Radical SAM Domain Research Articles

Fused radical SAM and αKG-HExxH domain proteins contain a distinct structural fold and catalyse cyclophane formation and β-hydroxylation.

Two of nature's recurring binding motifs in metalloproteins are the CxxxCxxC motif in radical SAM enzymes and the 2-His-1-carboxylate motif found both in zincins and α-ketoglutarate and non-haem iron enzymes. Here we show the confluence of these two domains in a single post-translational modifying enzyme containing an N-terminal radical S-adenosylmethionine domain fused to a C-terminal 2-His-1-carboxylate (HExxH) domain. The radical SAM domain catalyses three-residue cyclophane formation and is the signature modification of triceptides, a class of ribosomally synthesized and post-translationally modified peptides. The HExxH domain is a defining feature of zinc metalloproteases. Yet the HExxH motif-containing domain studied here catalyses β-hydroxylation and is an α-ketoglutarate non-haem iron enzyme. We determined the crystal structure for this HExxH protein at 2.8 Å, unveiling a distinct structural fold, thus expanding the family of α-ketoglutarate non-haem iron enzymes with a class that we propose to name αKG-HExxH. αKG-HExxH proteins represent a unique family of ribosomally synthesized and post-translationally modified peptide modifying enzymes that can furnish opportunities for genome mining, synthetic biology and enzymology.

Molecular and functional characterization of viperin in golden pompano, Trachinotus ovatus

The radical S-adenosyl methionine domain-containing protein 2 (RSAD2), also known as viperin, plays a momentous and multifaceted role in antiviral immunity. However, the function of viperin is uninvestigated in golden pompano, Trachinotus ovatus. In the present study, a viperin homolog, named To-viperin, was cloned and characterized from golden pompano, and its role in response to grouper iridovirus (SGIV) and nervous necrosis virus (NNV) infection was investigated. The whole open reading frame (ORF) of To-viperin was composed of 1050 bp and encoded a polypeptide of 349 amino acids with 70.66%–83.51% identity with the known viperin homologs from other fish species. A variable N-terminal domain, a highly conserved C-terminal domain, and a conserved middle radical SAM domain (aa 61–271) with the three-cysteine motif CxxCxxC was found in To-viperin sequence. Expression analysis showed that To-viperin was constitutively expressed in all tested organs and was located mainly in the ER of golden pompano cells. Treatments with SGIV, poly I: C, or NNV could induce the up-regulation of viperin to varying degrees. The ectopic expression of To-viperin in vitro significantly reduced the viral titer of SGIV and NNV. Furthermore, To-viperin overexpression enhanced the expression of IFNc, IRF3, and ISG15 genes as well as, to a lesser extent, the IL-6 gene. In summary, our results suggested that the function of viperin is likely to be conserved in fish specise, as observed in other vertebrates, shedding light on the evolutionary conservation of viperin.

In-silico analyses provide strong statistical evidence for intra-species recombination events of the gyrA and CmeABC operon loci contributing to the continued emergence of resistance to fluoroquinolones in natural populations of Campylobacter jejuni

The continued emergence of Campylobacter jejuni strains resistant to fluoroquinolones (FQs) has posed a significant threat to global public health, leading frequently to undesirable outcomes of human campylobacteriosis treatment. The molecular genetic mechanisms contributing to the increased retention of resistance to FQs in natural populations of this species, especially in antibiotic-free environments, are not clearly understood. This study aimed to determine whether genetic recombination could be such a mechanism. We applied a large array of algorithms, imbedded in the SplitsTree and RDP4 software packages, to analyse the DNA sequences of the chromosomal loci, including the gyrA gene and the CmeABC operon, to identify events of their genetic recombination between C. jejuni strains. The SplitsTree analyses of the above genetic loci resulted in several parallelograms with the bootstrap values being in a range of 94.7 to 100, with the high fit estimates being 99.3 to 100. These analyses were further strongly supported by the Phi test results (P ≤ 0.02715) and the RDP4-generated statistics (P ≤ 0.04005). The recombined chromosomal regions, along with the gyrA gene and CmeABC operon loci, were also found to contain the genetic loci that included, but were not limited to, the genes encoding for phosphoribosyltransferase, lipoprotein, outer membrane motility protein, and radical SAM domain protein. These findings strongly suggest that the genetic recombination of the chromosomal regions involving gyrA, CmeABC, and their adjacent loci may be an additional mechanism underlying the constant emergence of epidemiologically successful FQ-resistant strains in natural populations of C. jejuni.

Viperin, an IFN-Stimulated Protein, Delays Rotavirus Release by Inhibiting Non-Structural Protein 4 (NSP4)-Induced Intrinsic Apoptosis.

Viral infections lead to expeditious activation of the host’s innate immune responses, most importantly the interferon (IFN) response, which manifests a network of interferon-stimulated genes (ISGs) that constrain escalating virus replication by fashioning an ill-disposed environment. Interestingly, most viruses, including rotavirus, have evolved numerous strategies to evade or subvert host immune responses to establish successful infection. Several studies have documented the induction of ISGs during rotavirus infection. In this study, we evaluated the induction and antiviral potential of viperin, an ISG, during rotavirus infection. We observed that rotavirus infection, in a stain independent manner, resulted in progressive upregulation of viperin at increasing time points post-infection. Knockdown of viperin had no significant consequence on the production of total infectious virus particles. Interestingly, substantial escalation in progeny virus release was observed upon viperin knockdown, suggesting the antagonistic role of viperin in rotavirus release. Subsequent studies unveiled that RV-NSP4 triggered relocalization of viperin from the ER, the normal residence of viperin, to mitochondria during infection. Furthermore, mitochondrial translocation of NSP4 was found to be impeded by viperin, leading to abridged cytosolic release of Cyt c and subsequent inhibition of intrinsic apoptosis. Additionally, co-immunoprecipitation studies revealed that viperin associated with NSP4 through regions including both its radical SAM domain and its C-terminal domain. Collectively, the present study demonstrated the role of viperin in restricting rotavirus egress from infected host cells by modulating NSP4 mediated apoptosis, highlighting a novel mechanism behind viperin’s antiviral action in addition to the intricacy of viperin–virus interaction.

Viperin_sv1 promotes RIG-I expression and suppresses SVCV replication through its radical SAM domain

SVCV infection is known to activate the host's innate immune responses, including the production of interferon (IFN) and interferon-stimulated genes (ISGs). Viperin_sv1 is a novel splice variant of viperin, which is induced during SVCV infection and proves to positively regulate the IFN activation and production. However, the underlying mechanism remains unsolved. In this study, the P protein of SVCV was identified to be the key to induce the mRNA modification and production of viperin_sv1 during the virus infection. Besides, Viperin_sv1 was able to trigger the RLR signaling cascades to activate type-1 interferon response. Additional analysis revealed that viperin_sv1 promoted the stability and function of RIG-I, which result in the production of IFN and ISGs. Moreover, the central SAM domain of viperin_sv1 was demonstrated to be essential for regulating RIG-I protein expression and inducing IFN production. Furthermore, this study also showed that SVCV replication could be inhibited by the viperin_sv1 SAM domain. In conclusion, our study demonstrates that viperin_sv1 reduces the replication of SVCV by promoting the RIG-I protein expression. Our findings identified the antiviral function played by the SAM domain of viperin_sv1 and suggested an antiviral mechanism that is conserved among different species.

Viperin inhibits classical swine fever virus replication by interacting with viral nonstructural 5A protein.

Classical swine fever virus (CSFV) is a single-stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon-inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK-15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co-immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N-terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV-induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.

Characterization and Transcript Expression Analyses of Atlantic Cod Viperin.

Viperin is a key antiviral effector in immune responses of vertebrates including the Atlantic cod (Gadus morhua). Using cloning, sequencing and gene expression analyses, we characterized the Atlantic cod viperin at the nucleotide and hypothetical amino acid levels, and its regulating factors were investigated. Atlantic cod viperin cDNA is 1,342 bp long, and its predicted protein contains 347 amino acids. Using in silico analyses, we showed that Atlantic cod viperin is composed of 5 exons, as in other vertebrate orthologs. In addition, the radical SAM domain and C-terminal sequences of the predicted Viperin protein are highly conserved among various species. As expected, Atlantic cod Viperin was most closely related to other teleost orthologs. Using computational modeling, we show that the Atlantic cod Viperin forms similar overall protein architecture compared to mammalian Viperins. qPCR revealed that viperin is a weakly expressed transcript during embryonic development of Atlantic cod. In adults, the highest constitutive expression of viperin transcript was found in blood compared with 18 other tissues. Using isolated macrophages and synthetic dsRNA (pIC) stimulation, we tested various immune inhibitors to determine the possible regulating pathways of Atlantic cod viperin. Atlantic cod viperin showed a comparable pIC induction to other well-known antiviral genes (e.g., interferon gamma and interferon-stimulated gene 15-1) in response to various immune inhibitors. The pIC induction of Atlantic cod viperin was significantly inhibited with 2-Aminopurine, Chloroquine, SB202190, and Ruxolitinib. Therefore, endosomal-TLR-mediated pIC recognition and signal transducers (i.e., PKR and p38 MAPK) downstream of the TLR-dependent pathway may activate the gene expression response of Atlantic cod viperin. Also, these results suggest that antiviral responses of Atlantic cod viperin may be transcriptionally regulated through the interferon-activated pathway.

The antiviral protein viperin regulates chondrogenic differentiation via CXCL10 protein secretion

Viperin (also known as radical SAM domain–containing 2 (RSAD2)) is an interferon-inducible and evolutionary conserved protein that participates in the cell's innate immune response against a number of viruses. Viperin mRNA is a substrate for endoribonucleolytic cleavage by RNase mitochondrial RNA processing (MRP) and mutations in the RNase MRP small nucleolar RNA (snoRNA) subunit of the RNase MRP complex cause cartilage-hair hypoplasia (CHH), a human developmental condition characterized by metaphyseal chondrodysplasia and severe dwarfism. It is unknown how CHH-pathogenic mutations in RNase MRP snoRNA interfere with skeletal development, and aberrant processing of RNase MRP substrate RNAs is thought to be involved. We hypothesized that viperin plays a role in chondrogenic differentiation. Using immunohistochemistry, real-time quantitative PCR, immunoblotting, ELISA, siRNA-mediated gene silencing, plasmid-mediated gene overexpression, label-free MS proteomics, and promoter reporter bioluminescence assays, we discovered here that viperin is expressed in differentiating chondrocytic cells and regulates their protein secretion and the outcome of chondrogenic differentiation by influencing transforming growth factor β (TGF-β)/SMAD family 2/3 (SMAD2/3) activity via C-X-C motif chemokine ligand 10 (CXCL10). Of note, we observed disturbances in this viperin–CXCL10–TGF-β/SMAD2/3 axis in CHH chondrocytic cells. Our results indicate that the antiviral protein viperin controls chondrogenic differentiation by influencing secretion of soluble proteins and identify a molecular route that may explain impaired chondrogenic differentiation of cells from individuals with CHH.

Abstract B163: Regulation of translation by the interferon-induced antiviral protein viperin

Abstract The innate immune response involves the induction of hundreds of interferon-stimulated genes (ISGs), many of which play a role in cancer immunosurveillance and antiviral immunity. Therefore, understanding how ISGs function and coordinate with the cellular network is of critical importance. We focus on one such ISG, viperin (virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). Viperin has been reported to inhibit a broad spectrum of DNA and RNA viruses, including many flaviviruses, human cytomegalovirus (HCMV), Chikungunya virus, influenza A virus, Sindbis virus, vesicular stomatitis virus, tick-borne encephalitis virus, HIV-1 and more. It is highly conserved in evolution, from protozoans to humans. Viperin is a peripheral membrane protein, associating on the cytosolic face of the ER via its N-terminal amphipathic helix. The cytosolic domain contains a radical SAM domain with a [4Fe-4S] cluster coordinated by three cysteine residues in the active site. Although the enzymatic function and substrate of viperin is still under debate, a recent study reveals that viperin catalyzes the conversion of cytidine triphosphate to 3′-deoxy-3′,4′-didehydro-CTP (ddhCTP) via its radical SAM activity. However, a general mechanism for viperin action and its potential roles within the innate immune response remain to be defined. Here, we demonstrate that viperin inhibits global protein translation via its radical SAM-dependent enzymatic activity. Using immortalized mouse macrophages from wild-type and viperin knockout mice we show that, although other ISGs are induced, type I IFN treatment of WT but not viperin KO mouse microphages leads to a global change in polysome profile with an increase in the ribosome fraction and a decrease in actively translating ribosomes. In parallel, the translational regulation function was evaluated in 293T-cells with transient transfection and doxycycline-inducible system. Despite no IFN stimulation and therefore no other ISG expression, viperin effectively represses translation. Furthermore, we find that viperin reduces global protein translation by ~30% in live cells using [35S]-methionine metabolic pulse-labeling or O-propargyl-puromycin (OPP) labelling. The radical SAM enzymatic activity is required for translational inhibition: site-directed mutagenesis of amino residues involved in iron-sulfur cluster and substrate binding abolishes the translational inhibition activity. We also demonstrate that ddhCTP, a recently identified vipern product, inhibits global protein translation in live cells. Viral replication requires the host translation machinery to make viral proteins, and our results suggest that much of its antiviral activity may be attributable to translational regulation. We show that viperin inhibits viral protein synthesis and viral replication of the Kunjin virus, a West Nile Virus variant, in tissue culture cells. Our study suggests a partially unifying mechanism for the broad antiviral function of viperin based on translational regulation. Citation Format: Chun-Chieh Hsu, Maudry Laurent-Rolle, Peter Cresswell. Regulation of translation by the interferon-induced antiviral protein viperin [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B163.

Sequence analysis and subcellular localization of crucian carp Carassius auratus viperin

Human viperin is known as an interferon (IFN)-inducible antiviral protein and localizes to endoplasmic reticulum (ER) via its N-terminal amphipathic α-helix. Little is known about subcellular localization of fish viperin. Herein, we characterized subcellular localization of a fish viperin from crucian carp Carassius auratus. Crucian carp viperin is nearly identical to the other viperin proteins in sequence, with the exception of the first N-terminal 70 amino acids that are defined as N-terminal variable domain including an amphipathic α-helix. In addition to N-terminal variable domain, crucian carp viperin protein harbors a conserved middle radical SAM domain and a conserved C-terminal domain. Subcellular localization analyses indicate that crucian carp viperin is a cytoplasmic protein associated with ER. Sequence analyses reveal that amino acids 1–74 forms an amphipathic α-helix domain that drives ER-localization of crucian carp viperin. In addition, Coimmunoprecipitation assays show that crucian carp viperin proteins are able to self-associate. These results together indicate that similar to mammalian homologs, fish viperins likely play important roles in IFN response.

Cloning and characterization of two genes coding for the histone acetyltransferases, Elp3 and Mof, in brown planthopper (BPH), Nilaparvata lugens (Stål)

Histone acetylation is a vital mechanism for the post-translational modifications of chromatin components. Histone acetyltransferases (HATs) are critical elements that determine histone acetylation and regulate chromatin dynamics and gene expression. While histone acetyltransferases have been well studied in mammals and Drosophila melanogaster, information from agriculturally important insect pests is still limited. In our effort to understand the epigenetic mechanisms regulating development in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a major rice pest in many parts of Asia, two full-length cDNA sequences encoding HAT members of the GNAT and MYST family, namely NlElp3 and NlMof, respectively, were isolated and structurally and phylogenetically characterized. The NlElp3 contains an open reading frame (ORF) of 1656bp encoding a protein of 551 amino acids. The NlMof contains a 1353bp ORF encoding a protein of 450 amino acids. Sequence analysis showed that NlElp3 contains GNAT-type HAT domain and Radical SAM domain, and NlMof contains chromodomain and MOZ-SAS acetyltransferase domain. Multiple sequence alignments showed that NlElp3 and NlMof have high amino acid sequence identity with other insect homologues. Expression analysis of the NlElp3 and NlMof revealed significant differences in mRNA expression levels among N. lugens developmental stages, suggesting that HAT activities of NlElp3 and NlMof may be controlled, at least in part, by their developmental regulation. Remarkably, the mRNA expression levels of NlElp3 and NlMof in female adults were significantly higher than that in male adults, supporting an important role for both genes in female reproductive function in N. lugens.

Biological Systems Discovery In Silico: Radical S -Adenosylmethionine Protein Families and Their Target Peptides for Posttranslational Modification

Data mining methods in bioinformatics and comparative genomics commonly rely on working definitions of protein families from prior computation. Partial phylogenetic profiling (PPP), by contrast, optimizes family sizes during its searches for the cooccurring protein families that serve different roles in the same biological system. In a large-scale investigation of the incredibly diverse radical S-adenosylmethionine (SAM) enzyme superfamily, PPP aided in building a collection of 68 TIGRFAMs hidden Markov models (HMMs) that define nonoverlapping and functionally distinct subfamilies. Many identify radical SAM enzymes as molecular markers for multicomponent biological systems; HMMs defining their partner proteins also were constructed. Newly found systems include five groupings of protein families in which at least one marker is a radical SAM enzyme while another, encoded by an adjacent gene, is a short peptide predicted to be its substrate for posttranslational modification. The most prevalent, in over 125 genomes, featuring a peptide that we designate SCIFF (six cysteines in forty-five residues), is conserved throughout the class Clostridia, a distribution inconsistent with putative bacteriocin activity. A second novel system features a tandem pair of putative peptide-modifying radical SAM enzymes associated with a highly divergent family of peptides in which the only clearly conserved feature is a run of His-Xaa-Ser repeats. A third system pairs a radical SAM domain peptide maturase with selenocysteine-containing targets, suggesting a new biological role for selenium. These and several additional novel maturases that cooccur with predicted target peptides share a C-terminal additional 4Fe4S-binding domain with PqqE, the subtilosin A maturase AlbA, and the predicted mycofactocin and Nif11-class peptide maturases as well as with activators of anaerobic sulfatases and quinohemoprotein amine dehydrogenases. Radical SAM enzymes with this additional domain, as detected by TIGR04085, significantly outnumber lantibiotic synthases and cyclodehydratases combined in reference genomes while being highly enriched for members whose apparent targets are small peptides. Interpretation of comparative genomics evidence suggests unexpected (nonbacteriocin) roles for natural products from several of these systems.

Differential proteomic analysis of Acidithiobacillus ferrooxidans cells maintained in contact with bornite or chalcopyrite: Proteins involved with the early bacterial response

Acidithiobacillus ferrooxidans is a chemoautotrophic bacterium capable of oxidizing ferrous iron or sulfides to obtain energy. Bornite and chalcopyrite are copper sulfides containing iron that present different susceptibilities to the bioleaching process. Here, the early bacterial response to these minerals was investigated using a differential proteomic approach. The protein profiles of cells kept in contact with bornite or chalcopyrite for 24 or 48h were compared to that of cells not exposed to the minerals. Response to bornite exposure involved thirteen proteins, mainly related to energy metabolism, detoxification and protein synthesis. We detected increases in the expression levels of the proteins chaperonin, antioxidant and aldehyde dehydrogenase, as well as decreases in the expression levels of the proteins radical SAM domain, fructose-1,6-bisphosphatase, PfkB domain, heat shock HslVU, ribulose bisphosphate carboxylase and ribosomal proteins. Chalcopyrite contact led to a distinct metabolic response of the bacterium, since no significant alteration in the level of protein expression was detected. These findings could help to understand the metabolic impact in A. ferrooxidans after the initial addition of the cells to bornite or chalcopyrite during bioleaching processes.

A role for the elongator complex in zygotic paternal genome demethylation

The life cycle of mammals begins when a sperm enters an egg. Immediately after fertilization, both the maternal and paternal genomes undergo dramatic reprogramming to prepare for the transition from germ cell to somatic cell transcription programs 1. One of the molecular events that takes place during this transition is the demethylation of the paternal genome 2,3. Despite extensive efforts, the factors responsible for paternal DNA demethylation have not been identified 4. To search for such factors, we developed a live cell imaging system that allows us to monitor the paternal DNA methylation state in zygotes. Through siRNA-mediated knockdown in zygotes, we identified Elp3/KAT9, a component of the elongator complex 5, to be important for paternal DNA demethylation. We demonstrate that knockdown of Elp3 impairs paternal DNA demethylation as indicated by reporter binding, immunostaining and bisulfite sequencing. Similar results were also obtained when other elongator components, Elp1 and Elp4, were knocked down. Importantly, injection of mRNA encoding the Elp3 radical SAM domain mutant, but not the HAT domain mutant, into MII oocytes before fertilization also impaired paternal DNA demethylation indicating that the SAM radical domain is involved in the demethylation process. Thus, our study not only establishes a critical role for the elongator in zygotic paternal genome demethylation, but also suggests that the demethylation process may be mediated through a reaction that requires an intact radical SAM domain.

Identification of Three Interferon-Inducible Cellular Enzymes That Inhibit the Replication of Hepatitis C Virus

Hepatitis C virus (HCV) infection is a common cause of chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. However, the underlying mechanism of IFN-alpha therapy remains to be elucidated. To identify the cellular proteins that mediate the antiviral effects of IFN-alpha, we created a HEK293-based cell culture system to inducibly express individual interferon-stimulated genes (ISGs) and determined their antiviral effects against HCV. By screening 29 ISGs that are induced in Huh7 cells by IFN-alpha and/or up-regulated in HCV-infected livers, we discovered that viperin, ISG20, and double-stranded RNA-dependent protein kinase (PKR) noncytolytically inhibited the replication of HCV replicons. Mechanistically, inhibition of HCV replication by ISG20 and PKR depends on their 3'-5' exonuclease and protein kinase activities, respectively. Moreover, our work, for the first time, provides strong evidence suggesting that viperin is a putative radical S-adenosyl-l-methionine (SAM) enzyme. In addition to demonstrating that the antiviral activity of viperin depends on its radical SAM domain, which contains conserved motifs to coordinate [4Fe-4S] cluster and cofactor SAM and is essential for its enzymatic activity, mutagenesis studies also revealed that viperin requires an aromatic amino acid residue at its C terminus for proper antiviral function. Furthermore, although the N-terminal 70 amino acid residues of viperin are not absolutely required, deletion of this region significantly compromises its antiviral activity against HCV. Our findings suggest that viperin represents a novel antiviral pathway that works together with other antiviral proteins, such as ISG20 and PKR, to mediate the IFN response against HCV infection.