to those of the parental virus at 33 degrees C, the ORF17 mutants grew to 20- to 35-fold-lower titers than parental virus at 37 degrees C. ORF62 protein was distributed in a different pattern in the nuclei and cytoplasm of cells infected with an ORF17 deletion mutant at 37 degrees C compared to 33 degrees C. Inoculation of cotton rats with the ORF17 deletion mutant resulted in a level of latent infection similar to that produced by inoculation with the parental virus. The importance of ORF17 protein for viral replication at 37 degrees C but not at 33 degrees C suggests that this protein may facilitate the growth of virus in certain tissues in vivo.     

The γ134.5 Protein of Herpes Simplex Virus 1 Is Required To Interfere with Dendritic Cell Maturation during Productive Infection

The γ₁34.5 protein of herpes simplex virus 1 is an essential factor for viral virulence. In infected cells, this viral protein prevents the translation arrest mediated by double-stranded RNA-dependent protein kinase R. Additionally, it associates with and inhibits TANK-binding kinase 1, an essential component of Toll-like receptor-dependent and -independent pathways that activate interferon regulatory factor 3 and cytokine expression. Here, we show that γ₁34.5 is required to block the maturation of conventional dendritic cells (DCs) that initiate adaptive immune responses. Unlike wild-type virus, the γ₁34.5 null mutant stimulates the expression of CD86, major histocompatibility complex class II (MHC-II), and cytokines such as alpha/beta interferon in immature DCs. Viral replication in DCs inversely correlates with interferon production. These phenotypes are also mirrored in a mouse ocular infection model. Further, DCs infected with the γ₁34.5 null mutant effectively activate naïve T cells whereas DCs infected with wild-type virus fail to do so. Type I interferon-neutralizing antibodies partially reverse virus-induced upregulation of CD86 and MHC-II, suggesting that γ₁34.5 acts through interferon-dependent and -independent mechanisms. These data indicate that γ₁34.5 is involved in the impairment of innate immunity by inhibiting both type I interferon production and DC maturation, leading to defective T-cell activation.Herpes simplex virus 1 (HSV-1) is a human pathogen responsible for localized mucocutaneous lesions and encephalitis (51). Following primary infection, HSV-1 establishes a latent or lytic infection in which the virus interacts with host cells, which include dendritic cells (DCs), required to initiate adaptive immune responses (36). Immature DCs, which reside in almost all peripheral tissues, are able to capture and process viral antigens (15). In this process, DCs migrate to lymph nodes, where they mature and present antigens to T cells. Mature DCs display high levels of major histocompatibility complex class II (MHC-II) and costimulatory molecules such as CD40, CD80, and CD86. Additionally, DCs release proinflammatory cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha, alpha interferon (IFN-α), and IFN-β, which promote DC maturation and activation. An important feature of functional DCs is to activate naïve T cells, and myeloid submucosal and lymph node resident DCs are responsible for HSV-specific T-cell activation (2, 45, 52). Moreover, DCs play a direct role in innate antiviral immunity by secreting type I IFN.HSV-1 is capable of infecting both immature and mature DCs (20, 24, 34, 38, 42). A previous study suggested that HSV entry into DCs requires viral receptors HVEM and nectin-2 (42). Upon HSV infection, plasmacytoid DCs detect viral genome through Toll-like receptor 9 (TLR9) and produce high levels of IFN-α (16, 23, 30, 40). In contrast, myeloid DCs, which are major antigen-presenting cells, recognize viral components through distinct pathways, independently of TLR9 (16, 36, 40). It has been suggested previously that HSV proteins or RNA intermediates produced during viral replication trigger myeloid DCs (16, 40). Indeed, a protein complex that consists of HSV glycoproteins B, D, H, and L stimulates the expression of CD40, CD83, CD86, and cytokines in myeloid DCs (41). Hence, DCs sense HSV through TLR-dependent and -independent mechanisms (16, 40, 41). Nevertheless, HSV replication compromises DC functions and subsequent T-cell activation (3, 20, 24, 42). HSV-1 interaction with immature DCs results in the downregulation of costimulatory molecules and cytokines (20, 34, 38, 42). While HSV-2 induces rapid apoptosis, HSV-1 does so with a delayed kinetics in human DCs (4, 20, 38). HSV-1 is also reported to interfere with functions of mature DCs (24, 39). Upon infection, HSV-1 induces the degradation of CD83 but not CD80 or CD86 in mature DCs (24, 25). Additionally, HSV-1 reduces levels of the chemokine receptors CCR7 and CXCR4 on mature DCs and subsequently impairs DC migration to the respective chemokine ligands CCL19 and CXCL12 (39).Although HSV infection impairs DC functions, viral components responsible for this impairment have not been thoroughly investigated. It has been suggested previously that the virion host shut-off protein (vhs) of HSV-1 contributes partially to the viral block of DC activation (43). This activity is thought to stem from the ability of vhs to destabilize host mRNA. Emerging evidence suggests that ICP0 perturbs the function of mature DCs, where it mediates CD83 degradation via cellular proteasomes (25). Findings from related studies show that ICP0 inhibits the induction of IFN-stimulated genes mediated by IFN regulatory factor 3 (IRF3) or IRF7 in other cell types (11, 27, 32, 33). However, the link of ICP0 activities to DC maturation remains to be established. Recently, we found that γ₁34.5, an HSV virulence factor, associates with and inhibits TANK-binding kinase 1 (TBK1), an essential component of TLR-dependent and -independent pathways that activates IRF3 and cytokine expression (49). Interestingly, an HSV mutant lacking γ₁34.5 stimulates MHC-II surface expression in glioblastoma cells (47). These observations raise the hypothesis that γ₁34.5 may modulate DC maturation during HSV infection.In this study, we report that γ₁34.5 is required to perturb DC maturation during HSV infection, leading to impaired T-cell activation. Wild-type virus, but not the γ₁34.5 null mutant, suppresses the expression of costimulatory molecules as well as cytokines in DCs. We provide evidence that the viral block of DC maturation is associated with reduced IFN-α/β secretion. Furthermore, the expression of γ₁34.5 inhibits DC-mediated allogeneic T-cell activation and IFN-γ production. IFN-neutralizing antibodies partially reverse DC maturation induced by the γ₁34.5 null mutant. These results shed light on the role of γ₁34.5 relevant to DC maturation and T-cell responses in HSV infection.     

Identification of dendritic cell subsets responding to genital infection by Chlamydia muridarum

Dendritic cells (DCs) are central for the induction of T-cell responses needed for chlamydial eradication. Here, we report the activation of two DC subsets: a classical CD11b+ (cDC) and plasmacytoid (pDC) during genital infection with Chlamydia muridarum . Genital infection induced an influx of cDC and pDC into the genital tract and its draining lymph node (iliac lymph nodes, ILN) as well as colocalization with T cells in the ILN. Genital infection with C. muridarum also stimulated high levels of costimulatory molecules on cDC central for the activation of naïve T cells in vivo . In contrast, pDC expressed low levels of most costimulatory molecules in vivo and did not secrete cytokines associated with the production of T helper (Th)1 cells in vitro . However, pDC upregulated inducible costimulatory ligand expression and produced IL-6 and IL-10 in response to chlamydial exposure in vitro . Our findings show that these two DC subsets likely have different functions in vivo . cDCs are prepared for induction of antichlamydial T-cell responses, whereas pDCs have characteristics associated with the differentiation of non-Th1 cell subsets.     

Expression and function of the equine herpesvirus 1 virion-associated host shutoff homolog.

The ability of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2, respectively) to repress host cell protein synthesis early in infection has been studied extensively and found to involve the activities of the UL41 gene product, the virion-associated host shutoff (vhs) protein. To date, UL41 homologs have been identified in the genomes of three other alphaherpesviruses: equine herpesvirus 1 (EHV-1), varicella-zoster virus, and pseudorabies virus, but very little is known about the putative products of these homologous genes. Our earlier observations that no rapid early host protein shutoff occurred in EHV-1-infected cells led us to test EHV-1 vhs activity more thoroughly and to examine the expression and function of the EHV-1 UL41 homolog, ORF19. In the present study, the effects of EHV-1 and HSV-1 infections on cellular protein synthesis and mRNA degradation were compared at various multiplicities of infection in several cell types under an actinomycin D block. No virion-associated inhibition of cellular protein synthesis or vhs-induced cellular mRNA degradation was detected in cells infected with any of three EHV-1 strains (Ab4, KyA, and KyD) at multiplicities of infection at which HSV-1 strain F exhibited maximal vhs activity. However, further analyses revealed that (i) the EHV-1 vhs homolog gene, ORF19, was transcribed and translated into a 58-kDa protein in infected cells; (ii) the ORF19 protein was packaged into viral particles in amounts detectable in Western blots (immunoblots) with monoclonal antibodies; (iii) in cotransfection vhs activity assays, transiently-expressed ORF19 protein had intrinsic vhs activity comparable to that of wild-type HSV-1 vhs; and (iv) this intrinsic vhs activity was ablated by in vitro site-directed mutations in which either the functionally inactive HSV-1 vhs1 UL41 mutation (Thr at position 214 replaced by Ile [Thr-214-->Ile]) was recreated within ORF19 or two conserved residues within the putative poly(A) binding region of the ORF19 sequence were altered (Tyr-190, 192-->Phe). From these results we conclude that EHV-1's low vhs activity in infected cells is not a reflection of the ORF19 protein's intrinsic vhs activity but may be due instead to the amount of ORF19 protein associated with viral particles or to modulation of ORF19 protein's intrinsic activity by another viral component(s).     

The herpes simplex virus virion host shutoff function.

The virion host shutoff (vhs) function of herpes simplex virus (HSV) limits the expression of genes in the infected cells by destabilizing both host and viral mRNAs. vhs function mutants have been isolated which are defective in their ability to degrade host mRNA. Furthermore, the half-life of viral mRNAs is significantly longer in cells infected with the vhs-1 mutant virus than in cells infected with the wild-type (wt) virus. Recent data have shown that the vhs-1 mutation resides within the open reading frame UL41. We have analyzed the shutoff of host protein synthesis in cells infected with a mixture of the wt HSV-1 (KOS) and the vhs-1 mutant virus. The results of these experiments revealed that (i) the wt virus shutoff activity requires a threshold level of input virions per cell and (ii) the mutant vhs-1 virus protein can irreversibly block the wt virus shutoff activity. These results are consistent with a stoichiometric model in which the wt vhs protein interacts with a cellular factor which controls the half-life of cell mRNA. This wt virus interaction results in the destabilization of both host and viral mRNAs. In contrast, the mutant vhs function interacts with the cellular factor irreversibly, resulting in the increased half-life of both host and viral mRNAs.     

Dendritic Cell Subtypes from Lymph Nodes and Blood Show Contrasted Gene Expression Programs upon Bluetongue Virus Infection

Human and animal hemorrhagic viruses initially target dendritic cells (DCs). It has been proposed, but not documented, that both plasmacytoid DCs (pDCs) and conventional DCs (cDCs) may participate in the cytokine storm encountered in these infections. In order to evaluate the contribution of DCs in hemorrhagic virus pathogenesis, we performed a genome-wide expression analysis during infection by Bluetongue virus (BTV), a double-stranded RNA virus that induces hemorrhagic fever in sheep and initially infects cDCs. Both pDCs and cDCs accumulated in regional lymph nodes and spleen during BTV infection. The gene response profiles were performed at the onset of the disease and markedly differed with the DC subtypes and their lymphoid organ location. An integrative knowledge-based analysis revealed that blood pDCs displayed a gene signature related to activation of systemic inflammation and permeability of vasculature. In contrast, the gene profile of pDCs and cDCs in lymph nodes was oriented to inhibition of inflammation, whereas spleen cDCs did not show a clear functional orientation. These analyses indicate that tissue location and DC subtype affect the functional gene expression program induced by BTV and suggest the involvement of blood pDCs in the inflammation and plasma leakage/hemorrhage during BTV infection in the real natural host of the virus. These findings open the avenue to target DCs for therapeutic interventions in viral hemorrhagic diseases.     

Ebolavirus VP35 suppresses IFN production from conventional but not plasmacytoid dendritic cells

Ebolaviruses naturally infect a wide variety of cells including macrophages and dendritic cells (DCs), and the resulting cytokine and interferon-α/β (IFN) responses of infected cells are thought to influence viral pathogenesis. The VP35 protein impairs RIG-I-like receptor-dependent signaling to inhibit IFN production, and this function has been suggested to promote the ineffective host immune response characteristic of ebolavirus infection. To assess the impact of VP35 on innate immunity in biologically relevant primary cells, we used a recombinant Newcastle disease virus encoding VP35 (NDV/VP35) to infect macrophages and conventional DCs, which primarily respond to RNA virus infection via RIG-I-like pathways. VP35 suppressed not only IFN but also tumor necrosis factor (TNF)-α secretion, which are normally produced from these cells upon NDV infection. Additionally, in cells susceptible to the activity of VP35, IRF7 activation is impaired. In contrast, NDV/VP35 infection of plasmacytoid DCs, which activate IRF7 and produce IFN through TLR-dependent signaling, leads to robust IFN production. When plasmacytoid DCs deficient for TLR signaling were infected, NDV/VP35 was able to inhibit IFN production. Consistent with this, VP35 was less able to inhibit TLR-dependent versus RIG-I-dependent signaling in vitro. These data demonstrate that ebolavirus VP35 suppresses both IFN and cytokine production in multiple primary human cell types. However, cells that utilize the TLR pathway can circumvent this inhibition, suggesting that the presence of multiple viral sensors enables the host to overcome viral immune evasion mechanisms.