RIG-I, MDA5 and TLR3 synergistically play an important role in restriction of dengue virus infection

Dengue virus (DV) infection is one of the most common mosquito-borne viral diseases in the world. The innate immune system is important for the early detection of virus and for mounting a cascade of defense measures which include the production of type 1 interferon (IFN). Hence, a thorough understanding of the innate immune response during DV infection would be essential for our understanding of the DV pathogenesis. A recent application of the microarray to dengue virus type 1 (DV1) infected lung carcinoma cells revealed the increased expression of both extracellular and cytoplasmic pattern recognition receptors; retinoic acid inducible gene-I (RIG-I), melanoma differentiation associated gene-5 (MDA-5) and Toll-like receptor-3 (TLR3). These intracellular RNA sensors were previously reported to sense DV infection in different cells. In this study, we show that they are collectively involved in initiating an effective IFN production against DV. Cells silenced for these genes were highly susceptible to DV infection. RIG-I and MDA5 knockdown HUH-7 cells and TLR3 knockout macrophages were highly susceptible to DV infection. When cells were silenced for only RIG-I and MDA5 (but not TLR3), substantial production of IFN-β was observed upon virus infection and vice versa. High susceptibility to virus infection led to ER-stress induced apoptosis in HUH-7 cells. Collectively, our studies demonstrate that the intracellular RNA virus sensors (RIG-I, MDA5 and TLR3) are activated upon DV infection and are essential for host defense against the virus.     

Human skin Langerhans cells are targets of dengue virus infection

Dengue virus (DV), an arthropod-borne flavivirus, causes a febrile illness for which there is no antiviral treatment and no vaccine. Macrophages are important in dengue pathogenesis; however, the initial target cell for DV infection remains unknown. As DV is introduced into human skin by mosquitoes of the genus Aedes, we undertook experiments to determine whether human dendritic cells (DCs) were permissive for the growth of DV. Initial experiments demonstrated that blood-derived DCs were 10-fold more permissive for DV infection than were monocytes or macrophages. We confirmed this with human skin DCs (Langerhans cells and dermal/interstitial DCs). Using cadaveric human skin explants, we exposed skin DCs to DV ex vivo. Of the human leukocyte antigen DR-positive DCs that migrated from the skin, emigrants from both dermis and epidermis, 60-80% expressed DV antigens. These observations were supported by histologic findings from the skin rash of a human subject who received an attenuated tetravalent dengue vaccine. Immunohistochemistry of the skin showed CD1a-positive DCs double-labeled with an antibody against DV envelope glycoprotein. These data demonstrate that human skin DCs are permissive for DV infection, and provide a potential mechanism for the transmission of DV into human skin.     

Phagocytosis of picornavirus-infected cells induces an RNA-dependent antiviral state in human dendritic cells

Dendritic cells (DCs) play a central role in instructing antiviral immune responses. DCs, however, can become targeted by different viruses themselves. We recently demonstrated that human DCs can be productively infected with echoviruses (EVs), but not coxsackie B viruses (CVBs), both of which are RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. We now show that phagocytosis of CVB-infected, type I interferon-deficient cells induces an antiviral state in human DCs. Uptake of infected cells increased the expression of the cytoplasmic RNA helicases retinoic acid-inducible gene I and melanoma differentiation-associated gene 5 as well as other interferon-stimulated genes and protected DCs against subsequent infection with EV9. These effects depended on recognition of viral RNA and could be mimicked by exposure to the synthetic double-stranded RNA analogue poly(I:C) but not other Toll-like receptor (TLR) ligands. Blocking endosomal acidification abrogated protection, suggesting a role for TLRs in the acquisition of an antiviral state in DCs. In conclusion, recognition of viral RNA rapidly induces an antiviral state in human DCs. This might provide a mechanism by which DCs protect themselves against viruses when attracted to an environment with ongoing infection.     

Up‐regulation of galectin‐9 induces cell migration in human dendritic cells infected with dengue virus

Galectin‐9 (Gal‐9) exerts immunosuppressive effects by inducing apoptosis in T cells that produce interferon‐γ and interleukin (IL)‐17. However, Gal‐9 can be pro‐inflammatory in lipopolysaccharide‐stimulated monocytes. Using microarray analysis, we observed that Gal‐9 was up‐regulated in human dendritic cells (DCs) after dengue virus (DV) infection. The investigation into the immunomodulatory effects and mechanisms of Gal‐9 in DCs exposed to DV revealed that DV infection specifically increased mRNA and protein levels of Gal‐9 but not those of Gal‐1 or Gal‐3. Blocking p38, but not c‐Jun N‐terminal kinase or extracellular signal‐regulated kinase (ERK), inhibited DV‐induced expression of Gal‐9. Reduction in Gal‐9 by small interference RNA treatment suppressed DV‐stimulated migration of DCs towards the chemoattractants CCL19 and CCL21. In addition, DV‐induced IL‐12p40 production was reduced after knockdown of Gal‐9 in DCs. Furthermore, Gal‐9 deficiency suppressed DV‐induced activation of nuclear factor‐κB. Inhibition of DV‐induced DC migration under conditions of Gal‐9 deficiency was mediated through suppressing ERK activation but not by regulating the expression of CCR7, the receptor for CCL19 and CCL21. Both the reduction in IL‐12 production and the suppression of ERK activity might account for the inhibition of DV‐induced DC migration after knockdown of Gal‐9. In summary, this study reveals the roles of Gal‐9 in DV‐induced migration of DCs. The findings indicate that Gal‐9 might be a therapeutic target for preventing immunopathogenesis induced by DV infection.     

The nitric oxide pathway provides innate antiviral protection in conjunction with the type I interferon pathway in fibroblasts

The innate host response to virus infection is largely dominated by the production of type I interferon and interferon stimulated genes. In particular, fibroblasts respond robustly to viral infection and to recognition of viral signatures such as dsRNA with the rapid production of type I interferon; subsequently, fibroblasts are a key cell type in antiviral protection. We recently found, however, that primary fibroblasts deficient for the production of interferon, interferon stimulated genes, and other cytokines and chemokines mount a robust antiviral response against both DNA and RNA viruses following stimulation with dsRNA. Nitric oxide is a chemical compound with pleiotropic functions; its production by phagocytes in response to interferon-γ is associated with antimicrobial activity. Here we show that in response to dsRNA, nitric oxide is rapidly produced in primary fibroblasts. In the presence of an intact interferon system, nitric oxide plays a minor but significant role in antiviral protection. However, in the absence of an interferon system, nitric oxide is critical for the protection against DNA viruses. In primary fibroblasts, NF-κB and interferon regulatory factor 1 participate in the induction of inducible nitric oxide synthase expression, which subsequently produces nitric oxide. As large DNA viruses encode multiple and diverse immune modulators to disable the interferon system, it appears that the nitric oxide pathway serves as a secondary strategy to protect the host against viral infection in key cell types, such as fibroblasts, that largely rely on the type I interferon system for antiviral protection.     

Differential effects of dengue virus on infected and bystander dendritic cells

Dendritic cells (DCs) play a central role as major targets of dengue virus (DV) infections and initiators of antiviral immune responses. Previous observations showed that DCs are activated by infection, presumably acquiring the capacity to promote cell-mediated immunity. However, separate evaluations of the maturation profiles of infected and uninfected bystander cells show that infection impairs the ability of DCs to upregulate cell surface expression of costimulatory, maturation, and major histocompatibility complex molecules, resulting in reduced T-cell stimulatory capacity. Infected DCs failed to respond to tumor necrosis factor alpha as an additional maturation stimulus and were apoptotic. Interleukin 10 (IL-10) was detected in supernatants from cultures of DV-infected DCs and cocultures of DCs and T cells. Taken together, these results constitute an immune evasion strategy used by DV that directly impairs antigen-presenting cell function by maturation blockade and induction of apoptosis.     

The herpes simplex virus ICP0 RING finger domain inhibits IRF3- and IRF7-mediated activation of interferon-stimulated genes

Virus infection induces a rapid cellular response in cells characterized by the induction of interferon. While interferon itself does not induce an antiviral response, it activates a number of interferon-stimulated genes that collectively function to inhibit virus replication and spread. Previously, we and others reported that herpes simplex virus type 1 (HSV-1) induces an interferon -independent antiviral response in the absence of virus replication. Here, we report that the HSV-1 proteins ICP0 and vhs function in concert to disable the host antiviral response. In particular, we show that ICP0 blocks interferon regulatory factor IRF3- and IRF7-mediated activation of interferon-stimulated genes and that the RING finger domain of ICP0 is essential for this activity. Furthermore, we demonstrate that HSV-1 modifies the IRF3 pathway in a manner different from that of the small RNA viruses most commonly studied.     

Human MxA Protein Confers Resistance to Semliki Forest Virus and Inhibits the Amplification of a Semliki Forest Virus-Based Replicon in the Absence of Viral Structural Proteins

Mx proteins form a small family of interferon (IFN)-induced GTPases with potent antiviral activity against various negative-strand RNA viruses. To examine the antiviral spectrum of human MxA in homologous cells, we stably transfected HEp-2 cells with a plasmid directing the expression of MxA cDNA. HEp-2 cells are permissive for many viruses and are unable to express endogenous MxA in response to IFN. Experimental infection with various RNA and DNA viruses revealed that MxA-expressing HEp-2 cells were protected not only against influenza virus and vesicular stomatitis virus (VSV) but also against Semliki Forest virus (SFV), a togavirus with a single-stranded RNA genome of positive polarity. In MxA-transfected cells, viral yields were reduced up to 1,700-fold, and the degree of inhibition correlated well with the expression level of MxA. Furthermore, expression of MxA prevented the accumulation of 49S RNA and 26S RNA, indicating that SFV was inhibited early in its replication cycle. Very similar results were obtained with MxA-transfected cells of the human monocytic cell line U937. The results demonstrate that the antiviral spectrum of MxA is not restricted to negative-strand RNA viruses but also includes SFV, which contains an RNA genome of positive polarity. To test whether MxA protein exerts its inhibitory activity against SFV in the absence of viral structural proteins, we took advantage of a recombinant vector based on the SFV replicon. The vector contains only the coding sequence for the viral nonstructural proteins and the bacterial LacZ gene, which was cloned in place of the viral structural genes. Upon transfection of vector-derived recombinant RNA, expression of the β-galactosidase reporter gene was strongly reduced in the presence of MxA. This finding indicates that viral components other than the structural proteins are the target of MxA action.     

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2′-5′-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.     

The interferon-inducible RNA helicase, mda-5, is involved in measles virus-induced expression of antiviral cytokines

Activation of host cell antiviral responses is mediated by receptors detecting the presence of viruses. Here we have studied the role of double-stranded RNA (dsRNA) binding molecules melanoma differentiation-associated gene 5 (mda-5), retinoic acid inducible gene I (RIG-I), and Toll-like receptor 3 (TLR3) in measles virus (MV)-induced expression of antiviral cytokines and chemokines in human A549 lung epithelial cells and human umbilical vein endothelial cells (HUVECs). We show that MV infection results in the activation of mda-5, RIG-I, and TLR3 gene expression that is followed by high expression of interferon (IFN)-beta, interleukin (IL)-28 and IL-29, CCL5, and CXCL10 genes. We also demonstrate that IFN-alpha and IFN-beta upregulate mda-5, RIG-I, and TLR3 gene expression in epithelial and endothelial cell lines. Forced expression of mda-5, but not that of RIG-I or TLR3, leads to enhanced IFN-beta promoter activity in MV-infected A549 cells. Our results suggest that IFN-inducible mda-5 is involved in MV-induced expression of antiviral cytokines.     

Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses

Hantaviruses represent important human pathogens and can induce hemorrhagic fever with renal syndrome (HFRS), which is characterized by endothelial dysfunction. Both pathogenic and nonpathogenic hantaviruses replicate without causing any apparent cytopathic effect, suggesting that immunopathological mechanisms play an important role in pathogenesis. We compared the antiviral responses triggered by Hantaan virus (HTNV), a pathogenic hantavirus associated with HFRS, and Tula virus (TULV), a rather nonpathogenic hantavirus, in human umbilical vein endothelial cells (HUVECs). Both HTNV- and TULV-infected cells showed increased levels of molecules involved in antigen presentation. However, TULV-infected HUVECs upregulated HLA class I molecules more rapidly. Interestingly, HTNV clearly induced the production of beta interferon (IFN-beta), whereas expression of this cytokine was barely detectable in the supernatant or in extracts from TULV-infected HUVECs. Nevertheless, the upregulation of HLA class I on both TULV- and HTNV-infected cells could be blocked by neutralizing anti-IFN-beta antibodies. Most strikingly, the antiviral MxA protein, which interferes with hantavirus replication, was already induced 16 h after infection with TULV. In contrast, HTNV-infected HUVECs showed no expression of MxA until 48 h postinfection. In accordance with the kinetics of MxA expression, TULV replicated only inefficiently in HUVECs, whereas HTNV-infected cells produced high titers of virus particles that decreased after 48 h postinfection. Both hantavirus species, however, could replicate equally well in Vero E6 cells, which lack an IFN-induced MxA response. Thus, delayed induction of antiviral MxA in endothelial cells after infection with HTNV could allow viral dissemination and contribute to the pathogenesis leading to HFRS.     

Severe impairment of dendritic cell allostimulatory activity by Sendai virus vectors is overcome by matrix protein gene deletion

Delivery of Ags to dendritic cells (DCs) plays a pivotal role in the induction of efficient immune responses ranging from immunity to tolerance. The observation that certain viral pathogens are able to infect DCs has led to a concept in which applications of recombinant viruses are used for Ag delivery with the potential benefit of inducing potent Ag-specific T cell responses directed against multiple epitopes. As a prerequisite for such an application, the infection of DCs by recombinant viruses should not interfere with their stimulatory capacity. In this context, we could show that an emerging negative-strand RNA viral vector system based on the Sendai virus (SeV) is able to efficiently infect monocyte-derived human DCs (moDCs). However, after infection with SeV wild type, both the response of DCs to bacterial LPS as a powerful mediator of DC maturation and the allostimulatory activity were severely impaired. Interestingly, using various recombinant SeV vectors that were devoid of single viral genes, we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, use of M-deficient SeV vectors preserved the allostimulatory activity in infected moDCs despite an efficient expression of all other virally encoded genes, thereby identifying M-deficient vectors as a highly potent tool for the genetic manipulation of DCs.