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.

During the last few decades, the global incidence of dengue virus (DENV) has increased dramatically, and it is now endemic in more than 100 countries. To establish a productive infection in humans, DENV uses different strategies to inhibit or avoid the host innate immune system. Several DENV proteins have been shown to strategically target crucial components of the type I interferon system. Here, we report that the DENV NS2B protease cofactor targets the DNA sensor cyclic GMP-AMP synthase (cGAS) for lysosomal degradation to avoid the detection of mitochondrial DNA during infection. Such degradation subsequently results in the inhibition of type I interferon production in the infected cell. Our data demonstrate a mechanism by which cGAS senses cellular damage upon DENV infection.

Summary Viral protein homeostasis depends entirely on the machinery of the infected cell. Accordingly, viruses can illuminate the interplay between cellular proteostasis components and their distinct substrates. Here we define how the Hsp70 chaperone network mediates the dengue virus life cycle. Cytosolic Hsp70 isoforms are required at distinct steps of the viral cycle, including entry, RNA replication and virion biogenesis. Hsp70 function at each step is specified by nine distinct DNAJ cofactors. Of these, DnaJB11 relocalizes to virus-induced replication complexes to promote RNA synthesis, while DnaJB6 associates with capsid protein and facilitates virion biogenesis. Importantly, an allosteric Hsp70 inhibitor, JG40, potently blocks infection of different dengue serotypes in human primary blood cells without eliciting viral resistance or exerting toxicity to the host cells. JG40 also blocks replication of other medically-important flaviviruses including yellow fever, West Nile and Japanese encephalitis viruses. Thus, targeting host Hsp70 subnetworks provides a path for broad-spectrum antivirals.

Summary 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.

Dengue virus (DENV) is the most prevalent arthropod-borne human virus, able to infect and replicate in human dendritic cells (DCs), inducing their activation and the production of proinflammatory cytokines. However, DENV can successfully evade the immune response in order to produce disease in humans. Several mechanisms of immune evasion have been suggested for DENV, most of them involving interference with type I interferon (IFN) signaling. We recently reported that DENV infection of human DCs does not induce type I IFN production by those infected DCs, impairing their ability to prime naive T cells toward Th1 immunity. In this article, we report that DENV also reduces the ability of DCs to produce type I IFN in response to several inducers, such as infection with other viruses or exposure to Toll-like receptor (TLR) ligands, indicating that DENV antagonizes the type I IFN production pathway in human DCs. DENV-infected human DCs showed a reduced type I IFN response to Newcastle disease virus (NDV), Sendai virus (SeV), and Semliki Forest virus (SFV) infection and to the TLR3 agonist poly(I:C). This inhibitory effect is DENV dose dependent, requires DENV replication, and takes place in DENV-infected DCs as early as 2 h after infection. Expressing individual proteins of DENV in the presence of an IFN-␣/␤ production inducer reveals that a catalytically active viral protease complex is required to reduce type I IFN production significantly. These results provide a new mechanism by which DENV evades the immune system in humans.Dengue virus (DENV), a member of the Flaviviridae family and grouped within the Flavivirus genus (27), is the most prevalent arthropod-borne human virus with significant medical and biodefense importance (8,27). DENV is transmitted by mosquitoes, usually Aedes aegypti, with an estimated 100 million cases per year and 2.5 billion people at risk (53). Clinical manifestations include dengue fever, a febrile illness with rash, and dengue hemorrhagic fever, a severe and often lethal illness (53). There are four DENV serotypes (DENV1 to -4), and infection with one serotype confers life-long protection against that serotype at least by the induction of neutralizing antibodies (17). Currently there is no vaccine or effective antiviral treatment against this virus, and the most effective protective measures involve mosquito control.The DENV genome is a positive-strand RNA molecule of about 11 kb in length, with one single open reading frame (ORF) flanked by 5Ј and 3Ј nontranslated regions (27). After viral infection and the release of the viral nucleocapsid into the cytosol, a 3,391-amino-acid (aa)-long polyprotein is translated from the viral RNA at the surface of the endoplasmic reticulum (ER) (27). This polyprotein is cleaved into three structural (C, prM, and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins due to the combined and coordinated activities of host cell proteases in the ER and the viral protease complex (NS2B3) in the cytoplasm (27). The viral protease cle...

Dengue virus (DENV) infects human immune cells in vitro and likely infects dendritic cells (DCs) in vivo.DENV-2 productive infection induces activation and release of high levels of chemokines and proinflammatory cytokines in monocyte-derived DCs (moDCs), with the notable exception of alpha/beta interferon (IFN-␣/␤). Interestingly, DENV-2-infected moDCs fail to prime T cells, most likely due to the lack of IFN-␣/␤ released by moDCs, since this effect was reversed by addition of exogenous IFN-␤. Together, our data show that inhibition of IFN-␣/␤ production by DENV in primary human moDCs is a novel mechanism of immune evasion.Dengue virus (DENV) belongs to the family Flaviviridae and has great importance in the areas of medicine and biodefense (3,17). Transmitted by Aedes aegypti, DENV is the most prevalent arthropod-borne human virus (34). Generally, infected patients experience dengue fever, but 2 to 20% of cases manifest dengue hemorrhagic fever (DHF), a severe and often lethal illness (34). Dendritic cells (DCs) initiate immune responses and become activated upon contact with pathogens (2), with upregulation of costimulatory molecules and release of proinflammatory cytokines (2). Secretion of type I interferon (alpha/beta interferon [IFN-␣/␤]) by DCs contributes to the generation of antiviral innate and adaptive immune responses (7,16,18,32). DCs, together with other cell populations, have been suggested as target cells for DENV infection (12,14,15,35).We investigated the replication of DENV-2 in monocytederived DCs (moDCs) obtained by culturing CD14 ϩ cells, isolated from blood of healthy donors, for 6 days in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 4 (IL-4), and human serum (9). The purity obtained by flow cytometry was routinely 96 to 99% CD14 Ϫ CD11c ϩ HLA-DR low moDCs (7). DENV-2 (16681 and NGC strains) and DENV-3 (Hawaii strain) were grown in C6/36 insect cells (5) and were titrated by plaque assay (4). moDCs were infected with DENV-2 at different multiplicities of infection (MOIs), and viral replication was tested by quantitative reverse transcription-PCR (qRT-PCR) (9, 28). All infections in this study were performed in moDCs from at least three independent donors with three replicates per sample. DENV-2 replication peaked at 48 h after infection (Fig. 1A), and release of newly synthesized infectious particles had a similar kinetics (Fig. 1B), indicating productive infection of moDCs. As a measure of moDC activation, proinflammatory cytokine levels (IL-6 and tumor necrosis factor alpha [TNF-␣]) were quantified by enzyme-linked immunosorbent assay (ELISA) 24 h after DENV-2 infection (Fig. 1C). We observed high expression of those cytokines, comparable to cytokine expression after poly(I:C) treatment of moDCs. Interestingly, UV-inactivated DENV-2 did not induce the moDC activation, suggesting that DENV-2 replication is required to activate moDCs. Infection with influenza virus A/PR8/33 (PR8) showed low levels of cytokine induction due to the activity of the N...

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Chikungunya virus (CHIKV) is a mosquito-borne alphavirus known to cause epidemics resulting in predominantly symptomatic infections, which in rare cases cause long term debilitating arthritis and arthralgia. Significant progress has been made in understanding the roles of canonical RNA sensing pathways in the host recognition of CHIKV; however, less is known regarding antagonism of CHIKV by cytosolic DNA sensing pathways like that of cyclic GMP-AMP synthase (cGAS) and Stimulator of Interferon Genes (STING). With the use of cGAS or STING null cells we demonstrate that the pathway restricts CHIKV replication in fibroblasts and immune cells. We show that DNA accumulates in the cytoplasm of infected cells and that CHIKV blocks DNA dependent IFN-β transcription. This antagonism of DNA sensing is via an early autophagy-mediated degradation of cGAS and expression of the CHIKV capsid protein is sufficient to induce cGAS degradation. Furthermore, we identify an interaction of CHIKV nsP1 with STING and map the interaction to 23 residues in the cytosolic loop of the adaptor protein. This interaction stabilizes the viral protein and increases the level of palmitoylated nsP1 in cells. Together, this work supports previous publications highlighting the relevance of the cGAS-STING pathway in the early detection of (+)ssRNA viruses and provides direct evidence that CHIKV interacts with and antagonizes cGAS-STING signaling.