Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein

Hepatitis B virus (HBV) is regarded as a stealth virus, invading and replicating efficiently in human liver undetected by host innate antiviral immunity. Here, we show that type I interferon (IFN) induction but not its downstream signaling is blocked by HBV replication in HepG2.2.15 cells. This effect may be partially due to HBV X protein (HBx), which impairs IFNβ promoter activation by both Sendai virus (SeV) and components implicated in signaling by viral sensors. As a deubiquitinating enzyme (DUB), HBx cleaves Lys63-linked polyubiquitin chains from many proteins except TANK-binding kinase 1 (TBK1). It binds and deconjugates retinoic acid-inducible gene I (RIG I) and TNF receptor-associated factor 3 (TRAF3), causing their dissociation from the downstream adaptor CARDIF or TBK1 kinase. In addition to RIG I and TRAF3, HBx also interacts with CARDIF, TRIF, NEMO, TBK1, inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase epsilon (IKKi) and interferon regulatory factor 3 (IRF3). Our data indicate that multiple points of signaling pathways can be targeted by HBx to negatively regulate production of type I IFN.     

USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors

Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity.     

Sensitivity of rabies virus to type I interferon is determined by the phosphoprotein gene

The growth of a virulent strain of fixed rabies virus, Nishigahara, in mouse neuroblastoma NA cells treated with type I interferon (IFN) was compared with that of a derivative avirulent strain, Ni-CE. Nishigahara strain was slightly sensitive to IFN treatment but still grew more efficiently than did Ni-CE strain in IFN-treated NA cells. Furthermore, a virulent chimeric virus with the phosphoprotein gene from Nishigahara strain in the Ni-CE genome was less sensitive to IFN treatment than was Ni-CE strain, indicating that the IFN sensitivity is determined by the phosphoprotein gene of the virus.     

SUMOylation of RIG-I positively regulates the type I interferon signaling

Retinoic acid-inducible gene-I (RIG-I) functions as an intracellular pattern recognition receptor (PRR) that recognizes the 5’-triphosphate moiety of single-stranded RNA viruses to initiate the innate immune response. Previous studies have shown that Lys63-linked ubiquitylation is required for RIG-I activation and the downstream anti-viral type I interferon (IFN-I) induction. Herein we reported that, RIG-I was also modified by small ubiquitin-like modifier-1 (SUMO-1). Functional analysis showed that RIG-I SUMOylation enhanced IFN-I production through increased ubiquitylation and the interaction with its downstream adaptor molecule Cardif. Our results therefore suggested that SUMOylation might serve as an additional regulatory tier for RIG-I activation and IFN-I signaling.     

ISG15 modification of filamin B negatively regulates the type I interferon‐induced JNK signalling pathway

Interferon (IFN)‐induced signalling pathways have essential functions in innate immune responses. In response to type I IFNs, filamin B tethers RAC1 and a Jun N‐terminal kinase (JNK)‐specific mitogen‐activated protein kinase (MAPK) module—MEKK1, MKK4 and JNK—and thereby promotes the activation of JNK and JNK‐mediated apoptosis. Here, we show that type I IFNs induce the conjugation of filamin B by interferon‐stimulated gene 15 (ISG15). ISGylation of filamin B led to the release of RAC1, MEKK1 and MKK4 from the scaffold protein and thus to the prevention of sequential activation of the JNK cascade. By contrast, blockade of filamin B ISGylation by substitution of Lys 2467 with arginine or by knockdown of ubiquitin‐activating enzyme E1‐like (UBEL1) prevented the release of the signalling molecules from filamin B, resulting in persistent promotion of JNK activation and JNK‐mediated apoptosis. These results indicate that filamin B ISGylation acts as a negative feedback regulatory gate for the desensitization of type I IFN‐induced JNK signalling.     

Thermal biology of mosquito‐borne disease

Mosquito‐borne diseases cause a major burden of disease worldwide. The vital rates of these ectothermic vectors and parasites respond strongly and nonlinearly to temperature and therefore to climate change. Here, we review how trait‐based approaches can synthesise and mechanistically predict the temperature dependence of transmission across vectors, pathogens, and environments. We present 11 pathogens transmitted by 15 different mosquito species – including globally important diseases like malaria, dengue, and Zika – synthesised from previously published studies. Transmission varied strongly and unimodally with temperature, peaking at 23–29ºC and declining to zero below 9–23ºC and above 32–38ºC. Different traits restricted transmission at low versus high temperatures, and temperature effects on transmission varied by both mosquito and parasite species. Temperate pathogens exhibit broader thermal ranges and cooler thermal minima and optima than tropical pathogens. Among tropical pathogens, malaria and Ross River virus had lower thermal optima (25–26ºC) while dengue and Zika viruses had the highest (29ºC) thermal optima. We expect warming to increase transmission below thermal optima but decrease transmission above optima. Key directions for future work include linking mechanistic models to field transmission, combining temperature effects with control measures, incorporating trait variation and temperature variation, and investigating climate adaptation and migration.     

Sarbecovirus ORF6 proteins hamper induction of interferon signaling

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ATPase‐driven oligomerization of RIG‐I on RNA allows optimal activation of type‐I interferon

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.     

Inhibition of parathormone-stimulated bone resorption by type I interferon

The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation.     

ANXA1促进Ⅰ型干扰素表达抑制口蹄疫病毒的复制

【目的】研究膜联蛋白A1(Annexin A1,ANXA1)对Ⅰ型干扰素(I-IFN)表达及口蹄疫病毒(FMDV)复制的影响。【方法】开展过表达及Knockdown实验,检测ANXA1对FMDV复制的影响。利用双荧光素酶报告系统检测ANXA1对ISRE和IFN-β启动子元件活化的影响。双荧光素酶报告系统鉴定ANXA1调控Ⅰ-IFN通路活化靶分子。Western blotting检测ANXA1对干扰素调解因子3(IRF3)磷酸化的影响。Real-time PCR检测ANXA1对干扰素刺激基因(ISGs)的影响。【结果】过表达ANXA1显著抑制FMDV的复制;下调ANXA1表达促进FMDV复制(P0.01或P0.05);ANXA1促Ⅰ型干扰素通路活化,呈现剂量依赖性(P0.01)。ANXA1显著增强IRF3的磷酸化,促进ISGs的表达(P0.01或P0.05)。【结论】ANXA1促进Ⅰ-IFN表达,抑制FMDV的复制。     

The evolution of the type I interferon gene family in mammals

A phylogenetic analysis of mammalian type I interferon (IFN) genes showed: (1) that the three main subfamilies of these genes in mammals (IFN-, IFN-, and IFN-) diverged after the divergence of birds and mammals but before radiation of the eutherian orders and (2) that IFN- diverged first. Although apparent cases of interlocus recombination among mouse IFN- genes were identified, the hypothesis that coding regions of IFN- genes have been homogenized within species by interlocus recombination was not supported. Flanking regions as well as coding regions of IFN- were more similar within human and mouse than between these species; and reconstruction of the pattern of nucleotide substitution in IFN- coding regions of four mammalian species by the maximum parsimony method suggested that parallel substitutions have occurred far more frequently between species than within species. Therefore, it seems likely that IFN- genes have duplicated independently within different eutherian orders. In general, type I IFN genes are subject to purifying selection, which in the case of IFN- and IFN- is strongest in the putative receptor-binding domains. However, analysis of the pattern of nucleotide substitution among IFN- genes suggested that positive Darwinian selection may have acted in some cases to diversify members of this subfamily at the amino acid level.     

BST2 inhibits type I IFN (interferon) signaling by accelerating MAVS degradation through CALCOCO2-directed autophagy

Macroautophagy/autophagy is a conserved lysosomal degradation system that breaks down intracellular material through the formation of double-membrane autophagosomes in eukaryotic cells. Cargo receptors have been shown to play essential roles in capturing and delivering specific substrates into phagophores, the precursors to autophagosomes, for degradation. However, the detailed mechanism underlying selective recognition of the substrates for autophagic degradation remains poorly understood. Recently, we have revealed that IFN (interferon)-induced BST2 recruits the E3 ubiquitin ligase MARCH8 to catalyze the K27-linked ubiquitination of MAVS for CALCOCO2-directed autophagic degradation, hence inhibiting DDX58-mediated type I interferon signaling through a negative feedback loop.     

RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity

TANK‐binding kinase 1 (TBK1) activation is a central event in type I interferon production in anti‐virus innate immunity. However, the regulatory mechanism underlying TBK1 activation remains unclear. Here we report that Raf kinase inhibitory protein (RKIP) is essential for TBK1 activation and type I interferon production triggered by viral infection. Upon viral infection, RKIP is phosphorylated at serine 109 (S109) by TBK1. Phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation. Mutation of RKIP S109 to alanine abrogates the interaction between RKIP and TBK1, and the anti‐viral function of RKIP. RKIP deficiency inhibits intracellular double‐stranded RNA‐ or DNA‐induced type I interferon production. Consistently, RKIP deficiency renders the mice more susceptible to vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) infections. This study reveals a previously unrecognized positive feedback loop between RKIP and TBK1 that is essential for type I interferon production in anti‐viral innate immunity.     

汉坦病毒感染对Ⅰ型干扰素应答的调节机制

汉坦病毒主要感染人内皮细胞,细胞在病毒感染早期诱导生成的Ⅰ型干扰素(IFN-Ⅰ)可阻断汉坦病毒的复制,但不同的病毒蛋白和不同型别的汉坦病毒在IFN应答的调节机制上可能有所不同。就汉坦病毒感染对IFN应答的调节机制进行了综述。     

Ligand-induced assembling of the type I interferon receptor on supported lipid bilayers

Type I interferons (IFNs) elicit antiviral, antiproliferative and immuno-modulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons, in particular IFNalphas and IFNbeta, suggests different modes of receptor engagement. Using reflectometric interference spectroscopy (RIfS), we studied kinetics and affinities of the interactions between IFNs and the extracellular receptor domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC). For IFNalpha2, we determined a K(D) value of 3 nM and 5 microM for the interaction with ifnar2-EC and ifnar1-EC, respectively. As compared to IFNalpha2, IFNbeta formed complexes with ifnar2-EC as well as ifnar1-EC with substantially higher affinity. For neither IFNalpha2 nor IFNbeta was stabilization of the complex with ifnar1-EC in the presence of soluble ifnar2-EC observed. We investigated ligand-induced complex formation with ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers by RIfS and total internal reflection fluorescence spectroscopy. We observed very stable binding of IFNalpha2 at high receptor surface concentrations with an apparent k(d) value approximately 200 times lower than that for ifnar2-EC alone. The apparent k(d) value was strongly dependent on the surface concentration of the receptor components, suggesting kinetic stabilization. This was corroborated by the fast exchange of labeled IFNalpha2 bound to the receptor by unlabeled IFNalpha2. Taken together, our results indicate that IFN first binds to ifnar2 and subsequently recruits ifnar1 in a transient fashion. In particular, this second step is much more efficient for IFNbeta than for IFNalpha2, which could explain differential activities observed for these IFNs.     

Functional cartography of the ectodomain of the type I interferon receptor subunit ifnar1

Ligand-induced cross-linking of the type I interferon (IFN) receptor subunits ifnar1 and ifnar2 induces a pleiotrophic cellular response. Several studies have suggested differential signal activation by flexible recruitment of the accessory receptor subunit ifnar1. We have characterized the roles of the four Ig-like sub-domains (SDs) of the extracellular domain of ifnar1 (ifnar1-EC) for ligand recognition and receptor assembling. Various sub-fragments of ifnar1-EC were expressed in insect cells and purified to homogeneity. Solid phase binding assays with the ligands IFN(alpha)2 and IFN(beta) revealed that all three N-terminal SDs were required and sufficient for ligand binding, and that IFN(alpha)2 and IFN(beta) compete for this binding site. Cellular binding assays with different fragments, however, highlighted the key role of the membrane-proximal SD for the formation of an in situ IFN-receptor complex. Even substitution with the corresponding SD from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayers in vitro revealed that the membrane-proximal SD controls appropriate orientation of the receptor on the membrane, which is required for efficient association of ifnar1 into the ternary complex.     

Pterostilbene effectively inhibits influenza A virus infection by promoting the type I interferon production

With the prevalence of novel strains and drug-resistant influenza viruses, there is an urgent need to develop effective and low-toxicity anti-influenza therapeutics. Regulation of the type I interferon antiviral response is considered an attractive therapeutic strategy for viral infection. Pterostilbene, a 3,5-dimethoxy analog of resveratrol, is known for its remarkable pharmacological activity. Here, we found that pterostilbene effectively inhibited influenza A virus infection and mainly affected the late stages of viral replication. A mechanistic study showed that the antiviral activity of pterostilbene might promote the induction of antiviral type I interferon and expression of its downstream interferon-stimulated genes during viral infection. The same effect of pterostilbene was also observed in the condition of polyinosinic-polycytidylic acid (poly I:C) transfection. Further study showed that pterostilbene interacted with influenza non-structural 1 (NS1) protein, inhibited ubiquitination mediated degradation of RIG-I and activated the downstream antiviral pathway, orchestrating an antiviral state against influenza virus in the cell. Taken together, pterostilbene could be a promising anti-influenza agent for future antiviral drug exploitation and compounds with similar structures may provide new options for the development of novel inhibitors against influenza A virus (IAV).