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Retinoic acid-inducible gene-I (RIG-I) is a cytosolic pathogen sensor that is crucial against a number of viral infections. Many viruses have evolved to inhibit pathogen sensors to suppress host innate immune responses. In the case of influenza, nonstructural protein 1 (NS1) suppresses RIG-I function, leading to viral replication, morbidity, and mortality. We show that silencing NS1 with in-vitro-transcribed 5′-triphosphate containing NS1 short hairpin RNA (shRNA) (5′-PPP-NS1shRNA), designed using the conserved region of a number of influenza viruses, not only prevented NS1 expression but also induced RIG-I activation and type I interferon (IFN) expression, resulting in an antiviral state leading to inhibition of influenza virus replication in vitro. In addition, administration of 5′-PPP-NS1shRNA in prophylactic and therapeutic settings resulted in significant inhibition of viral replication following viral challenge in vivo in mice with corresponding increases of RIG-I, IFN-β, and IFN-λ, as well as a decrease in NS1 expression., Graphical Abstract

SummaryType I interferons (IFN-I) are essential antiviral cytokines produced upon microbial infection. IFN-I elicits this activity through the upregulation of hundreds of IFN-I-stimulated genes (ISGs). The full breadth of ISG induction demands activation of a number of cellular factors including the IκB kinase epsilon (IKKε). However, the mechanism of IKKε activation upon IFN receptor signaling has remained elusive. Here we show that TRIM6, a member of the E3-ubiquitin ligase tripartite motif (TRIM) family of proteins, interacted with IKKε and promoted induction of IKKε-dependent ISGs. TRIM6 and the E2-ubiquitin conjugase UbE2K cooperated in the synthesis of unanchored K48-linked polyubiquitin chains, which activated IKKε for subsequent STAT1 phosphorylation. Our work attributes a previously unrecognized activating role of K48-linked unanchored polyubiquitin chains in kinase activation and identifies the UbE2K-TRIM6-ubiquitin axis as critical for IFN signaling and antiviral response.

In the cytoplasm, the retinoic acid-inducible gene I (RIG-I) senses the RNA genomes of several RNA viruses. RIG-I binds to viral RNA, eliciting an antiviral response via the cellular adaptor MAVS. Crimean-Congo hemorrhagic fever virus (CCHFV), a negative-sense RNA virus with a 5′-monophosphorylated genome, is a highly pathogenic zoonotic agent with significant public health implications. We found that, during CCHFV infection, RIG-I mediated a type I interferon (IFN) response via MAVS. Interfering with RIG-I signaling reduced IFN production and IFN-stimulated gene expression and increased viral replication. Immunostimulatory RNA was isolated from CCHFV-infected cells and from virion preparations, and RIG-I coimmunoprecipitation of infected cell lysates isolated immunostimulatory CCHFV RNA. This report serves as the first description of a pattern recognition receptor for CCHFV and highlights a critical signaling pathway in the antiviral response to CCHFV. IMPORTANCE CCHFV is a tick-borne virus with a significant public health impact. In order for cells to respond to virus infection, they must recognize the virus as foreign and initiate antiviral signaling. To date, the receptors involved in immune recognition of CCHFV are not known. Here, we investigate and identify RIG-I as a receptor involved in initiating an antiviral response to CCHFV. This receptor initially was not expected to play a role in CCHFV recognition because of characteristics of the viral genome. These findings are important in understanding the antiviral response to CCHFV and support continued investigation into the spectrum of potential viruses recognized by RIG-I.

Cells are equipped with a large set of pattern recognition receptors or sensors that detect foreign molecules such as pathogenic nucleic acids and initiate proinflammatory and antimicrobial innate immune responses. RIG-I is a cytosolic sensor that detects 5'-triphosphate double-stranded RNAs produced during infection. RIG-I is responsible for mounting an antimicrobial response against a variety of viruses and intracellular bacteria. RIG-I contains an intricate structural architecture that allows for efficient signaling downstream in the pathway and autoregulation. The RIG-I-mediated antimicrobial pathway is highly regulated in cells requiring various cofactors, negative regulators, and posttranslational modifications. Modulation of RIG-I and RIG-I-mediated signaling in cells by pathogens to evade recognition and activation of the antimicrobial pathway highlights the essential nature of RIG-I in the innate immune response.

Influenza A virus (IAV), similar to other viruses, exploits the machinery of human host cells for its survival and replication. We identified α‐actinin‐4, a host cytoskeletal protein, as an interacting partner of IAV nucleoprotein (NP). We confirmed this interaction using co‐immunoprecipitation studies, first in a coupled in vitro transcription‐translation assay and then in cells either transiently co‐expressing the two proteins or infected with whole IAV. Importantly, the NP–actinin‐4 interaction was observed in several IAV subtypes, including the 2009 H1N1 pandemic virus. Moreover, immunofluorescence studies revealed that both NP and actinin‐4 co‐localized largely around the nucleus and also in the cytoplasmic region of virus‐infected A549 cells. Silencing of actinin‐4 expression resulted in not only a significant decrease in NP, M2 and NS1 viral protein expression, but also a reduction of both NP mRNA and viral RNA levels, as well as viral titers, 24 h post‐infection with IAV, suggesting that actinin‐4 was critical for viral replication. Furthermore, actinin‐4 depletion reduced the amount of NP localized in the nucleus. Treatment of infected cells with wortmannin, a known inhibitor of actinin‐4, led to a decrease in NP mRNA levels and also caused the nuclear retention of NP, further strengthening our previous observations. Taken together, the results of the present study indicate that actinin‐4, a novel interacting partner of IAV NP, plays a crucial role in viral replication and this interaction may participate in nuclear localization of NP and/or viral ribonucleoproteins. Structured digital abstract •http://www.uniprot.org/uniprot/P03466 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9512541, http://www.ebi.ac.uk/intact/interaction/EBI-9512553)•http://www.uniprot.org/uniprot/Q8JR21 and http://www.uniprot.org/uniprot/O43707 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0403 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0416 (http://www.ebi.ac.uk/intact/interaction/EBI-9514040)•http://www.uniprot.org/uniprot/Q91U50 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9514006)•http://www.uniprot.org/uniprot/Q5L4H4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007 (http://www.ebi.ac.uk/intact/interaction/EBI-9512166, http://www.ebi.ac.uk/intact/interaction/EBI-9512219)•http://www.uniprot.org/uniprot/C3W6D7 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513951)•http://www.uniprot.org/uniprot/Q5L4H4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007 (http://www.ebi.ac.uk/intact/interaction/EBI-9512237)•http://www.uniprot.org/uniprot/Q6DPG0 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513984) •http://www.uniprot.org/uniprot/B2BU63 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513930) •http://www.uniprot.org/uniprot/Q5L4H4 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0018 (http://www.ebi.ac.uk/intact/interaction/EBI-9512145, http://www.ebi.ac.uk/intact/interaction/EBI-9512095) •http://www.uniprot.org/uniprot/C9S3S8 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915 with http://www.uniprot.org/uniprot/O43707 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006 (http://www.ebi.ac.uk/intact/interaction/EBI-9513909), Human host proteins aiding the influenza A viral nucleoprotein in replication and transcription of virus and its intracellular shuttling are not well studied. The study presented here reports the dynamics of interaction between NP and actinin‐4 and the pivotal role of cytoskeletal protein in not only trafficking of NP, but also boosting its mRNA and vRNA levels, ultimately elevating viral titers.

Recognition of viral pathogens by the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family results in the activation of type I interferon (IFN) responses. To avoid this response, most viruses have evolved strategies that target different essential steps in the activation of host innate immunity. In this study, we report that the nonstructural protein NSs of the newly described severe fever with thrombocytopenia syndrome virus (SFTSV) is a potent inhibitor of IFN responses. The SFTSV NSs protein was found to inhibit the activation of the beta interferon (IFN-β) promoter induced by viral infection and by a RIG-I ligand. Astonishingly, we found that SFTSV NSs interacts with and relocalizes RIG-I, the E3 ubiquitin ligase TRIM25, and TANK-binding kinase 1 (TBK1) into SFTSV NSs-induced cytoplasmic structures. Interestingly, formation of these SFTSV NSs-induced structures occurred in the absence of the Atg7 gene, a gene essential for autophagy. Furthermore, confocal microscopy studies revealed that these SFTSV NSs-induced structures colocalize with Rab5 but not with Golgi apparatus or endoplasmic reticulum markers. Altogether, the data suggest that sequestration of RIG-I signaling molecules into endosome-like structures may be the mechanism used by SFTSV to inhibit IFN responses and point toward a novel mechanism for the suppression of IFN responses. IMPORTANCE The mechanism by which the newly described SFTSV inhibits host antiviral responses has not yet been fully characterized. In this study, we describe the redistribution of RIG-I signaling components into virus-induced cytoplasmic structures in cells infected with SFTSV. This redistribution correlates with the inhibition of host antiviral responses. Further characterization of the interplay between the viral protein and components of the IFN responses could potentially provide targets for the rational development of therapeutic interventions.

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.

The influenza virus neuraminidase (NA) protein primarily aids in the release of progeny virions from infected cells. Here, we demonstrate a novel role for NA in enhancing host cell survival by activating the Src/Akt signaling axis via an interaction with carcinoembryonic antigen-related cell adhesion molecule 6/cluster of differentiation 66c (C6). NA/C6 interaction leads to increased tyrosyl phosphorylation of Src, FAK, Akt, GSK3β, and Bcl-2, which affects cell survival, proliferation, migration, differentiation, and apoptosis. siRNA-mediated suppression of C6 resulted in a down-regulation of activated Src, FAK, and Akt, increased apoptosis, and reduced expression of viral proteins and viral titers in influenza virus-infected human lung adenocarcinoma epithelial and normal human bronchial epithelial cells. These findings indicate that influenza NA not only aids in the release of progeny virions, but also cell survival during viral replication.

+T-cell immunity a b s t r a c t The development of pre-pandemic influenza A H5N1 vaccines that confer both antigen-sparing and crossclade protection are a high priority given the limited worldwide capacity for influenza vaccine production, and the antigenic and genetic heterogeneity of circulating H5N1 viruses. The inclusion of potent adjuvants in vaccine formulations may achieve both of these aims. Here we show that the addition of JVRS-100, an adjuvant consisting of cationic liposome–DNA complexes (CLDC) to a clade 1-derived H5N1 split vaccine induced significantly higher virus-specific antibody than unadjuvanted formulations, with a >30fold dose-sparing effect and induction of increased antigen-specific CD4+ T-cell responses in mice. All mice that received one dose of adjuvanted vaccine and subsequent H5N1 viral challenges exhibited mild illness, lower lung viral titers, undetectable spleen and brain viral titers, and 100% survival after either homologous clade 1 or heterologous clade 2 H5N1 viral challenges, whereas unadjuvanted vaccine recipients showed significantly increased weight loss, viral titers, and mortality. The protective immunity induced by JVRS-100 adjuvanted H5N1 vaccine was shown to last for over one year without significant waning. Thus, JVRS-100 adjuvanted H5N1 vaccine elicited enhanced humoral and T-cell responses, dosesparing, and cross-clade protection in mice. CLDC holds promise as an adjuvant for human pre-pandemic inactivated H5N1 vaccines.

The cytosolic pathogen sensor RIG-I is activated by RNAs with exposed 5′-triphosphate (5′-ppp) and terminal double-stranded structures, such as those that are generated during viral infection. RIG-I has been shown to translocate on dsRNA in an ATP-dependent manner. However, the precise role of the ATPase activity in RIG-I activation remains unclear. Using in vitro-transcribed Sendai virus defective interfering RNA as a model ligand, we show that RIG-I oligomerizes on 5′-ppp dsRNA in an ATP hydrolysis-dependent and dsRNA length-dependent manner, which correlates with the strength of type-I interferon (IFN-I) activation. These results establish a clear role for the ligand-induced ATPase activity of RIG-I in the stimulation of the IFN response.

International audience; RIG-I is a cytosolic sensor of viral RNA, comprised of two N-terminal CARDs followed by helicase and C-terminal regulatory domains (helicase-CTD). Viral RNA binds to the helicase-CTD and "exposes" the CARDs for downstream signaling. The role of the second CARD (CARD2) is essential as RIG-I activation requires dephosphorylation of Thr170 followed by ubiquitination at Lys172. Here, we present the solution structure and dynamics of human RIG-I CARD2. Surprisingly, we find that Thr170 is mostly buried. Parallel studies on the phosphomimetic T170E mutant suggest that the loss of function upon Thr170 phosphorylation is likely associated with changes in the CARD1-CARD2 interface that may prevent Lys172 ubiquitination and/or binding to free K63-linked polyubiquitin. We also demonstrate a strong interaction between CARD2 and the helicase-CTD, and show that mutations at the interface result in constitutive activation of RIG-I. Collectively, our data suggests a close interplay between phosphorylation, ubiquitination, and activation of human RIG-I, all mediated by CARD2.

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., Author Summary Dengue virus (DENV) is a pathogen with a high impact in human health that replicates in a wide range of cells of the immune system. To efficiently infect humans, DENV must evade or inhibit fundamental elements of the innate immune system, namely the type I interferon response (IFN). Thus, DENV can inhibit type I IFN signaling (described by several groups), and type I IFN production (described by our group). We documented the inhibition of type I IFN production in human monocyte derived dendritic cells (MDDCs) with an otherwise strong cytokine and chemokine profile in those cells and that the NS2B3 protease complex of DENV functions as an antagonist of type I IFN production, and its proteolytic activity is necessary for this event. Here we identify the human adaptor molecule STING as a target of the NS2B3 protease complex and characterize the mechanism of inhibition of the type I IFN production in primary human MDDCs mediated by this viral factor. We also describe that DENV NS2B3 cannot degrade the mouse version of STING, a phenomenon that strictly 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.

Toll-like receptors (TLRs) play an important role in the induction of innate and adaptive immune response against influenza A virus (IAV) infection; however, the role of Toll-like receptor 7 (TLR7) during the innate immune response to IAV infection and the cell types affected by the absence of TLR7 are not clearly understood. In this study, we show that myeloid derived suppressor cells (MDSC) accumulate in the lungs of TLR7 deficient mice more so than in wild-type C57Bl/6 mice, and display increased cytokine expression. Furthermore, there is an increase in production of Th2 cytokines by TLR7(-/-) compared with wildtype CD4+ T-cells in vivo, leading to a Th2 polarized humoral response. Our findings indicate that TLR7 modulates the accumulation of MDSCs during an IAV infection in mice, and that lack of TLR7 signaling leads to a Th2-biased response.

To better understand the early virus-host interactions of the pandemic 2009 A(H1N1) viruses in humans, we examined early host responses following infection of human epithelial cell cultures with three 2009 A(H1N1) viruses (A/California/08/2009, A/Mexico/4108/2009, and A/Texas/15/2009), or a seasonal H1N1 vaccine strain (A/Solomon Islands/3/2006). We report here that infection with pandemic A/California/08/2009 and A/Mexico/4108/2009 viruses resulted in differences in virus infectivity compared to either pandemic A/Texas/15/2009 or the seasonal H1N1 vaccine strain. In addition, IFN-β levels were decreased in cell cultures infected with either the A/California/08/2009 or the A/Mexico/4108/2009 virus. Furthermore, infection with A/California/08/2009 and A/Mexico/4108/2009 viruses resulted in lower expression of four key proinflammatory markers (IL-6, RANTES, IP-10, and MIP-1β) compared with infection with either A/Texas/15/2009 or A/Solomon Islands/3/2006. Taken together, our results demonstrate that 2009 A(H1N1) viruses isolated during the Spring wave induced varying degrees of early host antiviral and inflammatory responses in human respiratory epithelial cells, highlighting the strain-specific nature of these responses, which play a role in clinical disease.

The role of the reactive oxygen species-producing NADPH oxidase family of enzymes in the pathology of influenza A virus infection remains enigmatic. Previous reports implicated NADPH oxidase 2 in influenza A virus-induced inflammation. In contrast, NADPH oxidase 1 (Nox1) was reported to decrease inflammation in mice within 7 days post-influenza A virus infection. However, the effect of NADPH oxidase 1 on lethality and adaptive immunity after influenza A virus challenge has not been explored. Here we report improved survival and decreased morbidity in mice with catalytically inactive NADPH oxidase 1 (Nox1*/Y) compared with controls after challenge with A/PR/8/34 influenza A virus. While changes in lung inflammation were not obvious between Nox1*/Y and control mice, we observed alterations in the T cell response to influenza A virus by day 15 post-infection, including increased interleukin-7 receptor-expressing virus-specific CD8+ T cells in lungs and draining lymph nodes of Nox1*/Y, and increased cytokine-producing T cells in lungs and spleen. Furthermore, a greater percentage of conventional and interstitial dendritic cells from Nox1*/Y draining lymph nodes expressed the co-stimulatory ligand CD40 within 6 days post-infection. Results indicate that NADPH oxidase 1 modulates the innate and adaptive cellular immune response to influenza virus infection, while also playing a role in host survival. Results suggest that NADPH oxidase 1 inhibitors may be beneficial as adjunct therapeutics during acute influenza infection.

Apoptosis induction is an antiviral host response, however, influenza A virus (IAV) infection promotes host cell death. The nucleoprotein (NP) of IAV is known to contribute to viral pathogenesis, but its role in virus-induced host cell death was hitherto unknown. We observed that NP contributes to IAV infection induced cell death and heterologous expression of NP alone can induce apoptosis in human airway epithelial cells. The apoptotic effect of IAV NP was significant when compared with other known proapoptotic proteins of IAV. The cell death induced by IAV NP was executed through the intrinsic apoptosis pathway. We screened host cellular factors for those that may be targeted by NP for inducing apoptosis and identified human antiapoptotic protein Clusterin (CLU) as a novel interacting partner. The interaction between IAV NP and CLU was highly conserved and mediated through β-chain of the CLU protein. Also CLU was found to interact specifically with IAV NP and not with any other known apoptosis modulatory protein of IAV. CLU prevents induction of the intrinsic apoptosis pathway by binding to Bax and inhibiting its movement into the mitochondria. We found that the expression of IAV NP reduced the association between CLU and Bax in mammalian cells. Further, we observed that CLU overexpression attenuated NP-induced cell death and had a negative effect on IAV replication. Collectively, these findings indicate a new function for IAV NP in inducing host cell death and suggest a role for the host antiapoptotic protein CLU in this process.

Activation of innate immunity pathways in susceptible cell types is critical for host defense against influenza-A viruses (IAV). Based on studies signifying a role for NOX1 in inflammation, we hypothesized that IAV of seasonal and pandemic potential may induce different isoforms of NOX enzymes in the respiratory tract, and that attenuation of virus strain-specific NOX enzymes can be exploited to prevent and/or treat severity of disease. Using three respiratory tract-relevant cell lines (epithelial/A549, endothelial/HULEC, and monoctytic/THP1) and three strains of IAV (H1N1/PR8, H3N2/X31, H1N1/WSN), we found significant upregulation of NOX1 in all three cell lines (e.g.-A549: PR8/55 fold; X31/35 fold; WSN/12,250 fold). Furthermore, in contrast to the epithelial cell line showing NOX1 expression by 4h with peak levels at 16h, the monocytic cell line showed delayed (8h) and sustained (48h) levels of NOX1. Interestingly, inhibition of NOX-isoforms in IAV-infected epithelial cells by diphenyleneiodonium (DPI), at a concentration (25μM), which did not impact cell death, showed significant decrease (75%) in virus triggered NOX1 levels. In addition, NOX-inhibition led to significant decrease in virus-induced chemokines (MCP1, MIP3, MIP1β, RANTES, and IP10) and proinflammatory cytokines (IL1α, IL6, IL8, and TNFα). Furthermore, decline in NOX1 correlated with a modest increase in viral NS1. Taken together, in addition to showing definitive evidence for upregulation of NOX1 in response to subtypes of IAV, our results strongly support a role for NOX1 in IAV-driven inflammation.