RNase L-activating 2'-5' oligoadenylates bind ABCF1, ABCF3 and Decr-1.

A notable signalling mechanism employed by mammalian innate immune signalling pathways uses nucleotide-based second messengers such as 2'3'-cGAMP and 2'-5'-oligoadenylates (OAs), which bind and activate STING and RNase L, respectively. Interestingly, the involvement of nucleotide second messengers to activate antiviral responses is evolutionarily conserved, as evidenced by the identification of an antiviral cGAMP-dependent pathway in Drosophila. Using a mass spectrometry approach, we identified several members of the ABCF family in human, mouse and Drosophila cell lysates as 2'-5' OA-binding proteins, suggesting an evolutionarily conserved function. Biochemical characterization of these interactions demonstrates high-affinity binding of 2'-5' OA to ABCF1, dependent on phosphorylated 2'-5' OA and an intact Walker A/B motif of the ABC cassette of ABCF1. As further support for species-specific interactions with 2'-5' OA, we additionally identified that the metabolic enzyme Decr1 from mouse, but not human or Drosophila cells, forms a high-affinity complex with 2'-5' OA. A 1.4 Å co-crystal structure of the mouse Decr1-2'-5' OA complex explains high-affinity recognition of 2'-5' OA and the mechanism of species specificity. Despite clear evidence of physical interactions, we could not identify profound antiviral functions of ABCF1, ABCF3 or Decr1 or 2'-5' OA-dependent regulation of cellular translation rates, as suggested by the engagement of ABCF proteins. Thus, although the biological consequences of the here identified interactions need to be further studied, our data suggest that 2'-5' OA can serve as a signalling hub to distribute a signal to different recipient proteins.

Ribose 2'-O-methylation provides a molecular signature for the distinction of self and non-self mRNA dependent on the RNA sensor Mda5.

The 5' cap structures of higher eukaryote mRNAs have ribose 2'-O-methylation. Likewise, many viruses that replicate in the cytoplasm of eukaryotes have evolved 2'-O-methyltransferases to autonomously modify their mRNAs. However, a defined biological role for 2'-O-methylation of mRNA remains elusive. Here we show that 2'-O-methylation of viral mRNA was critically involved in subverting the induction of type I interferon. We demonstrate that human and mouse coronavirus mutants lacking 2'-O-methyltransferase activity induced higher expression of type I interferon and were highly sensitive to type I interferon. Notably, the induction of type I interferon by viruses deficient in 2'-O-methyltransferase was dependent on the cytoplasmic RNA sensor Mda5. This link between Mda5-mediated sensing of viral RNA and 2'-O-methylation of mRNA suggests that RNA modifications such as 2'-O-methylation provide a molecular signature for the discrimination of self and non-self mRNA.

Structure of human IFIT1 with capped RNA reveals adaptable mRNA binding and mechanisms for sensing N1 and N2 ribose 2'-O methylations.

IFIT1 (IFN-induced protein with tetratricopeptide repeats-1) is an effector of the host innate immune antiviral response that prevents propagation of virus infection by selectively inhibiting translation of viral mRNA. It relies on its ability to compete with the translation initiation factor eIF4F to specifically recognize foreign capped mRNAs, while remaining inactive against host mRNAs marked by ribose 2'-O methylation at the first cap-proximal nucleotide (N1). We report here several crystal structures of RNA-bound human IFIT1, including a 1.6-Å complex with capped RNA. IFIT1 forms a water-filled, positively charged RNA-binding tunnel with a separate hydrophobic extension that unexpectedly engages the cap in multiple conformations (syn and anti) giving rise to a relatively plastic and nonspecific mode of binding, in stark contrast to eIF4E. Cap-proximal nucleotides encircled by the tunnel provide affinity to compete with eIF4F while allowing IFIT1 to select against N1 methylated mRNA. Gel-shift binding assays confirm that N1 methylation interferes with IFIT1 binding, but in an RNA-dependent manner, whereas translation assays reveal that N1 methylation alone is not sufficient to prevent mRNA recognition at high IFIT1 concentrations. Structural and functional analysis show that 2'-O methylation at N2, another abundant mRNA modification, is also detrimental for RNA binding, thus revealing a potentially synergistic role for it in self- versus nonself-mRNA discernment. Finally, structure-guided mutational analysis confirms the importance of RNA binding for IFIT1 restriction of a human coronavirus mutant lacking viral N1 methylation. Our structural and biochemical analysis sheds new light on the molecular basis for IFIT1 translational inhibition of capped viral RNA.

NSs Protein of Sandfly Fever Sicilian Phlebovirus Counteracts Interferon (IFN) Induction by Masking the DNA-Binding Domain of IFN Regulatory Factor 3.

Sandfly fever Sicilian virus (SFSV) is one of the most widespread and frequently identified members of the genus Phlebovirus (order Bunyavirales, family Phenuiviridae) infecting humans. Being transmitted by Phlebotomus sandflies, SFSV causes a self-limiting, acute, often incapacitating febrile disease ("sandfly fever," "Pappataci fever," or "dog disease") that has been known since at least the beginning of the 20th century. We show that, similarly to other pathogenic phleboviruses, SFSV suppresses the induction of the antiviral type I interferon (IFN) system in an NSs-dependent manner. SFSV NSs interfered with the TBK1-interferon regulatory factor 3 (IRF3) branch of the RIG-I signaling pathway but not with NF-κB activation. Consistently, we identified IRF3 as a host interactor of SFSV NSs. In contrast to IRF3, neither the IFN master regulator IRF7 nor any of the related transcription factors IRF2, IRF5, and IRF9 were bound by SFSV NSs. In spite of this specificity for IRF3, NSs did not inhibit its phosphorylation, dimerization, or nuclear accumulation, and the interaction was independent of the IRF3 activation or multimerization state. In further studies, we identified the DNA-binding domain of IRF3 (amino acids 1 to 113) as sufficient for NSs binding and found that SFSV NSs prevented the association of activated IRF3 with the IFN-ß promoter. Thus, unlike highly virulent phleboviruses, which either destroy antiviral host factors or sequester whole signaling chains into inactive aggregates, SFSV modulates type I IFN induction by directly masking the DNA-binding domain of IRF3.IMPORTANCE Phleboviruses are receiving increased attention due to the constant discovery of new species and the ongoing spread of long-known members of the genus. Outbreaks of sandfly fever were reported in the 19th century, during World War I, and during World War II. Currently, SFSV is recognized as one of the most widespread phleboviruses, exhibiting high seroprevalence rates in humans and domestic animals and causing a self-limiting but incapacitating disease predominantly in immunologically naive troops and travelers. We show how the nonstructural NSs protein of SFSV counteracts the upregulation of the antiviral interferon (IFN) system. SFSV NSs specifically inhibits promoter binding by IFN transcription factor 3 (IRF3), a molecular strategy which is unique among phleboviruses and, to our knowledge, among human pathogenic RNA viruses in general. This IRF3-specific and stoichiometric mechanism, greatly distinct from the ones exhibited by the highly virulent phleboviruses, correlates with the intermediate level of pathogenicity of SFSV.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Viral immune modulators perturb the human molecular network by common and unique strategies.

Viruses must enter host cells to replicate, assemble and propagate. Because of the restricted size of their genomes, viruses have had to evolve efficient ways of exploiting host cell processes to promote their own life cycles and also to escape host immune defence mechanisms. Many viral open reading frames (viORFs) with immune-modulating functions essential for productive viral growth have been identified across a range of viral classes. However, there has been no comprehensive study to identify the host factors with which these viORFs interact for a global perspective of viral perturbation strategies. Here we show that different viral perturbation patterns of the host molecular defence network can be deduced from a mass-spectrometry-based host-factor survey in a defined human cellular system by using 70 innate immune-modulating viORFs from 30 viral species. The 579 host proteins targeted by the viORFs mapped to an unexpectedly large number of signalling pathways and cellular processes, suggesting yet unknown mechanisms of antiviral immunity. We further experimentally verified the targets heterogeneous nuclear ribonucleoprotein U, phosphatidylinositol-3-OH kinase, the WNK (with-no-lysine) kinase family and USP19 (ubiquitin-specific peptidase 19) as vulnerable nodes in the host cellular defence system. Evaluation of the impact of viral immune modulators on the host molecular network revealed perturbation strategies used by individual viruses and by viral classes. Our data are also valuable for the design of broad and specific antiviral therapies.

Virulence factor NSs of rift valley fever virus recruits the F-box protein FBXO3 to degrade subunit p62 of general transcription factor TFIIH.

UNLABELLED: The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE: Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.

Sequestration by IFIT1 impairs translation of 2'O-unmethylated capped RNA.

Viruses that generate capped RNA lacking 2'O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as in vitro competition assays indicate that IFIT1 sequesters 2'O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2'O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2'O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2'O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.

TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium.

Infection with human coronavirus 229E (HCoV-229E) is associated with the common cold and may result in pneumonia in immunocompromised patients. The viral spike (S) protein is incorporated into the viral envelope and mediates infectious entry of HCoV-229E into host cells, a process that depends on the activation of the S-protein by host cell proteases. However, the proteases responsible for HCoV-229E activation are incompletely defined. Here we show that the type II transmembrane serine proteases TMPRSS2 and HAT cleave the HCoV-229E S-protein (229E-S) and augment 229E-S-driven cell-cell fusion, suggesting that TMPRSS2 and HAT can activate 229E-S. Indeed, engineered expression of TMPRSS2 and HAT rendered 229E-S-driven virus-cell fusion insensitive to an inhibitor of cathepsin L, a protease previously shown to facilitate HCoV-229E infection. Inhibition of endogenous cathepsin L or TMPRSS2 demonstrated that both proteases can activate 229E-S for entry into cells that are naturally susceptible to infection. In addition, evidence was obtained that activation by TMPRSS2 rescues 229E-S-dependent cell entry from inhibition by IFITM proteins. Finally, immunohistochemistry revealed that TMPRSS2 is coexpressed with CD13, the HCoV-229E receptor, in human airway epithelial (HAE) cells, and that CD13(+) TMPRSS2(+) cells are preferentially targeted by HCoV-229E, suggesting that TMPRSS2 can activate HCoV-229E in infected humans. In sum, our results indicate that HCoV-229E can employ redundant proteolytic pathways to ensure its activation in host cells. In addition, our observations and previous work suggest that diverse human respiratory viruses are activated by TMPRSS2, which may constitute a target for antiviral intervention.

Preclinical development of a first-in-class vaccine encoding HER2, Brachyury and CD40L for antibody enhanced tumor eradication.

The induction of antiviral innate immunity by systemic immunization with live virus can be employed to positively impact the response to therapeutic vaccination. We previously demonstrated that systemic immunization with a non-replicating MVA encoding CD40 ligand (CD40L) enhances innate immune cell activation and function, and triggers potent antitumor CD8+ T cell responses in different murine tumor models. Antitumor efficacy was increased when combined with tumor targeting antibodies. Here we report the development of TAEK-VAC-HerBy (TVH), a first-in-class human tumor antibody enhanced killing (TAEK) vaccine based on the non-replicating MVA-BN viral vector. It encodes the membrane bound form of human CD40L, HER2 and the transcription factor Brachyury. TVH is designed for therapeutic use in HER2- or Brachyury-expressing cancer patients in combination with tumor targeting antibodies. To preclude possible oncogenic activities in infected cells and to prevent binding of vaccine-encoded HER2 by monoclonal antibodies trastuzumab and pertuzumab, genetic modifications of HER2 were introduced in the vaccine. Brachyury was genetically modified to prevent nuclear localization of the protein thereby inhibiting its transcriptional activity. CD40L encoded in TVH enhanced human leukocyte activation and cytokine secretion in vitro. Lastly, TVH intravenous administration to non-human primates was proven immunogenic and safe in a repeat-dose toxicity study. Nonclinical data presented here highlight TVH as a first-in-class immunotherapeutic vaccine platform currently under clinical investigation.

Toscana virus induces interferon although its NSs protein reveals antagonistic activity.

Toscana virus (TOSV) is a phlebotomus-transmitted virus that belongs to the family Bunyaviridae and causes widespread infections in humans; about 30 % of these cases result in aseptic meningitis. In the present study, it was shown that TOSV is an inducer of beta interferon (IFN-ß), although its non-structural protein (NSs) could inhibit the induction of IFN-ß if expressed in a heterologous context. A recombinant Rift Valley fever virus expressing the TOSV NSs could suppress IFN-ß expression in infected cells. Moreover, in cells expressing NSs protein from a cDNA plasmid, IFN-ß transcripts were not inducible by poly(I : C). Unlike other members of the family Bunyaviridae, TOSV appears to express an NSs protein that is a weak antagonist of IFN induction. Characterization of the interaction of TOSV with the IFN system will help our understanding of virus-host cell interactions and may explain why the pathogenesis of this disease is mostly mild in humans.

Cross-species analysis of viral nucleic acid interacting proteins identifies TAOKs as innate immune regulators.

The cell intrinsic antiviral response of multicellular organisms developed over millions of years and critically relies on the ability to sense and eliminate viral nucleic acids. Here we use an affinity proteomics approach in evolutionary distant species (human, mouse and fly) to identify proteins that are conserved in their ability to associate with diverse viral nucleic acids. This approach shows a core of orthologous proteins targeting viral genetic material and species-specific interactions. Functional characterization of the influence of 181 candidates on replication of 6 distinct viruses in human cells and flies identifies 128 nucleic acid binding proteins with an impact on virus growth. We identify the family of TAO kinases (TAOK1, -2 and -3) as dsRNA-interacting antiviral proteins and show their requirement for type-I interferon induction. Depletion of TAO kinases in mammals or flies leads to an impaired response to virus infection characterized by a reduced induction of interferon stimulated genes in mammals and impaired expression of srg1 and diedel in flies. Overall, our study shows a larger set of proteins able to mediate the interaction between viral genetic material and host factors than anticipated so far, attesting to the ancestral roots of innate immunity and to the lineage-specific pressures exerted by viruses.

NSs protein of rift valley fever virus induces the specific degradation of the double-stranded RNA-dependent protein kinase.

Rift Valley fever virus (RVFV) continues to cause large outbreaks of acute febrile and often fatal illness among humans and domesticated animals in Africa, Saudi Arabia, and Yemen. The high pathogenicity of this bunyavirus is mainly due to the viral protein NSs, which was shown to prevent transcriptional induction of the antivirally active type I interferons (alpha/beta interferon [IFN-alpha/beta]). Viruses lacking the NSs gene induce synthesis of IFNs and are therefore attenuated, whereas the noninducing wild-type RVFV strains can only be inhibited by pretreatment with IFN. We demonstrate here in vitro and in vivo that a substantial part of the antiviral activity of IFN against RVFV is due to a double-stranded RNA-dependent protein kinase (PKR). PKR-mediated virus inhibition, however, was much more pronounced for the strain Clone 13 with NSs deleted than for the NSs-expressing strain ZH548. In vivo, Clone 13 was nonpathogenic for wild-type (wt) mice but could regain pathogenicity if mice lacked the PKR gene. ZH548, in contrast, killed both wt and PKR knockout mice indiscriminately. ZH548 was largely resistant to the antiviral properties of PKR because RVFV NSs triggered the specific degradation of PKR via the proteasome. The NSs proteins of the related but less virulent sandfly fever Sicilian virus and La Crosse virus, in contrast, had no such anti-PKR activity despite being efficient suppressors of IFN induction. Our data suggest that RVFV NSs has gained an additional anti-IFN function that may explain the extraordinary pathogenicity of this virus.

Species-independent bioassay for sensitive quantification of antiviral type I interferons.

BACKGROUND: Studies of the host response to infection often require quantitative measurement of the antiviral type I interferons (IFN-alpha/beta) in biological samples. The amount of IFN is either determined via its ability to suppress a sensitive indicator virus, by an IFN-responding reporter cell line, or by ELISA. These assays however are either time-consuming and lack convenient readouts, or they are rather insensitive and restricted to IFN from a particular host species. RESULTS: An IFN-sensitive, Renilla luciferase-expressing Rift Valley fever virus (RVFV-Ren) was generated using reverse genetics. Human, murine and avian cells were tested for their susceptibility to RVFV-Ren after treatment with species-specific IFNs. RVFV-Ren was able to infect cells of all three species, and IFN-mediated inhibition of viral reporter activity occurred in a dose-dependent manner. The sensitivity limit was found to be 1 U/ml IFN, and comparison with a standard curve allowed to determine the activity of an unknown sample. CONCLUSIONS: RVFV-Ren replicates in cells of several species and is highly sensitive to pre-treatment with IFN. These properties allowed the development of a rapid, sensitive, and species-independent antiviral assay with a convenient luciferase-based readout.

eIF2B as a Target for Viral Evasion of PKR-Mediated Translation Inhibition.

RNA-activated protein kinase (PKR) is a major innate immune factor that senses viral double-stranded RNA (dsRNA) and phosphorylates eukaryotic initiation factor (eIF) 2α. Phosphorylation of the α subunit converts the eIF2αßγ complex into a stoichiometric inhibitor of eukaryotic initiation factor eIF2B, thus halting mRNA translation. To escape this protein synthesis shutoff, viruses have evolved countermechanisms such as dsRNA sequestration, eIF-independent translation by an internal ribosome binding site, degradation of PKR, or dephosphorylation of PKR or of phospho-eIF2α. Here, we report that sandfly fever Sicilian phlebovirus (SFSV) confers such a resistance without interfering with PKR activation or eIF2α phosphorylation. Rather, SFSV expresses a nonstructural protein termed NSs that strongly binds to eIF2B. Although NSs still allows phospho-eIF2α binding to eIF2B, protein synthesis and virus replication are unhindered. Hence, SFSV encodes a unique PKR antagonist that acts by rendering eIF2B resistant to the inhibitory action of bound phospho-eIF2α.IMPORTANCE RNA-activated protein kinase (PKR) is one of the most powerful antiviral defense factors of the mammalian host. PKR acts by phosphorylating mRNA translation initiation factor eIF2α, thereby converting it from a cofactor to an inhibitor of mRNA translation that strongly binds to initiation factor eIF2B. To sustain synthesis of their proteins, viruses are known to counteract this on the level of PKR or eIF2α or by circumventing initiation factor-dependent translation altogether. Here, we report a different PKR escape strategy executed by sandfly fever Sicilian virus (SFSV), a member of the increasingly important group of phleboviruses. We found that the nonstructural protein NSs of SFSV binds to eIF2B and protects it from inactivation by PKR-generated phospho-eIF2α. Protein synthesis is hence maintained and the virus can replicate despite ongoing full-fledged PKR signaling in the infected cells. Thus, SFSV has evolved a unique strategy to escape the powerful antiviral PKR.

Interferon priming enables cells to partially overturn the SARS coronavirus-induced block in innate immune activation.

SARS coronavirus (SARS-CoV) is known to efficiently suppress the induction of antiviral type I interferons (IFN-alpha/beta) in non-lymphatic cells through inhibition of the transcription factor IRF-3. Plasmacytoid dendritic cells, in contrast, respond to infection with production of high levels of IFNs. Here, we show that pretreatment of non-lymphatic cells with small amounts of IFN-alpha (IFN priming) partially overturns the block in IFN induction imposed by SARS-CoV. IFN priming combined with SARS-CoV infection substantially induced genes for IFN induction, IFN signalling, antiviral effector proteins, ubiquitination and ISGylation, antigen presentation and other cytokines and chemokines, whereas each individual treatment had no major effect. Curiously, however, despite this typical IFN response, neither IRF-3 nor IRF-7 was transported to the nucleus as a sign of activation. Taken together, our results suggest that (i) IFN, as it is produced by plasmacytoid dendritic cells, could enable tissue cells to launch a host response to SARS-CoV, (ii) IRF-3 and IRF-7 may be active at subdetectable levels, and (iii) SARS-CoV does not activate IRF-7.

High-Throughput Screening Using a Whole-Cell Virus Replication Reporter Gene Assay to Identify Inhibitory Compounds against Rift Valley Fever Virus Infection.

Rift Valley fever virus (RVFV) is an emerging virus that causes serious illness in humans and livestock. There are no approved vaccines or treatments for humans. The purpose of the study was to identify inhibitory compounds of RVFV infection without any preconceived idea of the mechanism of action. A whole-cell-based high-throughput drug screening assay was developed to screen 28,437 small chemical compounds targeting RVFV infection. To accomplish both speed and robustness, a replication-competent NSs-deleted RVFV expressing a fluorescent reporter gene was developed. Inhibition of fluorescence intensity was quantified by spectrophotometry and related to virus infection in human lung epithelial cells (A549). Cell toxicity was assessed by the Resazurin cell viability assay. After primary screening, 641 compounds were identified that inhibited RVFV infection by ≥80%, with ≥50% cell viability at 50 µM concentration. These compounds were subjected to a second screening regarding dose-response profiles, and 63 compounds with ≥60% inhibition of RVFV infection at 3.12 µM compound concentration and ≥50% cell viability at 25 µM were considered hits. Of these, six compounds with high inhibitory activity were identified. In conclusion, the high-throughput assay could efficiently and safely identify several promising compounds that inhibited RVFV infection.

Cytoplasmic sensing of viral nucleic acids.

Viruses are the most abundant pathogens on earth. A fine-tuned framework of intervening pathways is in place in mammalian cells to orchestrate the cellular defence against these pathogens. Key for this system is sensor proteins that recognise specific features associated with nucleic acids of incoming viruses. Here we review the current knowledge on cytoplasmic sensors for viral nucleic acids. These sensors induce expression of cytokines, affect cellular functions required for virus replication and directly target viral nucleic acids through degradation or sequestration. Their ability to respond to a given nucleic acid is based on both the differential specificity of the individual proteins and the downstream signalling or adaptor proteins. The cooperation of these multiple proteins and pathways plays a key role in inducing successful immunity against virus infections.

mRNA export through an additional cap-binding complex consisting of NCBP1 and NCBP3.

The flow of genetic information from DNA to protein requires polymerase-II-transcribed RNA characterized by the presence of a 5'-cap. The cap-binding complex (CBC), consisting of the nuclear cap-binding protein (NCBP) 2 and its adaptor NCBP1, is believed to bind all capped RNA and to be necessary for its processing and intracellular localization. Here we show that NCBP1, but not NCBP2, is required for cell viability and poly(A) RNA export. We identify C17orf85 (here named NCBP3) as a cap-binding protein that together with NCBP1 forms an alternative CBC in higher eukaryotes. NCBP3 binds mRNA, associates with components of the mRNA processing machinery and contributes to poly(A) RNA export. Loss of NCBP3 can be compensated by NCBP2 under steady-state conditions. However, NCBP3 becomes pivotal under stress conditions, such as virus infection. We propose the existence of an alternative CBC involving NCBP1 and NCBP3 that plays a key role in mRNA biogenesis.

Incoming RNA virus nucleocapsids containing a 5'-triphosphorylated genome activate RIG-I and antiviral signaling.

Host defense to RNA viruses depends on rapid intracellular recognition of viral RNA by two cytoplasmic RNA helicases: RIG-I and MDA5. RNA transfection experiments indicate that RIG-I responds to naked double-stranded RNAs (dsRNAs) with a triphosphorylated 5' (5'ppp) terminus. However, the identity of the RIG-I stimulating viral structures in an authentic infection context remains unresolved. We show that incoming viral nucleocapsids containing a 5'ppp dsRNA "panhandle" structure trigger antiviral signaling that commences with RIG-I, is mediated through the adaptor protein MAVS, and terminates with transcription factor IRF-3. Independent of mammalian cofactors or viral polymerase activity, RIG-I bound to viral nucleocapsids, underwent a conformational switch, and homo-oligomerized. Enzymatic probing and superresolution microscopy suggest that RIG-I interacts with the panhandle structure of the viral nucleocapsids. These results define cytoplasmic entry of nucleocapsids as the proximal RIG-I-sensitive step during infection and establish viral nucleocapsids with a 5'ppp dsRNA panhandle as a RIG-I activator.