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Suppressing cellular signal transducers of transcription 2 (STAT2) is a common strategy that viruses use to establish infections, yet the detailed mechanism remains elusive, owing to a lack of structural information about the viral-cellular complex involved. Here, we report the cryo-EM and crystal structures of human STAT2 (hSTAT2) in complex with the non-structural protein 5 (NS5) of Zika virus (ZIKV) and dengue virus (DENV), revealing two-pronged interactions between NS5 and hSTAT2. First, the NS5 methyltransferase and RNA-dependent RNA polymerase (RdRP) domains form a conserved interdomain cleft harboring the coiled-coil domain of hSTAT2, thus preventing association of hSTAT2 with interferon regulatory factor 9. Second, the NS5 RdRP domain also binds the amino-terminal domain of hSTAT2. Disruption of these ZIKV NS5-hSTAT2 interactions compromised NS5-mediated hSTAT2 degradation and interferon suppression, and viral infection under interferon-competent conditions. Taken together, these results clarify the mechanism underlying the functional antagonism of STAT2 by both ZIKV and DENV.

Significance To successfully establish infection, viral pathogens have to overcome the interferon (IFN)-mediated antiviral response. Previous studies revealed that the viral accessory protein Orf6 of SARS-CoV and SARS-CoV-2 is able to inhibit STAT1 nuclear translocation to block IFN signaling. In this study, we report that Orf6 localizes at the nuclear pore complex (NPC) where it binds directly to the Nup98-Rae1 complex to target the nuclear import pathway and mediate this inhibition. A better understanding of the strategies used by viruses to subvert host immune responses is critical for the design of novel antivirals and vaccines., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic that is a serious global health problem. Evasion of IFN-mediated antiviral signaling is a common defense strategy that pathogenic viruses use to replicate and propagate in their host. In this study, we show that SARS-CoV-2 is able to efficiently block STAT1 and STAT2 nuclear translocation in order to impair transcriptional induction of IFN-stimulated genes (ISGs). Our results demonstrate that the viral accessory protein Orf6 exerts this anti-IFN activity. We found that SARS-CoV-2 Orf6 localizes at the nuclear pore complex (NPC) and directly interacts with Nup98-Rae1 via its C-terminal domain to impair docking of cargo-receptor (karyopherin/importin) complex and disrupt nuclear import. In addition, we show that a methionine-to-arginine substitution at residue 58 impairs Orf6 binding to the Nup98-Rae1 complex and abolishes its IFN antagonistic function. All together our data unravel a mechanism of viral antagonism in which a virus hijacks the Nup98-Rae1 complex to overcome the antiviral action of IFN.

The Toll/IL-1R domain-containing adaptor protein SARM1 is expressed primarily in the brain, where it mediates axonal degeneration. Additional roles for SARM1 in a number of other processes including TLR-signaling, viral infection, chemokine expression, and expression of the proapoptotic protein XAF1 have also been described. Much of the supporting evidence for SARM1 function has been generated by comparing WT C57BL/6 (B6) mice to SARM1-deficient mice backcrossed to the B6 background. Here we show that the Sarm1 gene lies in a gene-rich region encompassing XAF1, and the MIP and MCP chemokine family loci among other genes. Because gene-targeting of SARM1-deficient strains was done with 129 ES cells and these genes are too close to segregate, they remain 129 in sequence. As this could account for phenotypes attributed to SARM1, we generated new knockout mouse strains on a pure B6 background using CRISPR. Experiments in these new strains confirmed the role of SARM1 in axonal degeneration and susceptibility to WNV infection, but not in susceptibility to VSV or LACV infection, or chemokine or Xaf1 expression. Notably, the Xaf1 gene shows sequence variation between B6 and 129, resulting in coding changes and novel splice variants. Given its known role in apoptosis, XAF1 variants may account for some phenotypes described in previously made SARM1-deficient strains. RNAseq in the new strains reveal changes in the mitochondrial electron transport chain and ribosomal proteins, suggesting possible downstream targets of SARM1. Re-evaluation of described phenotypes in these new strains will be critical for defining the function of SARM1.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection. Influenza A virus (IAV) infection induces transcription termination defects in host cells, an effect modulated by SUMOylation of an intrinsically disordered region of the influenza NS1 protein expressed by the 1918 pandemic IAV strain.

Retinoic acid inducible gene (RIG-I)-mediated innate immunity plays a pivotal role in defence against virus infections. Previously we have shown that Sendai virus (SeV) defective interfering (DI) RNA functions as an exclusive and potent RIG-I ligand in DI-RNA-rich SeV-Cantell infected cells. To further understand how RIG-I is activated during SeV infection, we used a different interferon (IFN)-inducing SeV strain, recombinant SeVΔC, which, in contrast to SeV-Cantell is believed to stimulate IFN production due to the lack of the SeV IFN antagonist protein C. Surprisingly, we found that in SevΔC-infected cells, DI RNAs also functioned as an exclusive RIG-I ligand. Infections with wild-type SeV failed to generate any RIG-I-associated immunostimulatory RNA and this correlated with the lack of DI genomes in infected cells, as well as with the absence of cellular innate immune responses. Supplementation of the C protein in the context of SeVΔC infection led to a reduction in the number of DI RNAs, further supporting the potential role of the C protein as a negative regulator of DI generation and/or accumulation. Our findings indicate that limiting DI genome production is an important function of viral IFN antagonist proteins.

The RNA helicase LGP2 (Laboratory of Genetics and Physiology 2) is a non-signaling member of the retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), whose pivotal role on innate immune responses against RNA viruses is being increasingly uncovered. LGP2 is known to work in synergy with melanoma differentiation-associated gene 5 (MDA5) to promote the antiviral response induced by picornavirus infection. Here, we describe the activity of the foot-and-mouth disease virus (FMDV) Leader protease (Lpro) targeting LGP2 for cleavage. When LGP2 and Lpro were co-expressed, cleavage products were observed in an Lpro dose-dependent manner while co-expression with a catalytically inactive Lpro mutant had no effect on LGP2 levels or pattern. We further show that Lpro localizes and immunoprecipitates with LGP2 in transfected cells supporting their interaction within the cytoplasm. Evidence of LGP2 proteolysis was also detected during FMDV infection. Moreover, the inhibitory effect of LGP2 overexpression on FMDV growth observed was reverted when Lpro was co-expressed, concomitant with lower levels of IFN-β mRNA and antiviral activity in those cells. The Lpro target site in LGP2 was identified as an RGRAR sequence in a conserved helicase motif whose replacement to EGEAE abrogated LGP2 cleavage by Lpro. Taken together, these data suggest that LGP2 cleavage by the Leader protease of aphthoviruses may represent a novel antagonistic mechanism for immune evasion., Spanish Ministry of Economy, Industry and Competitiveness (MINECO) (MS) and P2013/ABI-2906 (co-financed by Autonomous Community of Madrid and EC FEDER funds) (FS)

Paramyxovirus V proteins are known antagonists of the RIG-I-like receptor (RLR)-mediated interferon induction pathway, interacting with and inhibiting the RLR MDA5. We report interactions between the Nipah virus V protein and both RIG-I regulatory protein TRIM25 and RIG-I. We also observed interactions between these host proteins and the V proteins of measles virus, Sendai virus, and parainfluenza virus. These interactions are mediated by the conserved C-terminal domain of the V protein, which binds to the tandem caspase activation and recruitment domains (CARDs) of RIG-I (the region of TRIM25 ubiquitination) and to the SPRY domain of TRIM25, which mediates TRIM25 interaction with the RIG-I CARDs. Furthermore, we show that V interaction with TRIM25 and RIG-I prevents TRIM25-mediated ubiquitination of RIG-I and disrupts downstream RIG-I signaling to the mitochondrial antiviral signaling protein. This is a novel mechanism for innate immune inhibition by paramyxovirus V proteins, distinct from other known V protein functions such as MDA5 and STAT1 antagonism. IMPORTANCE The host RIG-I signaling pathway is a key early obstacle to paramyxovirus infection, as it results in rapid induction of an antiviral response. This study shows that paramyxovirus V proteins interact with and inhibit the activation of RIG-I, thereby interrupting the antiviral signaling pathway and facilitating virus replication.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel human coronavirus that emerged in 2012, causing severe pneumonia and acute respiratory distress syndrome (ARDS), with a case fatality rate of ~36%. When expressed in isolation, CoV accessory proteins have been shown to interfere with innate antiviral signaling pathways. However, there is limited information on the specific contribution of MERS-CoV accessory protein 4b to the repression of the innate antiviral response in the context of infection. We found that MERS-CoV 4b was required to prevent a robust NF-κB dependent response during infection. In wild-type virus infected cells, 4b localized to the nucleus, while NF-κB was retained in the cytoplasm. In contrast, in the absence of 4b or in the presence of cytoplasmic 4b mutants lacking a nuclear localization signal (NLS), NF-κB was translocated to the nucleus leading to the expression of pro-inflammatory cytokines. This indicates that NF-κB repression required the nuclear import of 4b mediated by a specific NLS. Interestingly, we also found that both in isolation and during infection, 4b interacted with α-karyopherin proteins in an NLS-dependent manner. In particular, 4b had a strong preference for binding karyopherin-α4 (KPNA4), which is known to translocate the NF-κB protein complex into the nucleus. Binding of 4b to KPNA4 during infection inhibited its interaction with NF-κB-p65 subunit. Thereby we propose a model where 4b outcompetes NF-κB for KPNA4 binding and translocation into the nucleus as a mechanism of interference with the NF-κB-mediated innate immune response., This work was supported by grants from the Government of Spain (BIO2013-42869-R and BIO2016-75549-R AEI/FEDER, UE), the European Zoonotic anticipation and preparedness initiative ZAPI (IMI_115760), and U.S. National Institutes of Health (NIH) (contract #0258-3413/ HHSN266200700010C (LE), 2P01 AI060699 (LE and SP), and R01 AI129269 (SP). JC received a contract from the Government of Spain (BIO2013- 42869-R). ARF was supported by institutional NRSA training grant T32-AI007260 and an individual NIH NRSA grant (F32-AI113973).

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.

SummaryInnate immunity conferred by the type I interferon is critical for antiviral defense. To date only a limited number of tripartite motif (TRIM) proteins have been implicated in modulation of innate immunity and anti-microbial activity. Here we report the complementary DNA cloning and systematic analysis of all known 75 human TRIMs. We demonstrate that roughly half of the 75 TRIM-family members enhanced the innate immune response and that they do this at multiple levels in signaling pathways. Moreover, messenger RNA levels and localization of most of these TRIMs were found to be altered during viral infection, suggesting that their regulatory activities are highly controlled at both pre- and posttranscriptional levels. Taken together, our data demonstrate a very considerable dedication of this large protein family to the positive regulation of the antiviral response, which supports the notion that this family of proteins evolved as a component of innate immunity.

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.

Recombinant β-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant β-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant β-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals. Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Programmed cell death is essential to survival of multicellular organisms. Previously restricted to apoptosis, the concept of programmed cell death is now extended to other mechanisms, as programmed necrosis or necroptosis, autophagic cell death, pyroptosis and parthanatos, among others. Viruses have evolved to manipulate and take control over the programmed cell death response, and the infected cell attempts to neutralize viral infections displaying different stress signals and defensive pathways before taking the critical decision of self-destruction. Learning from viruses and their interplay with the host may help us to better understand the complexity of the self-defense death response that when altered might cause disorders as important as cancer. In addition, as the fields of immunotherapy and oncolytic viruses advance as promising novel cancer therapies, the programmed cell death response reemerges as a key point for the success of both therapeutic approaches. In this review we summarize the research of the multimodal cell death response induced by Newcastle disease viruses (NDV), considered nowadays a promising viral oncolytic therapeutic, and how the manipulation of the host programmed cell death response can enhance the NDV antitumor capacity.

© 2015, American Society for Microbiology. The N-terminal region of the foot-and-mouth disease virus (FMDV) 3D polymerase contains the sequence MRKTKLAPT (residues 16 to 24) that acts as a nuclear localization signal. A previous study showed that substitutions K18E and K20E diminished the transport to the nucleus of 3D and 3CD and severely impaired virus infectivity. These residues have also been implicated in template binding, as seen in the crystal structures of different 3D-RNA elongation complexes. Here, we report the biochemical and structural characterization of different mutant polymerases harboring substitutions at residues 18 and 20, in particular, K18E, K18A, K20E, K20A, and the double mutant K18A K20A (KAKA). All mutant enzymes exhibit low RNA binding activity, low processivity, and alterations in nucleotide recognition, including increased incorporation of ribavirin monophosphate (RMP) relative to the incorporation of cognate nucleotides compared with the wild-type enzyme. The structural analysis shows an unprecedented flexibility of the 3D mutant polymerases, including both global rearrangements of the closed-hand architecture and local conformational changes at loop β9-α11 (within the polymerase motif B) and at the template-binding channel. Specifically, in 3D bound to RNA, both K18E and K20E induced the opening of new pockets in the template channel where the downstream templating nucleotide at position +2 binds. The comparisons of free and RNA-bound enzymes suggest that the structural rearrangements may occur in a concerted mode to regulate RNA replication, processivity, and fidelity. Thus, the Nterminal region of FMDV 3D that acts as a nuclear localization signal (NLS) and in template binding is also involved in nucleotide recognition and can affect the incorporation of nucleotide analogues. IMPORTANCE : The study documents multifunctionality of a nuclear localization signal (NLS) located at the N-terminal region of the foot-andmouth disease viral polymerase (3D). Amino acid substitutions at this polymerase region can impair the transport of 3D to the nucleus, reduce 3D binding to RNA, and alter the relative incorporation of standard nucleoside monophosphate versus ribavirin monophosphate. Structural data reveal that the conformational changes in this region, forming part of the template channel entry, would be involved in nucleotide discrimination. The results have implications for the understanding of viral polymerase function and for lethal mutagenesis mechanisms., Research in Barcelona was supported by grant BIO2011-24333 from the Spanish MINECO and by SILVER Cooperation project GA number 260644 of the European Union, Seventh Framework Program. Work in Madrid was supported by grants BFU2011-23604 and BIO2011-24351 from MINECO, PLATESA-S2013/ABI-2906 from CAM, and Fundación Ramón Areces. Financial support was provided by the ESRF and Alba. C.F.-O. is a recipient of a JAE postdoctoral contract supported by the Fondo Social Europeo

Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5'-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

The cytoplasmic helicase RIG-I is an established sensor for viral 5′-triphosphorylated RNA species. Recently, RIG-I was also implicated in the detection of intracellular bacteria. However, little is known about the host cell specificity of this process and the bacterial pathogen-associated molecular pattern (PAMP) that activates RIG-I. Here we show that RNA of Salmonella enterica serovar Typhimurium activates production of beta interferon in a RIG-I-dependent fashion only in nonphagocytic cells. In phagocytic cells, RIG-I is obsolete for detection of Salmonella infection. We further demonstrate that Salmonella mRNA reaches the cytoplasm during infection and is thus accessible for RIG-I. The results from next-generation sequencing analysis of RIG-I-associated RNA suggest that coding bacterial mRNAs represent the activating PAMP., IMPORTANCE S. Typhimurium is a major food-borne pathogen. After fecal-oral transmission, it can infect epithelial cells in the gut as well as immune cells (mainly macrophages, dendritic cells, and M cells). The innate host immune system relies on a growing number of sensors that detect pathogen-associated molecular patterns (PAMPs) to launch a first broad-spectrum response to invading pathogens. Successful detection of a given pathogen depends on colocalization of host sensors and PAMPs as well as potential countermeasures of the pathogen during infection. RIG-I-like helicases were mainly associated with detection of RNA viruses. Our work shows that S. Typhimurium is detected by RIG-I during infection specifically in nonimmune cells.

We have experimentally tested whether the MRKTKLAPT sequence in FMDV 3D protein (residues 16 to 24) can act as a nuclear localization signal (NLS). Mutants with substitutions in two basic residues within this sequence, K18E and K20E, were generated. A decreased nuclear localization was observed in transiently expressed 3D and its precursor 3CD, suggesting a role of K18 and K20 in nuclear targeting. Fusion of MRKTKLAPT to the green fluorescence protein (GFP) increased the nuclear localization of GFP, which was not observed when GFP was fused to the 3D mutated sequences. These results indicate that the sequence MRKTKLAPT can be functionally considered as a NLS. When introduced in a FMDV full length RNA replacements K18E and K20E led to production of revertant viruses that replaced the acidic residues introduced (E) by K, suggesting that the presence of lysins at positions 18 and 20 of 3D is essential for virus multiplication. © 2013 Elsevier Inc., BIO2008-0447-C03-01 and BIO2011-24351; Fundación Ramón Areces

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.

The innate immune response relies on a set of Pathogen Recognition Receptors (PRRs) that sensor pathogen patterns (PAMPs). RIG-I is a cytosolic PRR that detects 5’-triphosphate double-stranded RNAs produced during infection. Once activated, the pathway leads to the induction of type I IFN and proinflammatory cytokines, leading to a cellular antiviral state. Upon recognition of the RNA, RIG-I hydrolyzes ATP and changes its conformation to an active state. The CARD domains are then exposed and become K63-linked polyubiquitinated by E3-ligases, such as TRIM25. The activation of this pathway is complex and well characterized, but most of the spatio-temporal events, and the subcellular localization where the essential proteins interact, are still under interrogation. Through different techniques, we analyzed how these proteins form complexes that are distributed and reorganized spatially within the cell in order to create an efficient antiviral state. RIG-I is the main sensor for recognition of viruses such as Paramyxoviruses, Flaviviruses, Rhabdoviruses and Orthomyxoviruses. Many of them have developed numerous and different strategies to overcome the activation of the RLR pathway. We will discuss and show new insights on how, where, and when, viral proteins can counteract the activation of the RLR pathway. NS1 of Influenza A virus, or NS3/4A of Hepatitis C Virus, as an example, are IFN antagonistic viral proteins that interact with specific complexes in very well defined areas in the host cell in order to inhibit the antiviral state in an infected cell.

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation. Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNβ promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6−/− mice may provide a feasible model to study the role of human HerC5 in antiviral responses.

Introduction The host innate immune system acts as the first line of defense to prevent viral invasion. Viral products are rapidly detected through several classes of pathogen recognition receptors (PRR), like the cytosolic RIG-I like receptors (RLRs), which recognize viral RNA products in the cytoplasm of infected cells. Once activated, RIG-I binds MAVS, leading to the production of type I IFN [1] . Some viruses have developed multiple mechanisms to interfere with the RLR pathway and evade the innate immune response in infected cells. Influenza A virus (IAV) blocks the synthesis of type I IFN by targeting host effector molecules involved in activation of the RIG-I pathway [2] , [3] . Methods In order to study the steps involved in the RLR pathway, we have developed a Bimolecular Fluorescence Complementation (BiFC) assay [4] . This method allow us to visualize the subcellular localization of protein interactions by microscopy, pull down selectively BiFC complexes and isolate specific interactive complexes from transfected cells. Results We were able to detect subcellular localizations associated with interactions between RIGI, MAVS, TRIM25 and other proteins of the RLR pathway in the cell. We have analyzed the effects on these interactions of two viral proteins known to antagonize the RLR pathway. The NS3/4A of Hepatitis C Virus (HCV) changes the localization of the complexes RIGI-MAVS and MAVS-MAVS. The non structural protein 1 (NS1) from IAV prevents the formation of the complexes RIGI-MAVS and TRIM25-TRIM25, but does not have any effect on the complex RIGI-TRIM25. These complexes are localized in different compartments in the host cells. Conclusion The use of the BiFC technique has provided us with a powerful tool to analyze known and novel protein interactions of the RLR pathway. We have been able to isolate and track directly protein interactions, analyze their localization in the host cell and the effects of viral IFN antagonist proteins on these complexes.