Critical roles for both STAT1-dependent and STAT1-independent pathways in the control of primary dengue virus infection in mice

Dengue virus (DEN), a flavivirus, causes dengue fever and dengue hemorrhagic fever/dengue shock syndrome, the most common mosquito-borne viral illnesses in humans worldwide. In this study, using STAT1(-/-) mice bearing two different mutant stat1 alleles in the 129/Sv/Ev background, we demonstrate that IFNR-dependent control of primary DEN infection involves both STAT1-dependent and STAT1-independent mechanisms. The STAT1 pathway is necessary for clearing the initial viral load, whereas the STAT1-independent pathway controls later viral burden and prevents DEN disease in mice. The STAT1-independent responses in mice with primary DEN infection included the early activation of B and NK cells as well as the up-regulation of MHC class I molecules on macrophages and dendritic cells. Infection of bone marrow-derived dendritic cell cultures with either DEN or Sindbis virus, another positive-strand RNA virus, confirmed the early vs late natures of the STAT1-dependent and STAT1-independent pathways. Collectively, these data begin to define the nature of the STAT1-dependent vs the STAT1-independent pathway in vivo.     

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.     

Maternal Antibody-Mediated Disease Enhancement in Type I Interferon-Deficient Mice Leads to Lethal Disease Associated with Liver Damage

Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals’ death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and therapeutic candidates.     

Dengue Virus Co-opts UBR4 to Degrade STAT2 and Antagonize Type I Interferon Signaling

An estimated 50 million dengue virus (DENV) infections occur annually and more than forty percent of the human population is currently at risk of developing dengue fever (DF) or dengue hemorrhagic fever (DHF). Despite the prevalence and potential severity of DF and DHF, there are no approved vaccines or antiviral therapeutics available. An improved understanding of DENV immune evasion is pivotal for the rational development of anti-DENV therapeutics. Antagonism of type I interferon (IFN-I) signaling is a crucial mechanism of DENV immune evasion. DENV NS5 protein inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation. Only proteolytically-processed NS5 can efficiently mediate STAT2 degradation, though both unprocessed and processed NS5 bind STAT2. Here we identify UBR4, a 600-kDa member of the N-recognin family, as an interacting partner of DENV NS5 that preferentially binds to processed NS5. Our results also demonstrate that DENV NS5 bridges STAT2 and UBR4. Furthermore, we show that UBR4 promotes DENV-mediated STAT2 degradation, and most importantly, that UBR4 is necessary for efficient viral replication in IFN-I competent cells. Our data underscore the importance of NS5-mediated STAT2 degradation in DENV replication and identify UBR4 as a host protein that is specifically exploited by DENV to inhibit IFN-I signaling via STAT2 degradation.     

Mouse STAT2 restricts early dengue virus replication

Dengue virus encodes several interferon antagonists. Among these the NS5 protein binds STAT2, a necessary component of the type I interferon signaling pathway, and targets it for degradation. We now demonstrate that the ability of dengue NS5 to associate with and degrade STAT2 is species specific. Thus, NS5 is able to bind and degrade human STAT2, but not mouse STAT2. This difference was exploited to demonstrate, absent manipulation of the viral genome, that NS5-mediated IFN antagonism is essential for efficient virus replication. Moreover, we demonstrate that differences in NS5 mediated binding and degradation between human and mouse STAT2 maps to a region within the STAT2 coiled-coil domain. By using STAT2(-/-) mice, we also demonstrate that mouse STAT2 restricts early dengue virus replication in?vivo. These results suggest that overcoming this restriction through transgenic mouse technology may help in the development of a long-sought immune-competent mouse model of dengue virus infection.     

Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8⁺ T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8⁺ T cells in preventing DENV-induced CNS disease.     

DENV Inhibits Type I IFN Production in Infected Cells by Cleaving Human STING

Dengue virus (DENV) is a pathogen with a high impact on human health. It replicates in a wide range of cells involved in the immune response. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response. DENV circumvents the host immune response by expressing proteins that antagonize the cellular innate immunity. We have recently documented the inhibition of type I IFN production by the proteolytic activity of DENV NS2B3 protease complex in human monocyte derived dendritic cells (MDDCs). In the present report we identify the human adaptor molecule STING as a target of the NS2B3 protease complex. We characterize the mechanism of inhibition of type I IFN production in primary human MDDCs by this viral factor. Using different human and mouse primary cells lacking STING, we show enhanced DENV replication. Conversely, mutated versions of STING that cannot be cleaved by the DENV NS2B3 protease induced higher levels of type I IFN after infection with DENV. Additionally, we show that DENV NS2B3 is not able to degrade the mouse version of STING, a phenomenon that severely restricts the replication of DENV in mouse cells, suggesting that STING plays a key role in the inhibition of DENV infection and spread in mice.     

Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins

Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I–like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.     

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.     

Adaptor Protein 1A Facilitates Dengue Virus Replication

Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication.     

ADAP2 Is an Interferon Stimulated Gene That Restricts RNA Virus Entry

Interferon stimulated genes (ISGs) target viruses at various stages of their infectious life cycles, including at the earliest stage of viral entry. Here we identify ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2) as a gene upregulated by type I IFN treatment in a STAT1-dependent manner. ADAP2 functions as a GTPase-activating protein (GAP) for Arf6 and binds to phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃) and PI(3,4)P₂. We show that overexpression of ADAP2 suppresses dengue virus (DENV) and vesicular stomatitis virus (VSV) infection in an Arf6 GAP activity-dependent manner, while exerting no effect on coxsackievirus B (CVB) or Sendai virus (SeV) replication. We further show that ADAP2 expression induces macropinocytosis and that ADAP2 strongly associates with actin-enriched membrane ruffles and with Rab8a- and LAMP1-, but not EEA1- or Rab7-, positive vesicles. Utilizing two techniques—light-sensitive neutral red (NR)-containing DENV and fluorescence assays for virus internalization—we show that ADAP2 primarily restricts DENV infection at the stage of virion entry and/or intracellular trafficking and that incoming DENV and VSV particles associate with ADAP2 during their entry. Taken together, this study identifies ADAP2 as an ISG that exerts antiviral effects against RNA viruses by altering Arf6-mediated trafficking to disrupt viral entry.     

Distinct requirements for IFNs and STAT1 in NK cell function

NK cell functions were examined in mice with a targeted mutation of the STAT1 gene, an essential mediator of IFN signaling. Mice deficient in STAT1 displayed impaired basal NK cytolytic activity in vitro and were unable to reject transplanted tumors in vivo, despite the presence of normal numbers of NK cells. IL-12 enhanced NK-mediated cytolysis, but poly(I:C) did not, and a similar phenotype occurred in mice lacking IFNalpha receptors. Molecules involved in activation and lytic function of NK cells (granzyme A, granzyme B, perforin, DAP10, and DAP12) were expressed at comparable levels in both wild-type and STAT1(-/-) mice, and serine esterase activity necessary for CTL function was normal, showing that the lytic machinery was intact. NK cells with normal cytolytic activity could be derived from STAT1(-/-) bone marrow progenitors in response to IL-15 in vitro, and enhanced NK lytic activity and normal levels of IFN-gamma were produced in response to IL-12 treatment in vivo. Despite these normal responses to cytokines, STAT1(-/-) mice could not reject the NK-sensitive tumor RMA-S, even following IL-12 treatment in vivo. Whereas in vitro NK cytolysis was also reduced in mice lacking both type I and type II IFN receptors, these mice resisted tumor challenge. These results demonstrate that both IFN-alpha and IFN-gamma are required to maintain NK cell function and define a STAT1-dependent but partially IFN-independent pathway required for NK-mediated antitumor activity.