Alpha and Beta Type 1 Interferon Signaling: Passage for Diverse Biologic Outcomes.

Type I interferon (IFN-I) elicits a complex cascade of events in response to microbial infection. Here, we review recent developments illuminating the large number of IFN-I species and describing their unique biologic functions.

Structural linkage between ligand discrimination and receptor activation by type I interferons.

Type I Interferons (IFNs) are important cytokines for innate immunity against viruses and cancer. Sixteen human type I IFN variants signal through the same cell-surface receptors, IFNAR1 and IFNAR2, yet they can evoke markedly different physiological effects. The crystal structures of two human type I IFN ternary signaling complexes containing IFNα2 and IFNω reveal recognition modes and heterotrimeric architectures that are unique among the cytokine receptor superfamily but conserved between different type I IFNs. Receptor-ligand cross-reactivity is enabled by conserved receptor-ligand "anchor points" interspersed among ligand-specific interactions that "tune" the relative IFN-binding affinities, in an apparent extracellular "ligand proofreading" mechanism that modulates biological activity. Functional differences between IFNs are linked to their respective receptor recognition chemistries, in concert with a ligand-induced conformational change in IFNAR1, that collectively control signal initiation and complex stability, ultimately regulating differential STAT phosphorylation profiles, receptor internalization rates, and downstream gene expression patterns.

Alum-anchored intratumoral retention improves the tolerability and antitumor efficacy of type I interferon therapies.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNß therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNß that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

Unique duplication of IFNh genes in Nile tilapia (Oreochromis niloticus) reveals lineage-specific evolution of IFNh in perciform fishes.

Fish appear to harbour a complex type I IFN repertoire containing subgroups a, b, c, d, e, f, and h, and IFNh is only reported in perciform fishes. However, no multiple copies of IFNh gene has been identified in fish to date. In this study, two IFNh genes named On-IFNh1 and On-IFNh2 were cloned from Nile tilapia, Oreochromis niloticus. The predicted proteins of On-IFNh1 and On-IFNh2 contain several structural features known in type I IFNs, and estimation of divergence time revealed that these two genes may have arisen from a much recent local duplication event. On-IFNh genes were constitutively expressed in all tissues examined, with the highest expression level observed in gill, and were rapidly induced in all organs/tissues tested following the stimulation of poly(I:C). In addition, both recombinant On-IFNh1 and On-IFNh2 trigger a relative delayed but sustained induction of interferon-stimulated genes (ISGs), whereas recombinant On-IFNc elicits a rapid and transient expression of ISGs in vivo. The present study thus contributes to a better understanding of the functional properties of tilapia interferons, and also provides a new insight into the evolution of IFNh in fish.

Identification of type I IFNs and their receptors in a cyprinid fish, the topmouth culter Culter alburnus.

In fish, type I IFNs are classified into three groups, i.e. group one, group two and group three, and further separated into seven subgroups based on the number of conserved cysteines and phylogenetic relationships. In the present study, four type I IFNs, named as IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, as reported in zebrafish, were identified in a cyprinid, the topmouth culter, Culter alburnus, a species introduced recently into China's aquaculture. These IFNs may be classified as IFNa, IFNc, IFNc and IFNd in a recent nomenclature, with IFNa and IFNd having two cysteines in group one, and IFNc four cysteines in group two. These IFNs, together with their possible receptors, IFNϕ1, IFNϕ2, IFNϕ3, IFNϕ4, and CRFB1, CRFB2 and CRFB5 have an open reading frame (ORF) of 540, 552, 567, 516 bp, and 1572, 1392, 1125 bp, respectively. These IFNs have high amino acid sequence identities, being 91.1-93.6% and 66.9-77.3%, with those in grass carp and zebrafish, respectively, and are expressed constitutively in organs/tissues examined in the fish. The expression of these IFNs can be further induced following poly (I:C) stimulation. However, the possible function of these IFNs and their signalling pathway are of interest for further research.

Ctenopharyngodon idellus DDX41 initiates IFN I and ISG15 expression in response to GCRV infection.

As a member of DExD/H-box helicase family, DDX41 (DEAD box polypeptide 41) acts as an intracellular DNA sensor that induces type I IFN expression in mammals. Fish DDX41 shares some similar properties with the mammalian counterparts. In this study, a DDX41 orthologous gene from grass carp (Ctenopharyngodon idellus) (CiDDX41) was cloned and characterized. The ORF of CiDDX41 encodes a polypeptide of 614 amino acids. Multiple alignments showed that DDX41 is highly conserved among different species. Phylogenetic tree analysis revealed that CiDDX41 shares a high degree of homology with Sinocyclocheilus rhinocerous DDX41. CiDDX41 is highly expressed in kidney, intestines, liver and spleen. Their expressions are up-regulated more obviously after the treatment with GCRV. Over-expression of CiDDX41 in CIK cells increases the transcription level of grass carp IFN I and ISG15. On the contrary, knockdown of CiDDX41 inhibits the IFN I and ISG15 transcription. Moreover, a part of CiDDX41 translocates from the nuclear to cytoplasm to interact with grass carp STING (CiSTING). In CIK cells, overexpression of CiDDX41 and CiSTING can promote the phosphorylation and nuclear-cytoplasm translocation of grass carp IRF7 (CiIRF7) and then acutely up-regulate the IFN I and ISG15 expression. However, the knockdown of CiDDX41 inhibits the phosphorylation IRF7. Taken together, all these results above suggested that CiDDX41 performs as an activator for innate immune through STING-IRF7 mediated signaling pathway.

Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3.

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

Defining the distinct, intrinsic properties of the novel type I interferon, IFNϵ.

The type I interferons (IFNs) are a family of cytokines with diverse biological activities, including antiviral, antiproliferative, and immunoregulatory functions. The discovery of the hormonally regulated, constitutively expressed IFNϵ has suggested a function for IFNs in reproductive tract homeostasis and protection from infections, but its intrinsic activities are untested. We report here the expression, purification, and functional characterization of murine IFNϵ (mIFNϵ). Recombinant mIFNϵ (rmIFNϵ) exhibited an α-helical fold characteristic of type I IFNs and bound to IFNα/ß receptor 1 (IFNAR1) and IFNAR2, but, unusually, it had a preference for IFNAR1. Nevertheless, rmIFNϵ induced typical type I IFN signaling activity, including STAT1 phosphorylation and activation of canonical type I IFN signaling reporters, demonstrating that it uses the JAK-STAT signaling pathway. We also found that rmIFNϵ induces the activation of T, B, and NK cells and exhibits antiviral, antiproliferative, and antibacterial activities typical of type I IFNs, albeit with 100-1000-fold reduced potency compared with rmIFNα1 and rmIFNß. Surprisingly, although the type I IFNs generally do not display cross-species activities, rmIFNϵ exhibited high antiviral activity on human cells, suppressing HIV replication and inducing the expression of known HIV restriction factors in human lymphocytes. Our findings define the intrinsic properties of murine IFNϵ, indicating that it distinctly interacts with IFNAR and elicits pathogen-suppressing activity with a potency enabling host defense but with limited toxicity, appropriate for a protein expressed constitutively in a sensitive mucosal site, such as the reproductive tract.

Identification, expression profiles and antiviral activities of a type I IFN from gibel carp Carassius auratus gibelio.

Type I interferons, as a class of multipotent cytokines, play a key role in host antiviral immune responses. In this study, a type I IFN coding gene of gibel carp, Carassius auratus gibelio, CagIFNa was cloned and sequenced. The full-length cDNA sequence of CagIFNa consists of 724 nucleotides that encode a predicted protein of 183 amino acids. CagIFNa has two highly conserved cysteine residues in the deduced protein, which is mostly conserved in the fish group I type I IFNs. CagIFNa was identified as a member of the IFNa subgroup of group I type I IFNs by phylogenetic analysis. CagIFNa transcripts were detected in all investigated tissues with higher levels in the liver, intestine, spleen and head kidney of gibel carp. Following injection with Cyprinid herpesvirus 2 (CyHV-2), CagIFNa gene expression was significantly inhibited in the spleen but delayed and then increased in head kidneys. Similarly, while CagIFNa expression was rapidly induced in gibel carp brain (GiCB) cells by poly I:C stimulation and its high induction level was delayed following CyHV-2 infection. CagIFNa overexpression in GiCB cells drastically reduced virus CPE and titer. Furthermore, several genes associated with type I IFN signaling pathway including IRF3, IRF7, IRF9, STAT1, Mx1 and PKR were induced in GiCB cells overexpressing CagIFNa upon CyHV-2 infection. These results show that CagIFNa plays a role in antiviral immune system in gibel carp.

Characterization and function of a group II type I interferon in the perciform fish, large yellow croaker (Larimichthys crocea).

Teleost fish possess two groups of type I interferons (IFNs) with two (group I IFNs) or four (group II IFNs) conserved cysteines, which are further classified into seven subgroups. In our previous study, two group I type I IFNs, LcIFNd and LcIFNh (a new subgroup member), were identified in the perciform fish, large yellow croaker (Larimichthys crocea). Here, we identified a group II type I IFN, LcIFNc, in this species. The deduced LcIFNc contained six cysteines, four of which are highly conserved (C1: C28, C2:C53, C3: C130, and C4:C159) in the fish group II type I IFNs, and a typical type I IFN signature motif was also found in it. Phylogenetic analysis indicated that LcIFNc belongs to the IFNc subgroup of fish group II type I IFNs. LcIFNc was constitutively expressed in all examined tissues, and was rapidly up-regulated in spleen and head kidney by poly(I:C) and Aeromonas hydrophila. Recombinant LcIFNc protein (rLcIFNc) could increase the expression of antiviral genes, Mx1, PKR and ISG15, in large yellow croaker peripheral blood leukocytes (PBLs). The rLcIFNc also exhibited obvious antiviral activity based on less cytopathic effect (CPE) and decreased expression levels of several viral genes in the rLcIFNc-treated grouper spleen (GS) cells following Singapore grouper iridovirus (SGIV) infection. Additionally, rLcIFNc was able to induce the expression of LcIFNc, as well as LcIFNd and LcIFNh in the PBLs and primary head kidney cells (HKCs) from large yellow croaker. These results therefore indicated that LcIFNc not only had antiviral activity, but also mediated the regulation of type I IFN response.

Sequence analysis and characterization of type I interferon and type II interferon from the critically endangered sturgeon species, A. dabryanus and A. sinensis.

In the present study, we identify three type I interferon (IFN) genes (Ad/AsIFNe1-3) and a type II IFN gene (Ad/AsIFNγ) from the Dabry's sturgeon (Acipenser dabryanus) and the Chinese sturgeon (Acipenser sinensis). Sequence analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ contain several conserved characteristics, including signal peptides, interferon alpha, beta, and delta (IFabd) domains, and N-glycosylation sites. Ad/AsIFNe1-3 belongs to the type I IFN group I subgroup, possessing two conserved cysteines residues (C1 and C3), and Ad/AsIFNγ contained a conserved nuclear localization sequence (NLS) motif. Ad/AsIFNe1-3 and Ad/AsIFNγ contain signature motifs indicative of their corresponding IFN group. The Ad/AsIFNe1-3 and Ad/AsIFNγ genes were found to consist of 5 exons/4 introns and 4 exons/3 introns, respectively. These IFNs were separated by four phase 0 introns (type I IFN) and three phase 0 introns (type II IFN). The sequences of IFNe1-3 and IFNγ from the Dabry's sturgeon and the Chinese sturgeon were closely aligned, suggested that these two species are closely related. Phylogenetic analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ clustered together with the corresponding homologous proteins from other fish species. AdIFNe1-3 were found to be high expressed in early embryonic development, suggesting that AdIFNe1-3 might indicate maternal transmission, while AdIFNγ may not mediate embryonic development. Tissue distribution analysis revealed that AdIFNe1-3 and AdIFNγ carry out biological functions in immune and non-immune tissues compartments. AdIFNe1-3 and AdIFNγ can be stimulated by polyinosinic-polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). AdIFNe1-3 have stronger antiviral activity than AdIFNγ, and AdIFNγ has a stronger antibacterial activity than AdIFNe1-3. The differential responses of these genes to poly I:C and LPS suggest differences in the mechanisms of defense against viruses and bacteria.

Denaturation and Aggregation of Interferon-τ in Aqueous Solution.

PURPOSE: To evaluate the different degrees of residual structure in the unfolded state of interferon-τ using chemical denaturation as a function of temperature by both urea and guanidinium hydrochloride. METHODS: Asymmetrical flow field-flow fractionation (AF4) using both UV and multi-angle laser light scattering (MALLS). Flow Microscopy. All subvisible particle imaging measurements were made using a FlowCAM flow imaging system. RESULTS: The two different denaturants provided different estimates of the conformational stability of the protein when extrapolated back to zero denaturant concentration. This suggests that urea and guanidinium hydrochloride (GnHCl) produce different degrees of residual structure in the unfolded state of interferon-τ. The differences were most pronounced at low temperature, suggesting that the residual structure in the denatured state is progressively lost when samples are heated above 25°C. The extent of expansion in the unfolded states was estimated from the m-values and was also measured using AF4. In contrast, the overall size of interferon-τ was determined by AF4 to decrease in the presence of histidine, which is known to bind to the native state, thereby providing conformational stabilization. Addition of histidine as the buffer resulted in formation of fewer subvisible particles over time at 50°C. Finally, the thermal aggregation was monitored using AF4 and the rate constants were found to be comparable to those determined previously by SEC and DLS. The thermal aggregation appears to be consistent with a nucleation-dependent mechanism with a critical nucleus size of 4 ± 1. CONCLUSION: Chemical denaturation of interferon-τ by urea or GnHCl produces differing amounts of residual structure in the denatured state, leading to differing estimates of conformational stability. AF4 was used to determine changes in size, both upon ligand binding as well as upon denaturation with GnHCl. Histidine appears to be the preferred buffer for interferon-τ, as shown by slower formation of soluble aggregates and reduced levels of subvisible particles when heated at 50°C.

Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

Interferons (IFNs) play a key role in the innate immunity of vertebrates against viral infections by inducing hundreds of IFN-stimulated genes (ISGs), such as isg15. Isg15 is an ubiquitin-like protein, which can conjugate cellular and viral proteins in a process called ISGylation, although it can also act as a cytokine-like protein. Gilthead seabream (Sparus aurata L.) is an important asymptomatic carrier of viral haemorrhagic septicaemia virus (VHSV) and nodavirus, representing a threat to other co-cultivated susceptible species. In order to better understand virus-host interactions in this fish species, this study addresses the identification and molecular characterization of seabream isg15 (sb-isg15). In addition, the modulation of transcript levels of sb-isg15 was analysed in SAF-1 cells and seabream acidophilic granulocytes (AGs) stimulated in vitro with different pathogen-associated molecular patterns (PAMPs) or inoculated with VHSV and striped jack nervous necrosis virus (SJNNV). The full-length cDNA of sb-isg15 gene, encoding a predicted protein of 155 amino acids, was identified and seen to share the same characteristics as other fish and mammalian isg15 genes. Here we report the clear induction of sb-isg15 transcript levels in SAF-1 cells and AGs stimulated with toll-like receptor (TLR) ligands, such as polyinosinic:polycytidylic acid (poly I:C) or genomic DNA from Vibrio anguillarum (VaDNA), respectively. Furthermore, VHSV and SJNNV inoculation induced a significant degree of sb-isg15 transcription in SAF-1 cells and AGs. However, the relative levels of viral RNA transcription showed that SJNNV replication seems to be more efficient than VHSV in both in vitro systems. Interestingly, sb-isg15 transcript induction elicited by VaDNA was reduced in VHSV- and SJNNV-inoculated AGs, suggesting an interference prompted by the viruses against the type I IFN system. Taken together, these findings support the use of seabream AGs as a valuable experimental system to study virus-host interactions, in which sb-isg15 seems to play an important role.

Identification and characterization of a type I interferon induced by cyprinid herpesvirus 2 infection in crucian carp Carassius auratus gibelio.

Crucian carp (Carassius auratus gibelio) is a popular food fish in Asia, and cyprinid herpesvirus 2 (CyHV-2) is the only known viral pathogen for crucian carp. Type I interferon genes are induced up on host cell recognition of viral nucleic acids and well recognized for their crucial roles in providing local or systemic protection against the viruses in various organisms. In a transcriptome analysis to uncover differentially expressed genes in crucian carp in response to CyHV-2 challenge, a partial interferon transcript was identified to be significantly up-regulated in the kidney of infected fish, which was named as crucian carp IFNc (ccIFNc). The complete ORF of ccIFNc was further determined by RACE technique, which spanned over 546 bp and encoded a polypeptide containing 182 amino acids. Phylogenetic analysis revealed that ccIFNc clustered with known type I IFN genes from other aquatic organisms. Quantitative RT-PCR analysis demonstrated that ccIFNc was constitutively expressed in all investigated tissues with a comparably higher expression level in spleen, gill, kidney, and muscle. Following challenge with CyHV-2, the transcriptional levels of ccIFNc were dramatically up-regulated in all of the tested tissues, especially in the spleen and gill with increased folds of 436 and 158, respectively. The intramuscular (i.m.) injection of a eukaryotic expression plasmid encoding ccIFNc (pEGFP-cIFNc) resulted in increased ccIFNc expression and reduced the mortality after the CyHV-2 challenge significantly. In summary, our data suggested that the ccIFNc belongs to the type I interferon family with a potential role in countering CyHV-2 infection in crucian carp.

Identification of 2 novel type I IFN genes in Japanese flounder, Paralichthys olivaceus.

Two novel type I interferon genes (JfIFN3 and JfIFN4) have been identified in Japanese flounder Paralichthys olivaceus. Open reading frames of JfIFN3 and JfIFN4 were 555bp and 528bp, encoding 184aa and 175aa, respectively. The genomic structures of JfIFN3 and JfIFN4 are composed of 5 exons and 4 introns. JfIFN4 has 2 conserved cysteine residues, while JfIFN3 has 4. JfIFN3 and JfIFN4 showed the highest amino acid sequence identities to turbot IFN1 (74%) and IFN2 (62%), respectively. Interestingly, JfIFN3 and JfIFN4 were clustered in distinct branches with JfIFN1 and JfIFN2, which have reported so far. The mRNA levels of JfIFN4 were apparently increased in the kidney and spleen at 3 h after ployI:C injection, while JfIFN1-3 were not detected by RT-PCR.

HBsAg blocks TYPE I IFN induced up-regulation of A3G through inhibition of STAT3.

Interferon (IFN) is a regularly utilized therapeutic for the treatment of chronic hepatitis B and appears to induce superior HBeAg seroconversion comparing nucleos/tide analogs. However, the mechanisms underlying IFN inhibition of HBV replication, as well as poor responses to IFN are unclear. Apobec3G has been reported to be involved in regulating HBV replication. In this study, we investigated Apobec3G expression and regulatory pathways during HBV infection. We show that over-expression of A3G leads to inhibition of HBV replication. We also show that IFN induces a significant increase in A3G protein expression, which is associated with STAT3 activation. We further show that A3G expression in HBV patients is lower compared to non-infected controls, possibly by HBsAg which inhibits IFN induced A3G up-regulation in a dose dependent manner. This process is likely mediated through inhibition of STAT3-Ser727 phosphorylation. The results presented in this study indicate that STAT3 plays an important role in IFN-induced A3G production, and HBsAg may correlated with poor response to IFN treatment.

Structural and dynamic determinants of type I interferon receptor assembly and their functional interpretation.

Type I interferons (IFNs) form a network of homologous cytokines that bind to a shared, heterodimeric cell surface receptor and engage signaling pathways that activate innate and adaptive immune responses. The ability of IFNs to mediate differential responses through the same cell surface receptor has been subject of a controversial debate and has important medical implications. During the past decade, a comprehensive insight into the structure, energetics, and dynamics of IFN recognition by its two-receptor subunits, as well as detailed correlations with their functional properties on the level of signal activation, gene expression, and biological responses were obtained. All type I IFNs bind the two-receptor subunits at the same sites and form structurally very similar ternary complexes. Differential IFN activities were found to be determined by different lifetimes and ligand affinities toward the receptor subunits, which dictate assembly and dynamics of the signaling complex in the plasma membrane. We present a simple model, which explains differential IFN activities based on rapid endocytosis of signaling complexes and negative feedback mechanisms interfering with ternary complex assembly. More insight into signaling pathways as well as endosomal signaling and trafficking will be required for a comprehensive understanding, which will eventually lead to therapeutic applications of IFNs with increased efficacy.

Expression and purification of buffalo interferon-tau and efficacy of recombinant buffalo interferon-tau for in vitro embryo development.

The aim of our study was to optimize growth and induction parameters, for expression and large scale purification of functionally active buffalo interferon tau, and to study its possible impact on in vitro blastocyst development. The buffalo interferon-tau gene (BuIFN-T1) bearing gene bank accession No. JX481984, with signal sequence, was obtained through polymerase chain reaction (PCR) from bovine early embryos and was cloned into pJET vector. After being verified, the fragments without signal sequence, were inserted into the expression vector pET-22b and the recombinant plasmid was induced to express the recombinant protein in a prokaryotic expression system. The recombinant BuIFN-T was confirmed by SDS-PAGE and Western blot and subjected to three steps of large scale purification using His Affinity chromatography, Anion Exchange chromatography and Gel Filtration chromatography. The purified recombinant BuIFN-T protein was validated by mass spectroscopy analysis. To examine the effect of recombinant BuIFN-T protein on developmental competency of buffalo embryos, purified recombinant BuIFN-T protein was added to in vitro embryo culture medium (at concentration of 0, 1µg/ml, 2µg/ml, 4µg/ml) for 9days. Addition of recombinant BuIFN-T (2µg/ml) significantly improved the rate of blastocyst production, 45.55% against 31.1% control (p<0.01). Here we conclude that the recombinant BuIFN-T was successfully purified to homogeneity from a prokaryotic expression system and it significantly increased the blastocyst production rate in buffalo. These findings suggest a potential impact of IFN-T in promoting embryonic growth and development.

Identification of STAT2 serine 287 as a novel regulatory phosphorylation site in type I interferon-induced cellular responses.

STAT2 is a positive modulator of the transcriptional response to type I interferons (IFNs). STAT2 acquires transcriptional function by becoming tyrosine phosphorylated and imported to the nucleus following type I IFN receptor activation. Although most STAT proteins become dually phosphorylated on specific tyrosine and serine residues to acquire full transcriptional activity, no serine phosphorylation site in STAT2 has been reported. To find novel phosphorylation sites, mass spectrometry of immunoprecipitated STAT2 was used to identify several phosphorylated residues. Of these, substitution of serine 287 with alanine (S287A) generated a gain-of-function mutant that enhanced the biological effects of IFN-α. S287A-STAT2 increased cell growth inhibition, prolonged protection against vesicular stomatitis virus infection and enhanced transcriptional responses following exposure of cells to IFN-α. In contrast, a phosphomimetic STAT2 mutant (S287D) produced a loss-of-function protein that weakly activated IFN-induced ISGs. Our mechanistic studies suggest that S287A-STAT2 likely mediates its gain-of-function effects by prolonging STAT2/STAT1 dimer activation and retaining it in transcriptionally active complexes with chromatin. Altogether, we have uncovered that in response to type I IFN, STAT2 is serine phosphorylated in the coiled-coil domain that when phosphorylated can negatively regulate the biological activities of type I IFNs.

Crystal structure of Zebrafish interferons I and II reveals conservation of type I interferon structure in vertebrates.

Interferons (IFNs) play a major role in orchestrating the innate immune response toward viruses in vertebrates, and their defining characteristic is their ability to induce an antiviral state in responsive cells. Interferons have been reported in a multitude of species, from bony fish to mammals. However, our current knowledge about the molecular function of fish IFNs as well as their evolutionary relationship to tetrapod IFNs is limited. Here we establish the three-dimensional (3D) structure of zebrafish IFNϕ1 and IFNϕ2 by crystallography. These high-resolution structures offer the first structural insight into fish cytokines. Tetrapods possess two types of IFNs that play an immediate antiviral role: type I IFNs (e.g., alpha interferon [IFN-α] and beta interferon [IFN-ß]) and type III IFNs (lambda interferon [IFN-λ]), and each type is characterized by its specific receptor usage. Similarly, two groups of antiviral IFNs with distinct receptors exist in fish, including zebrafish. IFNϕ1 and IFNϕ2 represent group I and group II IFNs, respectively. Nevertheless, both structures reported here reveal a characteristic type I IFN architecture with a straight F helix, as opposed to the remaining class II cytokines, including IFN-λ, where helix F contains a characteristic bend. Phylogenetic trees derived from structure-guided multiple alignments confirmed that both groups of fish IFNs are evolutionarily closer to type I than to type III tetrapod IFNs. Thus, these fish IFNs belong to the type I IFN family. Our results also imply that a dual antiviral IFN system has arisen twice during vertebrate evolution.