Cytokine-independent upregulation of MDA5 in viral infection

The RNA helicases RIG-I and MDA5 detect virus infection of dendritic cells (DCs) leading to cytokine induction. Maximal sensitivity for virus detection by these helicases is obtained after their upregulation, which is thought to occur primarily through type I interferon (IFN) signaling. Here we demonstrate that in response to paramyxovirus infection, RIG-I upregulation requires type I IFN whereas MDA5 expression is increased by Sendai virus infection independently of signaling mediated by type I IFN, STAT1, tumor necrosis factor alpha, or NF-kappaB. This MDA5 upregulation is largely lost in IRF3 knockout DCs and is achieved in type I IFN-deficient cells expressing constitutively active IRF3.     

Crystal structure of human IPS-1/MAVS/VISA/Cardif caspase activation recruitment domain

Background  

IPS-1/MAVS/VISA/Cardif is an adaptor protein that plays a crucial role in the induction of interferons in response to viral infection. In the initial stage of the intracellular antiviral response two RNA helicases, retinoic acid inducible gene-I (RIG-I) and melanoma differentiation-association gene 5 (MDA5), are independently able to bind viral RNA in the cytoplasm. The 62 kDa protein IPS-1/MAVS/VISA/Cardif contains an N-terminal caspase activation and recruitment (CARD) domain that associates with the CARD regions of RIG-I and MDA5, ultimately leading to the induction of type I interferons. As a first step towards understanding the molecular basis of this important adaptor protein we have undertaken structural studies of the IPS-1 MAVS/VISA/Cardif CARD region.     

RIG-I-mediated antiviral signaling is inhibited in HIV-1 infection by a protease-mediated sequestration of RIG-I

The rapid induction of type I interferon (IFN) is essential for establishing innate antiviral responses. During infection, cytoplasmic viral RNA is sensed by two DExD/H box RNA helicases, RIG-I and MDA5, ultimately driving IFN production. Here, we demonstrate that purified genomic RNA from HIV-1 induces a RIG-I-dependent type I IFN response. Both the dimeric and monomeric forms of HIV-1 were sensed by RIG-I, but not MDA5, with monomeric RNA, usually found in defective HIV-1 particles, acting as a better inducer of IFN than dimeric RNA. However, despite the presence of HIV-1 RNA in the de novo infection of monocyte-derived macrophages, HIV-1 replication did not lead to a substantial induction of IFN signaling. We demonstrate the existence of an evasion mechanism based on the inhibition of the RIG-I sensor through the action of the HIV-1 protease (PR). Indeed, the ectopic expression of PR resulted in the inhibition of IFN regulatory factor 3 (IRF-3) phosphorylation and decreased expression of IFN and interferon-stimulated genes. A downregulation of cytoplasmic RIG-I levels occurred in cells undergoing a single-cycle infection with wild-type provirus BH10 but not in cells transfected with a protease-deficient provirus, BH10-PR(-). Cellular fractionation and confocal microscopy studies revealed that RIG-I translocated from the cytosol to an insoluble fraction during the de novo HIV-1 infection of monocyte-derived macrophages, in the presence of PR. The loss of cytoplasmic RIG-I was prevented by the lysosomal inhibitor E64, suggesting that PR targets RIG-I to the lysosomes. This study reveals a novel PR-dependent mechanism employed by HIV-1 to counteract the early IFN response to viral RNA in infected cells.     

RIG-I mediates the co-induction of tumor necrosis factor and type I interferon elicited by myxoma virus in primary human macrophages

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.     

Coxsackievirus Cloverleaf RNA Containing a 5′ Triphosphate Triggers an Antiviral Response via RIG-I Activation

Upon viral infections, pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate an antiviral state associated with the production of type I interferons (IFNs) and inflammatory markers. Type I IFNs play crucial roles in innate antiviral responses by inducing expression of interferon-stimulated genes and by activating components of the adaptive immune system. Although pegylated IFNs have been used to treat hepatitis B and C virus infections for decades, they exert substantial side effects that limit their use. Current efforts are directed toward the use of PRR agonists as an alternative approach to elicit host antiviral responses in a manner similar to that achieved in a natural infection. RIG-I is a cytosolic PRR that recognizes 5′ triphosphate (5′ppp)-containing RNA ligands. Due to its ubiquitous expression profile, induction of the RIG-I pathway provides a promising platform for the development of novel antiviral agents and vaccine adjuvants. In this study, we investigated whether structured RNA elements in the genome of coxsackievirus B3 (CVB3), a picornavirus that is recognized by MDA5 during infection, could activate RIG-I when supplied with 5′ppp. We show here that a 5′ppp-containing cloverleaf (CL) RNA structure is a potent RIG-I inducer that elicits an extensive antiviral response that includes induction of classical interferon-stimulated genes, as well as type III IFNs and proinflammatory cytokines and chemokines. In addition, we show that prophylactic treatment with CVB3 CL provides protection against various viral infections including dengue virus, vesicular stomatitis virus and enterovirus 71, demonstrating the antiviral efficacy of this RNA ligand.     

Regulation of RLR-mediated innate immune signaling--it is all about keeping the balance

The current view of cytoplasmic RNA-mediated innate immune signaling involves the differential activation of the RNA helicases retinoic acid-inducible gene 1 (RIG-I), melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology-2 (LGP2) by distinct RNA viruses. RIG-I, MDA5 and LGP2 form the RIG-I like receptor family (RLR). Since the initial characterization of the RLRs rapid progress has been made in the understanding of the molecular mechanisms that upon virus infection lead to the activation of downstream signaling cascades and the subsequent induction of type I interferon (IFN) and proinflammatory cytokines by these receptors. However, antiviral responses must be tightly regulated in order to prevent uncontrolled production of type I IFN that might have deleterious effects on the host. Exploring the structural and molecular mechanisms that underlie RLR signaling thus was accompanied by the discovery of how RLR-dependent antiviral responses are modulated. This article summarizes the current understanding of endogenous regulation in RLR signaling by various intrinsic molecules that exert their regulatory function in both the steady state or upon viral infection by targeting multiple steps of the signaling cascade.     

Mechanism of mda-5 Inhibition by Paramyxovirus V Proteins

The RNA helicases encoded by melanoma differentiation-associated gene 5 (mda-5) and retinoic acid-inducible gene I (RIG-I) detect foreign cytoplasmic RNA molecules generated during the course of a virus infection, and their activation leads to induction of type I interferon synthesis. Paramyxoviruses limit the amount of interferon produced by infected cells through the action of their V protein, which binds to and inhibits mda-5. Here we show that activation of both mda-5 and RIG-I by double-stranded RNA (dsRNA) leads to the formation of homo-oligomers through self-association of the helicase domains. We identify a region within the helicase domain of mda-5 that is targeted by all paramyxovirus V proteins and demonstrate that they inhibit activation of mda-5 by blocking dsRNA binding and consequent self-association. In addition to this commonly targeted domain, some paramyxovirus V proteins target additional regions of mda-5. In contrast, V proteins cannot bind to RIG-I and consequently have no effect on the ability of RIG-I to bind dsRNA or to form oligomers.     

RIG-I is required for the inhibition of measles virus by retinoids

Vitamin A can significantly decrease measles-associated morbidity and mortality. Vitamin A can inhibit the replication of measles virus (MeV) in vitro through an RARα- and type I interferon (IFN)-dependent mechanism. Retinoid-induced gene I (RIG-I) expression is induced by retinoids, activated by MeV RNA and is important for IFN signaling. We hypothesized that RIG-I is central to retinoid-mediated inhibition of MeV in vitro. We demonstrate that RIG-I expression is increased in cells treated with retinoids and infected with MeV. The central role of RIG-I in the retinoid-anti-MeV effect was demonstrated in the Huh-7/7.5 model; the latter cells having non-functional RIG-I. RAR-dependent retinoid signaling was required for the induction of RIG-I by retinoids and MeV. Retinoid signaling was also found to act in combination with IFN to induce high levels of RIG-I expression. RIG-I promoter activation required both retinoids and MeV, as indicated by markers of active chromatin. IRF-1 is known to be regulated by retinoids and MeV, but we found recruitment of IRF-1 to the RIG-I promoter by retinoids alone. Using luciferase expression constructs, we further demonstrated that the IRF-1 response element of RIG-I was required for RIG-I activation by retinoids or IFN. These results reveal that retinoid treatment and MeV infection induces significant RIG-I. RIG-I is required for the retinoid-MeV antiviral response. The induction is dependent on IFN, retinoids and IRF-1.     

Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate

Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation.     

RIG-I family RNA helicases: cytoplasmic sensor for antiviral innate immunity

Viral infection is detected by cellular sensors as foreign nucleic acid and initiates innate antiviral responses, including the activation of type I interferon (IFN) and proinflammatory cytokines. Recent advances in cytoplasmic virus sensors highlight their essential role in the induction of innate immunity. Moreover, it is intriguing to understand how they can discriminate innate RNA from viral foreign RNA. In this mini-review, we focus on these cytoplasmic virus sensors, termed retinoic acid inducible gene-I (RIG-I)-like receptors (RLRs), and discuss their function in the innate immune system.     

RIG-I Self-Oligomerization Is Either Dispensable or Very Transient for Signal Transduction

Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments in vitro upon activation, RIG-I is believed to oligomerize after RNA binding in order to transduce a signal. Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales (Ebola, rabies and measles viruses) leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.     

Suppression of RNA interference by adenovirus virus-associated RNA

We show that human adenovirus inhibits RNA interference (RNAi) at late times of infection by suppressing the activity of two key enzyme systems involved, Dicer and RNA-induced silencing complex (RISC). To define the mechanisms by which adenovirus blocks RNAi, we used a panel of mutant adenoviruses defective in virus-associated (VA) RNA expression. The results show that the virus-associated RNAs, VA RNAI and VA RNAII, function as suppressors of RNAi by interfering with the activity of Dicer. The VA RNAs bind Dicer and function as competitive substrates squelching Dicer. Further, we show that VA RNAI and VA RNAII are processed by Dicer, both in vitro and during a lytic infection, and that the resulting short interfering RNAs (siRNAs) are incorporated into active RISC. Dicer cleaves the terminal stem of both VA RNAI and VA RNAII. However, whereas both strands of the VA RNAI-specific siRNA are incorporated into RISC, the 3' strand of the VA RNAII-specific siRNA is selectively incorporated during a lytic infection. In summary, our work shows that adenovirus suppresses RNAi during a lytic infection and gives insight into the mechanisms of RNAi suppression by VA RNA.     

Paramyxovirus V proteins interact with the RNA Helicase LGP2 to inhibit RIG-I-dependent interferon induction

RIG-I and mda-5 are activated by viral RNA and stimulate type I interferon production. Laboratory of genetics and physiology 2 (LGP2) shares homology with RIG-I and mda-5 but lacks the CARD domains required for signaling. The V proteins of paramyxoviruses limit interferon induction by binding mda-5 and preventing its activation; however, they do not bind RIG-I and have not been considered inhibitors of RIG-I signaling. Here we uncover a novel mechanism of RIG-I inhibition in which the V protein of parainfluenzavirus type 5 (PIV5; formerly known as simian virus type 5 [SV5]) interacts with LGP2 and cooperatively inhibits induction by RIG-I ligands. A complex between RIG-I and LGP2 is observed in the presence of PIV5-V, and we propose that this complex is refractory to activation by RIG-I ligands. The V proteins from other paramyxoviruses also bind LGP2 and demonstrate LGP2-dependent inhibition of RIG-I signaling. This is significant, because it demonstrates a general mechanism for the targeting of the RIG-I pathway by paramyxoviruses.     

Characterization of a low-molecular-weight virus-associated (VA) RNA encoded by simian adenovirus type 7 which functionally can substitute for adenovirus type 5 VA RNAI.

Human adenoviruses (Ads), like Ad type 2 (Ad2) and Ad5, encode a low-molecular-weight RNA (designated virus-associated [VA] RNAI) which is required for the efficient translation of viral mRNAs late after infection. We cloned and characterized a VA RNA gene from simian adenovirus type 7 (SA7) which appears to have biological activity analogous to that of Ad2 VA RNAI. Thus, SA7 VA RNA stimulates protein synthesis in a transient expression assay and can also functionally substitute for VA RNAI during lytic growth of human Ad5. The SA7 genome encodes only one VA RNA species, in contrast to human Ad2, which encodes two distinct species. This RNA is transcribed by RNA polymerase III in the rightward direction from a gene located at about coordinate 30 on the viral genome, like its Ad2 counterparts. SA7 VA RNA shows only a limited primary sequence homology with the Ad2 VA RNAs (approximately 55%); the flanking sequences, in fact, are better conserved than the VA RNA gene itself. The predicted secondary structure of SA7 VA RNA is, however, very similar to that of Ad2 VA RNAI, inferring that the double-stranded nature rather than the primary sequence of VA RNA is important for its biological activity.     

The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I

The paramyxovirus Sendai (SV), is a well-established inducer of IFN-alphabeta gene expression. In this study we show that SV induces IFN-alphabeta gene expression normally in cells from mice with targeted deletions of the Toll-IL-1 resistance domain containing adapters MyD88, Mal, Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF), and TRIF-related adaptor molecule TLR3, or the E3 ubiquitin ligase, TNFR-associated factor 6. This TLR-independent induction of IFN-alphabeta after SV infection is replication dependent and mediated by the RNA helicase, retinoic acid-inducible gene-I (RIG-I) and not the related family member, melanoma differentiation-associated gene 5. Furthermore, we characterize a RIG-I-like RNA helicase, Lgp2. In contrast to RIG-I or melanoma differentiation-associated gene 5, Lgp2 lacks signaling caspase recruitment and activation domains. Overexpression of Lgp2 inhibits SV and Newcastle disease virus signaling to IFN-stimulated regulatory element- and NF-kappaB-dependent pathways. Importantly, Lgp2 does not prevent TLR3 signaling. Like RIG-I, Lgp2 binds double-stranded, but not single-stranded, RNA. Quantitative PCR analysis demonstrates that Lgp2 is present in unstimulated cells at a lower level than RIG-I, although both helicases are induced to similar levels after virus infection. We propose that Lgp2 acts as a negative feedback regulator of antiviral signaling by sequestering dsRNA from RIG-I.