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The phosphatase Cdc25A plays an important role in cell cycle regulation by dephosphorylating its substrates, such as cyclin-dependent kinases. In this study, we demonstrate that Cdc25A negatively regulates RIG-I-mediated antiviral signaling. We found that ectopic expression of Cdc25A in 293T cells inhibits the activation of beta interferon (IFN-β) induced by Sendai virus and poly(I·C), while knockdown of Cdc25A enhances the transcription of IFN-β stimulated by RNA virus infection. The inhibitory effect of Cdc25A on the antiviral immune response is mainly dependent on its phosphatase activity. Data from a luciferase assay indicated that Cdc25A can inhibit TBK1-mediated activation of IFN-β. Further analysis indicated that Cdc25A can interact with TBK1 and reduce the phosphorylation of TBK1 at S172, which in turn decreases the phosphorylation of its downstream substrate IRF3. Consistently, knockdown of Cdc25A upregulates the phosphorylation of both TBK1-S172 and IRF3 in Sendai virus-infected or TBK1-transfected 293T cells. In addition, we confirmed that Cdc25A can directly dephosphorylate TBK1-S172-p. These results demonstrate that Cdc25A inhibits the antiviral immune response by reducing the active form of TBK1. Using herpes simplex virus 1 (HSV-1) infection, an IFN-β reporter assay, and reverse transcription-quantitative PCR (RT-qPCR), we demonstrated that Cdc25A can also inhibit DNA virus-induced activation of IFN-β. Using a vesicular stomatitis virus (VSV) infection assay, we confirmed that Cdc25A can repress the RIG-I-like receptor (RLR)-mediated antiviral immune response and influence the antiviral status of cells. In conclusion, we demonstrate that Cdc25A negatively regulates the antiviral immune response by inhibiting TBK1 activity. IMPORTANCE The RLR-mediated antiviral immune response is critical for host defense against RNA virus infection. However, the detailed mechanism for balancing the RLR signaling pathway in host cells is not well understood. We found that the phosphatase Cdc25A negatively regulates the RNA virus-induced innate immune response. Our studies indicate that Cdc25A inhibits the RLR signaling pathway via its phosphatase activity. We demonstrated that Cdc25A reduces TBK1 activity and consequently restrains the activation of IFN-β transcription as well as the antiviral status of nearby cells. We showed that Cdc25A can also inhibit DNA virus-induced activation of IFN-β. Taken together, our findings uncover a novel function and mechanism for Cdc25A in regulating antiviral immune signaling. These findings reveal Cdc25A as an important negative regulator of antiviral immunity and demonstrate its role in maintaining host cell homeostasis following viral infection., (Copyright © 2018 American Society for Microbiology.)

MicroRNAs (miRNAs) are small noncoding RNAs that are crucial posttranscriptional regulators for host mRNAs. Recent studies indicate that miRNAs may modulate host response during RNA virus infection. However, the role of miRNAs in immune response against H5N1 infection is not clearly understood. In this study, we showed that expression of cellular miRNA miR-324-5p was downregulated in A549 cells in response to infection with RNA viruses H5N1, A/PR8/H1N1, and Newcastle disease virus (NDV) and transfection with poly(I·C). We found that miR-324-5p inhibited H5N1 replication by targeting the PB1 viral RNA of H5N1 in host cells. In addition, transcriptome analysis revealed that miR-324-5p enhanced the expression of type I interferon, type III interferon, and interferon-inducible genes (ISGs) by targeting CUEDC2, the negative regulator of the JAK1-STAT3 pathway. Together, these findings highlight that the miR-324-5p plays a crucial role in host defense against H5N1 by targeting viral PB1 and host CUEDC2 to inhibit H5N1 replication. IMPORTANCE Highly pathogenic influenza A virus (HPAIV) continues to pose a pandemic threat globally. From 2003 to 2017, H5N1 HPAIV caused 453 human deaths, giving it a high mortality rate (52.74%). This work shows that miR-324-5p suppresses H5N1 HPAIV replication by directly targeting the viral genome (thereby inhibiting viral gene expression) and cellular CUEDC2 gene, the negative regulator of the interferon pathway (thereby enhancing antiviral genes). Our study enhances the knowledge of the role of microRNAs in the cellular response to viral infection. Also, the study provides help in understanding how the host cells utilize small RNAs in controlling the viral burden., (Copyright © 2018 American Society for Microbiology.)

We compared the HIV-1-specific immune responses generated by targeting HIV-1 envelope protein (Env gp140) to either CD40 or LOX-1, two endocytic receptors on dendritic cells (DCs), in rhesus macaques primed with a poxvirus vector (NYVAC-KC) expressing Env gp140. The DC-targeting vaccines, humanized recombinant monoclonal antibodies fused to Env gp140, were administered as a boost with poly-ICLC adjuvant either alone or coadministered with the NYVAC-KC vector. All the DC-targeting vaccine administrations with poly-ICLC increased the low-level serum anti-Env IgG responses elicited by NYVAC-KC priming significantly more (up to a P value of 0.01) than in a group without poly-ICLC. The responses were robust and cross-reactive and contained antibodies specific to multiple epitopes within gp140, including the C1, C2, V1, V2, and V3, C4, C5, and gp41 immunodominant regions. The DC-targeting vaccines also elicited modest serum Env-specific IgA responses. All groups gave serum neutralization activity limited to tier 1 viruses and antibody-dependent cytotoxicity responses (ADCC) after DC-targeting boosts. Furthermore, CD4 + and CD8 + T cell responses specific to multiple Env epitopes were strongly boosted by the DC-targeting vaccines plus poly-ICLC. Together, these results indicate that prime-boost immunization via NYVAC-KC and either anti-CD40.Env gp140/poly-ICLC or anti-LOX-1.Env gp140/poly-ICLC induced balanced antibody and T cell responses against HIV-1 Env. Coadministration of NYVAC-KC with the DC-targeting vaccines increased T cell responses but had minimal effects on antibody responses except for suppressing serum IgA responses. Overall, targeting Env to CD40 gave more robust T cell and serum antibody responses with broader epitope representation and greater durability than with LOX-1. IMPORTANCE An effective vaccine to prevent HIV-1 infection does not yet exist. An approach to elicit strong protective antibody development is to direct virus protein antigens specifically to dendritic cells, which are now known to be the key cell type for controlling immunity. In this study, we have tested in nonhuman primates two prototype vaccines engineered to direct the HIV-1 coat protein Env to dendritic cells. These vaccines bind to either CD40 or LOX-1, two dendritic cell surface receptors with different functions and tissue distributions. We tested the vaccines described above in combination with attenuated virus vectors that express Env. Both vaccines, but especially that delivered via CD40, raised robust immunity against HIV-1 as measured by monitoring potentially protective antibody and T cell responses in the blood. The safety and efficacy of the CD40-targeted vaccine justify further development for future human clinical trials., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: Hepatitis E virus (HEV) causes both the endemic and epidemic spread of acute hepatitis in many parts of the world. HEV open reading frame 3 (ORF3) encodes a 13-kDa multifunctional protein (vp13) that is essential for HEV infection of animals. The exact role of vp13 in HEV infection remains unclear. In this study, vp13 was found to enhance interferon (IFN) production induced by poly(I · C), a synthetic analog of double-stranded RNA. Poly(I · C) treatment induced a higher level of IFN-β mRNA in HeLa cells stably expressing vp13 than in control cells. Using a luciferase reporter construct driven by the IFN-β promoter, we demonstrated that vp13 enhanced retinoic acid-inducible gene I (RIG-I)-dependent luciferase expression. This enhancement was found to be due to both an increased level of RIG-I protein and its activation. The levels of both endogenous and exogenous RIG-I were increased by vp13 by extension of the half-life of RIG-I. Additionally, vp13 interacts with the RIG-I N-terminal domain and enhances its K63-linked ubiquitination, which is essential for RIG-I activation. Analysis of vp13 deletion constructs suggested that the C-terminal domain of vp13 was essential for the enhancement of RIG-I signaling. In HEV-infected hepatoma cells, wild-type HEV led to a higher level of RIG-I and more poly(I · C)-induced IFN-β expression than did ORF3-null mutants. Analysis of vp13 from four HEV genotypes showed that vp13 from genotype I and III strains boosted RIG-I signaling, while vp13 from genotype II and IV strains had a minimal effect. These results indicate that vp13 enhances RIG-I signaling, which may play a role in HEV invasion., Importance: Hepatitis E virus (HEV) is a significant pathogen causing hepatitis in many parts of the world, yet it is understudied compared with other viral hepatitis pathogens. Here we found that the HEV open reading frame 3 product, vp13, enhances interferon induction stimulated by a synthetic analog of double-stranded RNA. This enhancement may play a role in HEV invasion, as vp13 is essential for HEV infection in vivo. The results of this study provide insights into virus-cell interactions during HEV infection. In addition to revealing its possible roles in HEV interference with cellular signaling, these results suggest that the second half of the vp13 sequence can be ligated into the genomes of attenuated live viruses to induce an innate immune response for better protective immunity, as well as a marker for differentiation of vaccinated animals from those infected with the corresponding wild-type viruses., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Upon viral infection, pattern recognition receptors sense viral nucleic acids, leading to the production of type I interferons (IFNs), which initiate antiviral activities. Type I IFNs bind to their cognate receptor, IFNAR, resulting in the activation of signal-transducing activators of transcription 1 (STAT1). Thus, it has long been thought that double-stranded RNA (dsRNA)-induced STAT1 phosphorylation is mediated by the transactivation of type I IFN signaling. Foreign RNA, such as viral RNA, in cells is sensed by the cytoplasmic sensors retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). In this study, we explored the molecular mechanism responsible for STAT1 phosphorylation in response to the sensing of dsRNA by cytosolic RNA sensors. Polyinosinic-poly(C) [poly(I:C)], a synthetic dsRNA that is sensed by both RIG-I and MDA-5, induces STAT1 phosphorylation. We found that the poly(I:C)-induced initial phosphorylation of STAT1 is dependent on the RIG-I pathway and that MDA-5 is not involved in STAT1 phosphorylation. Furthermore, pretreatment of the cells with neutralizing antibody targeting the IFN receptor suppressed the initial STAT1 phosphorylation in response to poly(I:C), suggesting that this initial phosphorylation event is predominantly type I IFN dependent. In contrast, neither the known RIG-I pathway nor type I IFN is involved in the late phosphorylation of STAT1. In addition, poly(I:C) stimulated STAT1 phosphorylation in type I IFN receptor-deficient U5A cells with delayed kinetics. Collectively, our study provides evidence of a comprehensive regulatory mechanism in which dsRNA induces STAT1 phosphorylation, indicating the importance of STAT1 in maintaining very tight regulation of the innate immune system.

The retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) comprise three homologues: RIG-I, melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2). They activate the host interferon (IFN) system upon recognition of viral RNA pathogen-associated molecular patterns (PAMPs) in the cytoplasm. Bioinformatic analysis of the sequenced vertebrate genomes suggests that the cytosolic surveillance system is conserved in lower vertebrates, and recent functional studies have confirmed that RIG-I is important to fish antiviral immunity. In this study, we have identified MDA5 and LGP2 homologues from rainbow trout Oncorhynchus mykiss and an additional LGP2 variant with an incomplete C-terminal domain of RIG-I. Trout MDA5 and LGP2 were constitutively produced in fibroblast and macrophage cell lines and upregulated by poly(I:C), recombinant IFN, or infection by RNA viruses (viral hemorrhagic septicemia virus and salmon alphavirus) with a single-stranded positive or negative genome. Overexpression of MDA5 and LGP2 but not of the LGP2 variant resulted in significant accumulation of Mx transcripts in cultured cells, which correlated with a marked enhancement of protection against viral infection. These results demonstrate that both MDA5 and LGP2 are important RLRs in host surveillance against infection of both negative and positive viruses and that the LGP2 variant with a deletion of 54 amino acids at the C terminus acts as a negative regulator for LGP2-elicited antiviral signaling by competing for the viral RNA PAMPs. Interestingly, MDA5 expression was not affected by overexpressed LGP2 in transfected cells and vice versa, suggesting that they likely act in parallel as positive regulators for IFN production.

Profound type I interferon (IFN-I)-dependent attrition of memory CD8 and CD4 T cells occurs early during many infections. It is dramatic at 2 to 4 days following lymphocytic choriomeningitis virus (LCMV) infection of mice and can be elicited by the IFN-inducing Toll receptor agonist poly(I:C). We show that this attrition occurs in many organs, indicating that it is due to T cell loss rather than redistribution. This loss correlated with elevated intracellular staining of T cells ex vivo for activated caspases but with only low levels of ex vivo staining with annexin V, probably due to the rapid clearance of apoptotic cells in vivo. Instead, a high frequency of annexin V-reactive CD8alpha(+) dendritic cells (DCs), which are known to be highly phagocytic, accumulated in the spleen as the memory T cell populations disappeared. After short in vitro incubation, memory phenotype T cells isolated from LCMV-infected mice (day 3) or mice treated with poly(I:C) (12 h) displayed substantial DNA fragmentation, as detected by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, compared to T cells isolated from uninfected mice, indicating a role for apoptosis in the memory T cell attrition. This apoptosis of memory CD8 T cells early during LCMV infection was reduced in mice lacking the proapoptotic molecule Bim. Evidence is presented showing that high levels of T cell attrition, as found in young mice, correlate with reduced immunodomination by cross-reactive memory cells.

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-D-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34(+) hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE(D)) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-alpha), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rgamma(-/-) mouse model engrafted with human CD34(+) hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-alpha effectively suppressed poly(I:C)-induced TNF-alpha production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.

Interferon (IFN) signaling is initiated by the recognition of viral components by host pattern recognition receptors. Dengue virus (DEN) triggers IFN-beta induction through a molecular mechanism involving the cellular RIG-I/MAVS signaling pathway. Here we report that the MAVS protein level is reduced in DEN-infected cells and that caspase-1 and caspase-3 cleave MAVS at residue D429. In addition to its well-known function in IFN induction, MAVS is also a proapoptotic molecule that triggers disruption of the mitochondrial membrane potential and activation of caspases. Although different domains are required for the induction of cytotoxicity and IFN, caspase cleavage at residue 429 abolished both functions of MAVS. The apoptotic role of MAVS in viral infection and double-stranded RNA (dsRNA) stimulation was demonstrated in cells with reduced endogenous MAVS expression induced by RNA interference. Even though IFN-beta promoter activation was largely suppressed, DEN production was not affected greatly in MAVS knockdown cells. Instead, DEN- and dsRNA-induced cell death and caspase activation were delayed and attenuated in the cells with reduced levels of MAVS. These results reveal a new role of MAVS in the regulation of cell death beyond its well-known function of IFN induction in antiviral innate immunity.

Exposure to aldrithiol-2-inactivated human immunodeficiency virus type 1 or gp120, but not gp41, triggered alpha interferon (IFN-alpha), CC chemokine ligand 2 (CCL2), CCL3, and CCL4 production in human plasmacytoid dendritic cells (DCs) but not in myeloid DCs (M-DCs) or monocyte-derived DCs from the same donors. The nonresponsiveness of M-DCs for IFN-alpha/beta production was a general feature specific to these cells, as they also failed to produce it in response to inactivated influenza virus, poly(I-C), lipopolysaccharide, Staphylococcus aureus Cowans I, or CD40L. The different capacities of circulating DC subsets to produce immune mediators in response to most stimuli argue for a different role for these cells in the regulation of innate immunity to pathogens.

Vertebrates mount a strong innate immune response against viruses, largely by activating the interferon system. Double-stranded RNA (dsRNA), a common intermediate formed during the life cycle of many viruses, is a potent trigger of this response. In contrast, no general inducible antiviral defense mechanism has been reported in any invertebrate. Here we show that dsRNA induces antiviral protection in the marine crustacean Litopenaeus vannamei. When treated with dsRNA, shrimp showed increased resistance to infection by two unrelated viruses, white spot syndrome virus and Taura syndrome virus. Induction of this antiviral state is independent of the sequence of the dsRNA used and therefore distinct from the sequence-specific dsRNA-mediated genetic interference phenomenon. This demonstrates for the first time that an invertebrate immune system, like its vertebrate counterparts, can recognize dsRNA as a virus-associated molecular pattern, resulting in the activation of an innate antiviral response.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.

An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I)-poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I)-poly(C) as well as or better than poly(I)-poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrose-gradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.