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CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target–crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target–crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target–crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA–target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes.

CRISPR systems elicit interference when a foreign nucleic acid is detected by its ability to base-pair to crRNA. Understanding what degree of complementarity between a foreign nucleic acid and crRNA is required for interference is a central question in the study of CRISPR systems. A clear description of which target-crRNA mismatches abrogate interference in type III, Cas10-containing, CRISPR systems has proved elusive due to the complexity of the system which utilizes three distinct interference activities. We characterized the effect of target-crRNA mismatches on in vitro cyclic oligoadenylate (cOA) synthesis and in vivo in an interference assay that depends on cOA synthesis. We found that sequence context affected whether a mismatched target was recognized by crRNA both in vitro and in vivo. We also investigated how the position of a mismatch within the target-crRNA duplex affected recognition by crRNA. Our data provide support for the hypothesis that a Cas10-activating region exists in the crRNA-target duplex, that the Cas10-proximal region of the duplex is the most critical in regulating cOA synthesis. Understanding the rules governing target recognition by type III CRISPR systems is critical: as one of the most prevalent CRISPR systems in nature, it plays an important role in the survival of many genera of bacteria. Recently, type III systems were re-purposed as a sensitive and accurate molecular diagnostic tool. Understanding the rules of target recognition in this system will be critical as it is engineered for biotechnology purposes. [ABSTRACT FROM AUTHOR]

The effects of zeatin, salicylic acid, and plant oligoadenylates on susceptibility of wheat seedlings to powdery mildew and the shape of dose-response curves for immunomodulation were studied. Salicylic acid and zeatin produced complex dose-responses with upregulation or downregulation of plant susceptibility. Under different experimental conditions, the dose-response curve for zeatin varied in shape from a curve with a maximum to a curve with a minimum of susceptibility to powdery mildew. More complicated curves with a resistance region framed with regions of increased susceptibility were also found. Plant oligoadenylates inhibited the development of powdery mildew at optimal concentrations. The increasing concentrations of salicylic acid and plant oligoadenylates, when combined with zeatin, led to a gradual change in the shape of the zeatin concentration curve. Treatment with salicylic acid and plant oligoadenylates in these experiments may simulate plant responses under biotic and abiotic stresses. Thus, exogenous zeatin was applied to model changes in the phytohormone metabolism. The complex multiphase nature of dose-response is proposed to cause a kind of random-number generator that produces variability in the physiological and immunological status of plants in natural conditions. Variation in susceptibility of individual plants may be crucial for the survival of infected plants and the stabilization of plant-pathogen interactions. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Synthetic development of modified nucleotides improves DMPK and pharmacodynamics. • Understanding cellular uptake mechanisms will improve targeted drug delivery. • Preclinical animal data scale well to humans, supporting dose-setting in clinical trials. • Improved bioanalytical sensitivity is needed to establish long elimination half-lives. • Mastering oral delivery will fuel interest from the industry and boost science. Oligonucleotide and modified mRNA therapeutics have great potential to treat diseases that are currently challenging to cure and are expanding into global and chronic disease areas such as cancer and various cardiovascular diseases. Advanced drug delivery systems or ligand–drug conjugates are utilized to achieve 'the right dose to the right target' to benefit efficacy and safety in patients. Chemistry and ADME characteristics distinguish these therapeutics from small molecules. Understanding the scalability and translatability between species and compound properties is crucial for robust nonclinical PKPD predictions to support clinical study design. Although the field has been developing for three decades, there is still room for innovation but also a need for nonclinical regulatory guidance to address these new modalities. Teaser Oligonucleotide-based therapeutics show great clinical promise and drug metabolism and pharmacokinetic understanding will be key to expand into broader patient populations. [ABSTRACT FROM AUTHOR]

p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38β; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300 μg/10 μl) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38β MAPK ASO. Intrathecal injection of p38α or p38β ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38β mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38β MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions. [ABSTRACT FROM AUTHOR]

Context Toll-like receptor 4 (TLR4) activation contributes to obesity-associated insulin resistance in skeletal muscles (SM). TLR4 signaling involves two pathways: the myeloid differentiation primary response gene 88 (MyD88) leading to inflammatory cytokines production and the toll/interleukin-1 receptor domain-containing adapter-inducing interferon (IFN) I (TRIF)-dependent pathways leading to type 1 interferon (IFNI) and interferon stimulated genes (ISG) expression. The E3 ubiquitin ligase RNF41 allows the preferential activation of the TRIF-IFNI pathway; however, its role in insulin response has not been reported. Methods We measured RNF41 level and IFNI pathway activation (ISG expression) in SM biopsies of obese insulin sensitive (OIS) and obese insulin resistant (OIR) women. Then we isolated and differentiated in myotubes, primary human SM cell progenitors from OIS and OIR SM biopsies. We modulated RNF41 and ISG expression in these myotubes and investigated their effects on insulin response. Results RNF41 expression is down-regulated in vivo in OIR SM and myotubes compared to OIS SM and myotubes. TLR4 activation with palmitate induces TRIF-IFNI pathway and ISG in OIS myotubes but not in OIR myotubes. Inhibition of RNF41 expression with siRNF41 in OIS myotubes treated with palmitate attenuates insulin response, IFNI pathway activation and ISG induction, mimicking OIR phenotype. Further, overexpression of RNF41 in OIR myotubes increases insulin response and ISG expression. Exposure to IFNI or to its inducer polyinosinic-polycytidylic acid, restores ISG expression and insulin sensitivity in OIR myotubes and OIS myotubes transfected with siRNF41. Conclusion Our results identify RNF41 as essential to IFNI pathway activation in order to maintain muscle insulin sensitivity during human obesity. [ABSTRACT FROM AUTHOR]

Prevalent single cell RNA amplification and sequencing chemistries mainly focus on polyadenylated RNAs in eukaryotic cells by using oligo(dT) primers for reverse transcription. We develop a new RNA amplification method, "easier-sea", to reverse transcribe and amplify the total RNAs, both with and without polyadenylate tails, from a single cell for transcriptome sequencing with high efficiency, reproducibility, and accuracy. By distributing the reverse transcribed cDNA molecules into 1.5 X 10[sup 5] aqueous droplets in oil, the cDNAs are isothermally amplified using random primers in each of these 65-pL reactors separately. This new method greatly improves the ease of single-cell RNA sequencing by reducing the experimental steps. Meanwhile, with less chance to induce errors, this method can easily maintain the quality of single-cell sequencing. In addition, this polyadenylate-tail-independent method can be seamlessly applied to prokaryotic cell RNA sequencing. [ABSTRACT FROM AUTHOR]

Infections with viral pathogens impose considerable selective pressure on host defensive genes. Those genes at the forefront, responsible for identifying and binding exogenous molecular viral components, will carry the hallmarks of this struggle. Oligoadenylate synthetase (OAS) enzymes play a major role in the innate defense against a large number of viruses by acting as sensors of viral infections. Following their up-regulation by the interferon pathway, OASs bind viral dsRNA and then signal ribonuclease L (RNase L) to degrade RNA, shutting down viral and host protein synthesis. We have investigated the evolution of OAS1 in twenty-two Old World monkey species. We identified a total of 35 codons with the earmarks of positive selection and we performed a comprehensive analysis of their functional significance using in silico modeling of the OAS1 protein. Subdividing OAS1 into functional domains revealed intense purifying selection in the active domain but significant positive directional selection in the RNA-binding domain (RBD), the region where OAS1 binds viral dsRNA. The modeling analysis revealed a concentration of rapidly evolving residues in one region of the RBD suggestive of the sub-functionalization of different regions of the RBD. This analysis also identified several positively selected residues circumscribing the entry to the active site suggesting adaptive evasion of viral antagonism and/or selection for production of oligoadenylate of different length. [ABSTRACT FROM AUTHOR]

RNase L is activated by 2′,5′-oligoadenylates (2-5A) at subnanomolar levels to cleave single-stranded RNA. We previously reported the hypothesis that the introduction of an 8-methyladenosine residue at the 2′-terminus of the 2-5A tetramer shifts the 2-5A binding site of RNase L. In this study, we synthesized various 5′-modified 2-5A analogs with 8-methyladenosine at the 2′-terminus. The doxifluridine-conjugated 8-methyladenosine-substituted 2-5A analog was significantly more effective as an activator of RNase L than the parent 5′-monophophorylated 2-5A tetramer and showed a tumor suppressive effect against human cervical cancer cells. [ABSTRACT FROM AUTHOR]

The innate immune response is the first line of defense of the host cell against a viral infection. In turn, viruses have evolved a wide variety of strategies to hide from, and to directly antagonize, the host innate immune pathways. One of these pathways is the 2'-5'-oligoadenylate synthetase (OAS)/RNase L pathway. OAS is activated by double-stranded RNA (dsRNA) to produce 2'-5' oligoadenylates, which are the activators of RNase L; this enzyme degrades viral and cellular RNAs, restricting viral infection. It has been recently found that the carboxy-terminal domain (CTD) of rotavirus VP3 has a 2'-5'-phosphodiesterase (PDE) activity that is able to functionally substitute for the PDE activity of the mouse hepatitis virus ns2 protein. This particular phosphodiesterase cleaves the 2'-5'-phosphodiester bond of the oligoadenylates, antagonizing the OAS/RNase L pathway. However, whether this activity of VP3 is relevant during the replication cycle of rotavirus is not known. Here, we demonstrate that after rotavirus infection the OAS/RNase L complex becomes activated; however, the virus is able to control its activity using at least two distinct mechanisms. A virus-cell interaction that occurs during or before rotavirus endocytosis triggers a signal that prevents the early activation of RNase L, while later on the control is taken by the newly synthesized VP3. Cosilencing the expression of VP3 and RNase L in infected cells yields viral infectious particles at levels similar to those obtained in control infected cells, where no genes were silenced, suggesting that the capping activity of VP3 is not essential for the formation of infectious viral particles. [ABSTRACT FROM AUTHOR]

Interferons (IFN), first described 1957 by Isaacs and Lindemann, are antiviral proteins generated in cells after viral infections. One of several interferon-induced effector mechanisms is the so called 2-5A / RNaseL system: Interferon is produced in the virus-affected cells and released. After binding to cell membrane receptors of adjacent cells, 2-5 A synthetase (oligoadenylate synthetase, OAS) is generated, attaches to dsRNA section areas of the viral RNA and catalyses the production of 2-5 oligoadenylates from ATP. In 2-5 oligoadenylates, several adenosine residues (3-4 and more) are combined via phosphodiester binding in the unusual 2'-5' positions of the riboses. 2-5 oligoadenylates activate a RNaseL which degrades the viral RNA. Recently, characteristic gene mutations and other disturbances concerning the interferon system were detected in patients with severe COVID-19, leading to problems of 2-5 oligoadenylate synthesis and the activation of RNAseL. In order to circumvent these problems, we hypothesize that a direct application of 2-5 oligoadenylates, included in an inhalation spray, may be effective in treatment of severe COVID-19 infections of the respiratory system. In contrast to some other anti-COVID-19 drugs, oligoadenylates act inside the cells (like e.g. Paxlovid) and are therefore independent of cell surface mutations of the virus. For confirmation of our hypothesis, proof of concept investigations in vitro are suggested, before a possible clinical application can be considered. [ABSTRACT FROM AUTHOR]

Chromosomal aberrations in the product of conceptions (POC) are detected in around 50% lost pregnancies. Karyotype analysis of the POC, limited by high failure rate of culture, is not always carried out. The oligonucleotide-based chromosomal microarray analysis (CMA) of POC from 303 pregnancies was carried out. The findings showed a high diagnostic value of the test and its ability to detect a large number of clinically relevant rearrangements compared to karyotype analysis and comparative genomic hybridization (CGH) arrays. [ABSTRACT FROM PUBLISHER]

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here, we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were expressed differentially during axon specification. Furthermore, 8-pCPT, an EPAC pharmacological activator, and genetic manipulations of EPAC in neurons induced supernumerary axons indicative of Rap1b activation. Moreover, 8-pCPT-treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock-out (KO) mice led to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8-pCPT treatments in wild-type neurons were not found in EPAC1 KO mice neurons. Altogether, these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP-dependent axon determination. [ABSTRACT FROM AUTHOR]

Warning and monitoring projects of harmful algal blooms require simple and rapid methods for simultaneous and accurate detection and identification of causative algae present in the environmental samples. Here, reverse dot blot hybridization (RDBH) was employed to simultaneously detect several harmful algae by using five representative bloom-forming microalgae along the Chinese coast. A set of specific probes for RDBH were developed by PCR, cloning, and sequencing of the internal transcribed spacer (ITS), alignment analysis, and probe design. Each probe was oligo (dT)-tailed and spotted onto positively charged nylon membrane to make up a low-density oligonucleotide array. Universal primers designed within the conserved regions were used to amplify the ITS sequences by using genomic DNA of target as templates. The digoxigenin (Dig)-labeled PCR products were denatured and then hybridized to the oligonucleotide array. The array produced a unique hybridization pattern for each target species differentiating them from each other. The preparations of oligonucleotide array and hybridization conditions were optimized. The developed RDBH demonstrated a detection limit up to 10 cells. The detection performance of RDBH was relatively stable and not affected by non-target species and the fixation time of target species over at least 30 days. The RDBH could recover all the target species from the simulated field samples and target species confirmed by the subsequent microscopy examination in the environmental samples. These results indicate that RDBH can be a new technical platform for parallel discrimination of harmful algae and is promising for environmental monitoring of these microorganisms. [ABSTRACT FROM AUTHOR]

The synthesis and a preliminary investigation of the base pairing properties of (6' → 4')-linked 1',5'-anhydro-L-ribo-hexitol nucleic acids (α-L-HNA) have herein been reported through the study of a model oligoadenylate system in the mirror image world. Despite its considerable preorganization due to the rigidity of the "all equatorial" pyranyl sugar backbone, α-L-HNA represents a versatile informational biopolymer, in view of its capability to cross-communicate with natural and unnatural complements in both enantiomeric forms. This seems the result of an inherent flexibility of the oligonucleotide system, as witnessed by the singular formation of iso- and heterochiral associations composed of regular, enantiomorphic helical structures. The peculiar properties of α-L-HNA (and most generally of the α-HNA system) provide new elements in our understanding of the structural prerequisites ruling the stereoselectivity of the hybridization processes of nucleic acids. [ABSTRACT FROM AUTHOR]

Low molecular weight natural mediators, 2'-5'-linked oligoadenylates, play an important role in interferon-based antiviral mechanism; participate in growth, apoptosis and other important cellular processes. Taking into account their concentration within living cells, the 2'-5'A3 oligoadenylates may act as additional biologically active substrates, capable of regulating the S100A1 protein functioning in vivo. Aim. Find the evidence for the interaction of human apo-S100A1 with 2'-5'-linked oligoadenylates. Methods. Using the circular dichroism (CD) and heteronuclear NMR spectroscopy. Results. The obtained results demonstrated the occurrence of the secondary structure changes in human S100A1 protein upon the interaction with 2'-5'-linked oligoadenylates as well as indicated specific residues involved in this process. Conclusions. Our study points to the 2'-5'-linked oligoadenylates as possible additional elements of the complex system of fine regulation of the Ca2+-signal transduction pathway in human cells. [ABSTRACT FROM AUTHOR]

The capability of current MD simulations to be used as a tool in rational design of agonists of medically interesting enzyme RNase L was tested. Dimerization and enzymatic activity of RNase L is stimulated by 2′,5′-linked oligoadenylates (pAAA; 2-5A). First, it was necessary to ensure that a complex of monomeric human RNase L and 25A was stable in MD simulations. It turned out that Glu131 had to be protonated. The non-protonated Glu131 caused dissociation of 2-5A from RNase L. Because of the atypical 2′-5′ internucleotide linkages and a specific spatial arrangement of the 25A trimer, when a single molecule carries all possible conformers of the glycosidic torsion angle, several versions of the AMBER force field were tested. One that best maintained functionally important interactions of 25A and RNase L was selected for subsequent MD simulations. Furthermore, we wonder whether powerful GPUs are able to produce MD trajectories long enough to convincingly demonstrate effects of subtle perturbations of interactions between 25A and RNase L. Detrimental impacts of various point mutations of RNase L (R155A, F126A, W60A, K89A) on 2-5A binding were observed on a time scale of 200 ns. Finally, 2-5A analogues with a bridged 3′-O,4′-C-alkylene linkage (B) introduced into the adenosine units (A) were used to assess ability of MD simulations to distinguish on the time scale of hundreds of nanoseconds between agonists of RNase L (pAAB, pBAA, pBAB) and inactive analogs (pABA, pABB, pBBA, pBBB). Agonists were potently bound to RNase L during 200 ns MD runs. For inactive 2-5A analogs, by contrast, significant disruptions of their interactions with RNase L already within 100 ns MD runs were found. [ABSTRACT FROM AUTHOR]

The interaction between 2′-5′А3oligonucleotides and recombinant α-interferon was studied by MALDI-TOF mass spectrometry and fluorescence spectroscopy methods. The ability of this key protein of antiviral cell defense system to bind up to 5 molecules of oligoadenylates depending on their structure was established. The presence of interaction in oligoadenylate-interferon system was also confirmed by fluorescence studies. Investigated compounds quenched the emission of α-interferon from 40 to 63%. The efficiency of quenching of the fluorescence of α-interferon also depends on the oligoadenylates structure. [ABSTRACT FROM PUBLISHER]

Abstract: 5′-Triphosphate 2′-5′-oligoadenylate (2–5A) is the central player in the 2–5A system that is an innate immunity pathway in response to the presence of infectious agents. Intracellular endoribonuclease RNase L activated by 2–5A cleaves viral and cellular RNA resulting in apoptosis. The major limitations of 2–5A for therapeutic applications is the short biological half-life and poor cellular uptake. Modification of 2–5A with biolabile and lipophilic groups that facilitate its uptake, increase its in vivo stability and release the parent 2–5A drug in an intact form offer an alternative approach to therapeutic use of 2–5A. Here we have synthesized the trimeric and tetrameric 2–5A species bearing hydrophobic and enzymolabile pivaloyloxymethyl groups at 3′-positions and a triphosphate at the 5′-end. Both analogs were able to activate RNase L and the production of the trimer 2–5A (the most active) was scaled up to the milligram scale for antiviral evaluation in cells infected by influenza virus or respiratory syncytial virus. The trimer analog demonstrated some significant antiviral activity. [Copyright &y& Elsevier]

New analogs of natural (2′-5′)-oligoadenylates containing ribavirin or BAP-riboside fragments in addition to adenine 2′,3′-anhydronucleoside units were obtained. The synthesized compounds were highly stable to enzymatic hydrolysis by snake venom phosphodiesterase. It was shown that ribavirin-containing (2′-5′)-oligonucleotides could be used for elimination of pathogenic raspberry ringspot virus in culture in vitro of microrunners of black current and cherry. [ABSTRACT FROM AUTHOR]

The use of lytic viruses to preferentially infect and eliminate cancer cells while sparing normal cells is a promising experimental therapeutic approach for treating cancer. However, the efficacy of oncolytic virotherapy is often limited by two innate immunity pathways, the protein kinase PKR and the 2′-5′-oligoadenylate (OAS)/RNase L systems, which are widely present in many but not all tumor cell types. Previously, we reported that the anticancer drug, sunitinib, an inhibitor of VEGF-R and PDGF-R, has off-target effects against both PKR and RNase L. Here we show that combining sunitinib treatments with infection by an oncolytic virus, vesicular stomatitis virus (VSV), led to the elimination of prostate, breast, and kidney malignant tumors in mice. In contrast, either virus or sunitinib alone slowed tumor progression but did not eliminate tumors. In prostate tumors excised from treated mice, sunitinib decreased levels of the phosphorylated form of translation initiation factor, eIF2-α, a substrate of PKR, by 10-fold while increasing median viral titers by 23-fold. The sunitinib/VSV regimen caused complete and sustained tumor regression in both immunodeficient and immunocompetent animals. Results indicate that transient inhibition of innate immunity with sunitinib enhances oncolytic virotherapy allowing the recovery of tumor-bearing animals.Molecular Therapy (2013); 21 9, 1749-1757. doi:10.1038/mt.2013.112 [ABSTRACT FROM AUTHOR]

It is a matter of common knowledge, that «core» 2'-5'-oligoadenylates and their analogues posses broader biological activity than it can be predicted within the traditional «interferon» hypothesis. They exhibit immunomodulatory effects and are effective immunosuppressive agents upon organ and tissue transplantation. Oligoadenylates render antiviral activity against a wide range of viruses both DNA and RNA origin. These drugs regulate apoptosis, cell proliferation and possess antitumor activity. Such polyvalent activity is based on their ability to bind signaling proteins, slightly altering their conformation and modulating their activity. They can also change the secondary structure of both viral DNA and RNA, making them accessible for enzymatic cleavage. The hypothesis forwarded within this review is aimed at making an attempt to explain the mechanism of 2'-5'-oligoadenylates antiviral action. Our recent papers, containing experimental data to support this hypothesis, are discussed in this review. [ABSTRACT FROM AUTHOR]

Tick-borne encephalitis virus (TBEV) is a zoonotic agent that causes fatal encephalitis in humans. 2'-5'-oligoadenylate synthetase 1b ( Oas1b) has been identified as a flavivirus resistance gene, but most inbred laboratory mice do not possess a functional Oas1b gene. In this study, a congenic strain carrying a functional Oas1b gene, B6.MSM- Oas, was used to evaluate the pathogenicity of Far-Eastern TBEV. Although intracerebral infection of B6.MSM- Oas mice by Oshima 5-10 resulted in limited signs of illness, infection by Sofjin-HO resulted in death with severe neurologic signs. While Oshima 5-10 was cleared from the brain, Sofjin-HO was not cleared despite a similar level of expression of the intact Oas1b gene. Necrotic neurons with viral antigens and inflammatory reactions were observed in the brain infected with Sofjin-HO. These data indicate that the different susceptibility to the antiviral activity of Oas1b resulted in a difference in neurovirulence in the two TBEV strains. [ABSTRACT FROM AUTHOR]

A-74528 is a C30 polyketide natural product that functions as an inhibitor of 2′,5′-oligoadenylate phosphodiesterase (2′-PDE), a key regulatory enzyme of the interferon pathway. Modulation of 2′-PDE represents a unique therapeutic approach for regulating viral infections. The gene cluster responsible for biosynthesis of A-74528 yields minute amounts of this natural product together with considerably larger quantities of a structurally dissimilar C30 cytotoxic agent, fredericamycin. Through construction and analysis of a series of knockout mutants, we identified the genes necessary for A-74528 biosynthesis. Remarkably, the formation of six stereocenters and the regiospecific formation of six rings in A-74528 appear to be catalyzed by only two tailoring enzymes, a cyclase and an oxygenase, in addition to the core polyketide synthase. The inferred pathway was genetically refactored in a heterologous host, Streptomyces coelicolor CH999, to produce 3 mg/L A-74528 in the absence of fredericamycin. [ABSTRACT FROM AUTHOR]

The recessive disorder poikiloderma with neutropenia (PN) is caused by mutations in the C16orf57 gene that encodes the highly conserved USB1 protein. Here, we present the 1.1 A resolution crystal structure of human USB1, defining it as a member of the Li gT-1 ike superfamily of 2H phosphoesterases. We show that human USB1 is a distributive 3'-5' exoribonuclease that posttranscriptionally removes uridine and adenosine nucleosides from the 3' end of spliceosomal U6 small nuclear RNA (snRNA), directly catalyzing terminal 2', 3' cyclic phosphate formation. USB1 measures the appropriate length of the U6 oligo(U) tail by reading the position of a key adenine nucleotide (A102) and pausing 5 uridine residues downstream. We show that the 3' ends of U6 snRNA in PN patient lymphoblasts are elongated and unexpectedly carry nontemplated 3' oligo(A) tails that are characteristic of nuclear RNA surveillancetargets. Thus, our study reveals a novel quality control pathway in which posttranscrtptional 3'-end processing by USB1 protects U6 snRNA from targeting and destruction by the nuclear exosome. Our data implicate aberrant oligoadenylation of U6 snRNA in the pathogenesis of the leukemia predisposition disorder PN. [ABSTRACT FROM AUTHOR]

The human sensor of double-stranded RNA (dsRNA) oligoadenylate synthetase 1 (hOAS1) polymerizes ATP into 2′,5′-linked iso-RNA (2-5A) involved in innate immunity, cell cycle, and differentiation. We report the crystal structure of hOAS1 in complex with dsRNA and 2′-deoxy ATP at 2.7 A resolution, which reveals the mechanism of cytoplasmic dsRNA recognition and activation of oligoadenylate synthetases. Human OAS1 recognizes dsRNA using a previously uncharacterized protein/RNA interface that forms via a conformational change induced by binding of dsRNA. The protein/RNA interface involves two minor grooves and has no sequence-specific contacts, with the exception of a single hydrogen bond between the -NH2 group of nucleobase G17 and the carbonyl oxygen of serine 56. Using a biochemical readout, we show that hOAS1 undergoes more than 20,000-fold activation upon dsRNA binding and that canonical or GU-wobble substitutions produce dsRNA mutants that retain either full or partial activity, in agreement with the crystal structure. Ultimately, the binding of dsRNA promotes an elaborate conformational rearrangement in the N-terminal lobe of hOAS1, which brings residues D75, D77. and D148 into proximity and creates coordination geometry for binding of two catalytic Mg2+ ions and ATP. The assembly of this critical active-site structure provides the gate that couples binding of dsRNA to the production and downstream functions of 2-5A. [ABSTRACT FROM AUTHOR]

Aim. To investigate the ability of «core» 2'-5'- and 3'-5'-oligo- adenylates (OA) to interact with α-interferon — a key protein of the 2'- 5'-OAS/RNAase L system responsible for antiviral cell defense. Methods. MALDI-TOF mass spectrometry was used in the studies on protein-ligand interactions. Results. It was shown that 2'-5'-A3 and its epoxy-modified analog 2'-5'-A3-epo can bind to α-interferon in vitro. 3'-5'-triadenylate is also capable of binding to this protein. One to five ligand molecules can bind simultaneously to the molecule of α-interferon. At the same time, all studied oligonucleotides do not bind to insulin. Conclusions. It was established that «core» 2'-5'- and 3'-5'-triadenylates are capable of multiple interaction with α-interferon to form stable complexes. However, they do not bind to insulin which is not involved in the 2'-5'-OAS/RNAase L system. [ABSTRACT FROM AUTHOR]

Summary: 2′,5′-linked oligoadenylates (2-5As) serve as conserved messengers of pathogen presence in the mammalian innate immune system. 2-5As induce self-association and activation of RNase L, which cleaves cytosolic RNA and promotes the production of interferons (IFNs) and cytokines driven by the transcription factors IRF-3 and NF-κB. We report that human RNase L is activated by forming high-order complexes, reminiscent of the mode of activation of the phylogenetically related transmembrane kinase/RNase Ire1 in the unfolded protein response. We describe crystal structures determined at 2.4 Å and 2.8 Å resolution, which show that two molecules of 2-5A at a time tether RNase L monomers via the ankyrin-repeat (ANK) domain. Each ANK domain harbors two distinct sites for 2-5A recognition that reside 50 Å apart. These data reveal a function for the ANK domain as a 2-5A-sensing homo-oligomerization device and describe a nonlinear, ultrasensitive regulation in the 2-5A/RNase L system poised for amplification of the IFN response. [ABSTRACT FROM AUTHOR]

Abstract: RNase L mediates critical cellular functions including antiviral, proapoptotic, antiproliferative and tumor suppressive activities. In this study, the expression and function of RNase L in lung cancer cells were examined. Interestingly we have found that the expression of RNase L in lung cancer cells was 3- and 9-fold higher in its mRNA and protein levels, but a significant decrease of its enzymatic activity when compared to that in corresponding normal lung cells. Further investigation revealed that 2-5A-induced dimerization of the RNase L protein, a necessary prerequisite for activation of RNase L, was inhibited, as a result of that RLI, a specific inhibitor of RNase L, was remarkably up-regulated in the cancer cells. Our findings provide new insight into how cancer cells escape normal growth-regulating mechanisms to form a tumor and the information may be useful for the design of novel strategies for treating lung cancer through regulating RNase L activity. [Copyright &y& Elsevier]

RNase L, a key enzyme in the host defense system, is activated by the binding of 2′–5′-linked oligoadenylates (2–5A) to the N-terminal ankyrin repeat domain, which causes the inactive monomer to form a catalytically active homodimer. We focused on the structural changes of human RNase L as a result of interactions with four different activators: natural 2–5 pA 4 and three tetramers with 3′-end AMP units replaced with ribo- , arabino- and xylo- configured phosphonate analogs of AMP (pA 3 X). The extent of the RNase L dimerization and its cleavage activity upon binding of all these activators were similar. A drop-coating deposition Raman (DCDR) spectroscopy possessed uniform spectral changes upon binding of all of the tetramers, which verified the same binding mechanism. The estimated secondary structural composition of monomeric RNase L is 44% α-helix, 28% β-sheet, 17% β-turns and 11% of unordered structures, whereas dimerization causes a slight decrease in α-helix and increase in β-sheet (ca. 2%) content. The dimerization affects at least three Tyr, five Phe and two Trp residues. The α–β structural switch may fix domain positions in the hinge region (residues ca. 336–363) during homodimer formation. [ABSTRACT FROM AUTHOR]

Abstract: The vertebrate 2-5A system is part of the innate immune response and central to cellular antiviral activities. Upon activation by viral double-stranded RNA, 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are synthesized by one of several 2′-5′ oligoadenylate synthetases. The 2-5As bind and activate RNase L, an unspecific endoribonuclease, resulting in viral and cellular RNA decay. Given that most endogenous RNAs are degraded by RNase L, continued enzyme activity will eventually lead to cell growth arrest and cell death. This is averted, when 2-5As and their 5′-dephosphorylated forms, the so-called 2-5A core molecules, are cleaved and thus inactivated by 2′-5′-specific nuclease(s), e.g. phosphodiesterase 12, thereby turning RNase L into its latent form. In this study, we have characterized the human phosphodiesterase 12 in vitro focusing on its ability to degrade 2-5As and 2-5A core molecules. We have found that the enzyme activity is distributive and is influenced by temperature, pH and divalent cations. This allowed us to determine V max and K m kinetic parameters for the enzyme. We have also identified a novel 2′-5′-oligoadenylate nuclease; the human plasma membrane-bound ectonucleotide pyrophosphatase/phosphodiesterase 1, suggesting that 2-5A catabolism may be a multienzyme-regulated process. [Copyright &y& Elsevier]

Prostate-specific membrane antigen (PSMA) is an ideal biomarker for prostate cancer. A previously reported 2-5A conjugate RBI1033 (3) showed binding affinity more than 10 times higher than the parent urea-based compound (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido) pentanedioic acid (1). The purpose of this work is to further optimize the structure of 3 to identify highly selective ligands of PSMA. It was found that conjugates having 2-5A in their structure showed extraordinary improved binding affinity to PSMA compared with compound 1. Removal of 2-5A significantly reduced its biological activity. The results will provide a path to agents for targeted imaging and treatment of prostate cancer. [ABSTRACT FROM AUTHOR]

Aim. To determine 2',5'-oligoadenylate synthetase (2',5'-OAS) activity and interferon (IFN) level in lymphocytes of rats irradiated with 0.25, 0.5, 0.75 and 1.0 Gy doses, as well as to study the effect of interferon inducers on these parameters. Methods. Micro method of IFN titer determination by antiviral activity testing and spectrophotometric method of 2',5'-OAS activity determination were used. Results. An increase in IFN titer in lymphocytes of rats after irradiation with 0.25 and 0.5 Gy doses was shown. Higher radiation doses of 0.75 and 1.0 Gy caused a decrease of this parameter. The 2',5'-OAS activity exceeded a control at all applied doses, the maximum level in splenocytes corresponded to 0.25 Gy dose whereas the highest values in thymocytes were revealed at 0.5-1.0 Gy doses. The IFN inducers stimulated cytokine synthesis and increased the enzyme activity in lymphocytes of irradiated rats. Conclusions. The increase of 2',5'-OAS activity and IFN titer in lymphocytes of irradiated animals may be regarded as a protective post radiation reaction of cells. Under irradiation with higher doses - 0.75 and 1.0 Gy - the studied parameters decreased, that may be associated with more serious damage of cells, for correction of which the resources of repairing systems are not enough. The increased 2',5'-OAS activity and IFN titer in lymphocytes after their pre-incubation with IFN inducers can be considered as intensification of cellular protective postradiation mechanisms. [ABSTRACT FROM AUTHOR]

Abstract: To determine the influence of internucleotide linkage and sugar ring conformation, and the role of 5′-terminal phosphate, on the activation of human RNase L, a series of 2′- and 5′-O-methylphosphonate-modified tetramers were synthesized from appropriate monomeric units and evaluated for their ability to activate human RNase L. Tetramers pAAApcX modified by ribo, arabino or xylo 5′-phosphonate unit pcX activated RNase L with efficiency comparable to that of natural activator. Moreover, incorporation of phosphonate linkages ensured the stability against cleavage by nucleases. The substitution of 5′-terminal phosphate for 5′-terminal phosphonate in tetramer pcXAAA afforded tetramers with excellent activation efficiency and with complete stability against cleavage by phosphomonoesterases. [Copyright &y& Elsevier]

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • While several clinical studies have been published on toll-like receptor 7 (TLR7) agonists, there is no information on the quantitative link between 2′,5′-oligoadenylate synthetase (OAS) and antiviral efficacy (viral load). WHAT THIS STUDY ADDS • This study provides the only quantitative dynamic clinical relationship between the pharmacokinetics (PK) of PF-04878691 and OAS and lymphocyte levels, together with the only published clinical relationship between markers of anti-viral pharmacology (OAS) and antiviral efficacy (viral load). This study highlights how modelling and simulation can be used to impact compound progression decisions in clinical development. AIM To use non-linear mixed effects modelling and simulation techniques to predict whether PF-04878691, a toll-like receptor 7 (TLR7) agonist, would produce sufficient antiviral efficacy while maintaining an acceptable side effect profile in a 'proof of concept' (POC) study in chronic hepatitis C (HCV) patients. METHODS A population pharmacokinetic-pharmacodynamic (PKPD) model was developed using available 'proof of pharmacology' (POP) clinical data to describe PF-04878691 pharmacokinetics (PK) and its relationship to 2′,5′-oligoadenylate synthetase (OAS; marker of pharmacology) and lymphocyte levels (marker of safety) following multiple doses in healthy subjects. A second model was developed to describe the relationship between change from baseline OAS expressed as fold change and HCV viral RNA concentrations using clinical data available in HCV patients for a separate compound, CPG-10101 (ACTILON™), a TLR9 agonist. Using these models the antiviral efficacy and safety profiles of PF-04878691 were predicted in HCV patients. RESULTS The population PKPD models described well the clinical data as assessed by visual inspection of diagnostic plots, visual predictive checks and precision of the parameter estimates. Using these relationships, PF-04878691 exposure and HCV viral RNA concentration was simulated in HCV patients receiving twice weekly administration for 4 weeks over a range of doses. The simulations indicated that significant reductions in HCV viral RNA concentrations would be expected at doses >6 mg. However at these doses grade ≥3 lymphopenia was also predicted. CONCLUSIONS The model simulations indicate that PF-04878691 is unlikely to achieve POC criteria and support the discontinuation of this compound for the treatment of HCV. [ABSTRACT FROM AUTHOR]

Targeted inhibition of the Bcl-2 family of antiapoptotic proteins is expected to improve outcomes in chronic lymphocytic leukemia. Antisense oligonucleotides and small molecule inhibitors (BH3 mimetics) are two approaches that have been used to target Bcl-2 proteins. In this review, we summarize the experience with oblimersen sodium, an 18-base oligonucleotide targeting the first six codons of Bcl-2 mRNA, with particular focus on chronic lymphocytic leukemia. Despite evidence of improved outcomes in randomized trials of combination with chemoimmunotherapy, further development of this antisense approach has been slow, likely because of the clinical development of small molecule inhibitors [ABSTRACT FROM AUTHOR]

BACKGROUND: The antiviral, proapoptotic, antiproliferative gene 2′-5′ oligoadenylate synthetase (2-5OAS1) converts adenosine triphosphate into a series of 2′-5′ oligoadenylates (2-5A). In turn, 2-5A activates latent ribonuclease (RNaseL), a candidate hereditary prostate cancer gene. OAS1 polymorphism (reference single nucleotide polymorphism [SNP] 2660 [rs2660]) has been associated with increased susceptibility to infections and various diseases. In general, the low-enzyme-activity adenine-adenine (AA) genotype promotes susceptibility, whereas the high-enzyme-activity guanosine-guanosine (GG) genotype confers protection. In this study, the authors investigated the association of this functional OAS1 polymorphism (rs2660) with prostate cancer. METHODS: Sample size and power were calculated using a power calculation software program for case-control genetic association analyses. Genomic DNA samples from a control group (n = 140) and from a case group of patients with prostate cancer (n = 164) were used for genotyping SNPs rs2660, rs1131454, and rs34137742 in all samples. Statistical analyses were performed using a logistic regression model. RESULTS: A significant association was observed between the rs2660 genotype (A/G) and prostate cancer. Genotype AA increased the risk, whereas genotype GG decreased the risk of prostate cancer. The GG genotype was not observed in the African American samples. The AA genotype also increased the risk of prostate cancer with age. CONCLUSIONS: The OAS1 SNP rs2660 AA genotype was associated significantly with prostate cancer, whereas the GG genotype protected against prostate cancer. OAS1 rs2660 may be a prostate cancer susceptibility polymorphism, which is a significant observation, especially in a context of the OAS1-RNaseL pathway. Thus, a functional defect in OAS1 because of the rs2660 SNP not only can attenuate RNaseL function but also can alter cell growth and apoptosis independent of RNaseL. Cancer 2011;. © 2011 American Cancer Society. [ABSTRACT FROM AUTHOR]

Abstract: Interferon alpha (IFNα) conjugated with polyethylene glycol (PEG) has been widely used for the treatment of hepatitis C virus (HCV) infection as a once-a-week injection formulation. However, the PEGylated IFNα has a low efficacy of ca. 39% and a side effect after repeated injections possibly due to the non-specific delivery with PEGylation. In this work, target specific long-acting hyaluronic acid–interferon alpha (HA–IFNα) conjugate was successfully developed for the treatment of HCV infection. HA–IFNα conjugate was synthesized by coupling reaction between aldehyde modified HA and the N-terminal group of IFNα. The IFNα content could be controlled in the range of 2–9 molecules per single HA chain with a bioconjugation efficiency higher than 95%. According to in vitro anti-proliferation assay using Daudi cells, HA–IFNα conjugate showed a comparable biological activity to PEG-Intron. In vivo real-time bioimaging confirmed the target specific delivery of near-infrared fluorescence (NIRF) dye labeled HA–IFNα conjugate to the liver in mice. In addition, pharmacokinetic analysis revealed the enhanced residence time longer than 4 days. After tail-vein injection, HA–IFNα conjugate induced ca. 60% higher expression of 2′,5′-oligoadenylate synthetase 1 (OAS 1) for innate immune responses to viral infection in the murine liver tissues than IFNα and PEG-Intron. [Copyright &y& Elsevier]

Abstract: Susceptibility of inbred strains to infection with West Nile virus (WNV) has been genetically associated with an arginine-to-a nonsense codon substitution at position 253 (R253X) in the predicted sequence of the murine 2′,5′-oligoadenylate synthetase 1B (OAS1B) protein. We introduced by transgenesis the Oas1b cDNA from MBT/Pas mice carrying the R253 codon (Oas1b MBT ) into BALB/c mice homozygous for the X253 allele (Oas1b BALB/c ). Overexpression of Oas1b MBT mRNA in the brain of transgenic mice prior and in the time course of infection provided protection against the neuroinvasive WNV strain IS-98-ST1. A 200-fold induction of Oas1b MBT mRNA in the brain of congenic BALB/c mice homozygous for a MBT/Pas segment encompassing the Oas1b gene was also efficient in reducing both viral growth and mortality, whereas a 200-fold induction of Oas1b BALB/c mRNA was unable to prevent virally-induced encephalitis, confirming the critical role of the R253X mutation on Oas1b activity in live mice. [Copyright &y& Elsevier]

Abstract: The 2′,5′-oligoadenylate synthetases (2–5A synthetases, OAS) form a family of proteins presented in many branches of Metazoa. The phylum Porifera (sponges) contains OAS proteins which are different from those in vertebrates and form a distinct OAS subfamily. In turn, OAS proteins from different genera of Demospongia show rather low similarities in their primary structures. To ascertain divergence of the OAS genes within a particular sponge genus, we identified the OAS genes from the marine sponge Geodia barretti and compared them with those from another member of the genus Geodia, Geodia cydonium. The identity and similarity of the OAS sequences found in G. barretti with those from G. cydonium were considerably higher than identities and similarities compared with those from other sponges, 75% and 85% versus 27–30% and 42–47%, respectively. We also established the presence of a transcriptionally active polymorphic OAS pseudogene in the genome of G. barretti. The transcripts of the OAS pseudogene(s) lack several internal exons encoding necessary motifs for OAS enzymatic activity. The maintenance and further diversification of OAS gene(s) and pseudogene(s) suggest the prevalence of gene duplication events over the loss of gene duplicates in Geodia genomes during the evolution. [Copyright &y& Elsevier]

Abstract: Type II interferon (IFN-γ) plays an important role in defense against viral infection. Although this cytokine is found during influenza virus infection, it seems to have no protective function against the virus, and the reasons for this are not clear. To determine how the influenza virus overcomes the antiviral effects of IFN-γ, we examined the effect of A/Puerto-Rico/8/34 (H1N1) (PR8) infection on the expression of various IFN-γ inducible genes involved in defense against virus infection. The results showed that PR8 selectively affects IFN-γ induced MHC-II and iNOS expression in both the murine macrophage-like cell line, Raw264.7, and in primary alveolar macrophages. Infection of IFN-γ treated macrophages with PR8 resulted in decreased expression of CIITA/MHC-II and increased production of iNOS/NO. These changes correlate with activation of NF-κB but not with JAK/STAT signaling. The data indicate one possible mechanism underlying the ineffectiveness of IFN-γ against influenza virus, and suggest that NF-κB may be a promising target for anti-influenza drugs. [Copyright &y& Elsevier]

Response to interferon therapy and disease progression in hepatitis C virus (HCV) infected patients differs among individuals, suggesting a possibility of a contribution of host genetic factors. 2′-5′-oligoadenylate synthetase 1 (OAS1), an important component of the innate immune system with a proven antiviral function, may therefore have a relationship with the response to interferon therapy and clinical course of HCV disease. Our aim was to determine the frequency of single nucleotide polymorphism (SNP) at exon 7 splice acceptor site (SAS) of the OAS1 gene in relation to the interferon response and status of HCV infection. A 203 bp fragment containing exon 7 SAS was amplified in 70 HCV chronic patients and 50 healthy controls. SNP was examined using restriction fragment length polymorphism (RFLP) genotyping method. Correlations of SNP genotypes with response to interferon and clinical status of patients were statistically analyzed. There was an increasing trend of response from AA to AG to GG genotypes ( P = 0.007). Genotype AA was associated with non-response to interferon and higher degree of liver fibrosis ( P = 0.05). Multivariate analysis showed this SNP as independent and a significant determinant of the outcome of interferon therapy (odds ratio 4.913 [95% confidence interval 1.365-8.2], P = 0.006). This is the first study to show a significant association between the functional SNP at exon 7 SAS of OAS1 gene and the viral response to interferon in chronic HCV patients. Patients with AA genotype were associated with progressive HCV disease and viral resistance to interferon therapy. This OAS SNP is a potential bio-marker to predict IFN response in chronic hepatitis C patients. [ABSTRACT FROM AUTHOR]

The article discusses a study on a West Nile virus (WNV) subgenomic replicon that expresses secreted alkaline phosphatase (SEAP) reporter gene rather than viral structural genes (repWNV/SEAP). The study found that viral replication efficiency can be evaluated easily with the help of repWNV/SEAP by directly measuring SEAP secretion in cell culture medium. The availability of repWNV/SEAP was validated using the anti-flavivirus gene, mouse oligoadenylate synthetase 1b (Oas1b). Oas1b-transfected cells showed higher resistance to repWNV/SEAP replication compared with the original cells. The authors conclude that repWNV/SEAP is a useful tool for identifying and evaluating the safety, ease of use and realibility of anti-flavivirus genes or drugs.

Abstract: To evaluate the role of host single nucleotide polymorphisms (SNPs) of 2′,5′-oligoadenylate synthetase (OAS) in predicting IFN response in patients with HBV infection, OAS gene and four SNPs were examined in 363 patients with chronic HBV infection (including 41 patients with HBsAg seroconversion) and 57 healthy controls. One SNP and three haplotypes were identified after adjustment for age, sex, HBV DNA. The frequency of OAS3T/C heterozygotes is 52.2% in responders (R) and 38.2% in non-responders (NR), with an odds ratio (OR) of 1.511 (P =0.018). For complete responders (CR) and NR, the OR reached 2.323(P =0.023). Haplotype analyses revealed significant association between three OAS haplotypes and response to IFN-α treatment. Genotype combination and interaction between gene–gene analyses disclosed that there was a positive interaction between OAS2/OAS3 and OAS3/OASL, and the rate of OR was 2.46 (likelihood test, P =0.004) and 4.46 (likelihood test, P =0.004), respectively. Our results suggest that OAS gene variations may play an important role in response to IFN-α and provide a novel strategy for the resolution of HBV infection. [Copyright &y& Elsevier]

Abstract: Resistance to flavivirus-induced disease in mice is conferred by the autosomal gene Flv, identified as 2′-5′ oligoadenylate synthetase 1b (Oas1b). Resistant mice express a full-length Oas1b protein while susceptible mice express the truncated Oas1btr. In this study, Oas1b was shown to be an inactive synthetase. Although the Oas/RNase L pathway was previously shown to have an antiviral role during flavivirus infections, Oas1b protein inhibited Oas1a in vitro synthetase activity in a dose-dependent manner and reduced 2-5A production in vivo in response to poly(I:C). These findings suggest that negative regulation of 2-5A by inactive Oas1 proteins may fine tune the RNase L response that if not tightly controlled could cause significant damage in cells. The results also indicate that flavivirus resistance conferred by Oas1b is not mediated by 2-5A. Instead, Oas1b inhibits flavivirus replication by an alternative mechanism that overrides the proviral effect of reducing 2-5A accumulation and RNase L activation. [Copyright &y& Elsevier]

The 2′-5′-oligoadenylate synthetase (2′-5′-OAS) family members are interferon-induced antiviral proteins. Twenty-three single nucleotide polymorphisms located within the OAS1, OAS2, OAS3, and OASL genes were analyzed in 142 patients with Russian tick-borne encephalitis. Statistically significant differences in genotype, allele, and haplotype frequencies for 3 OAS2 single nucleotide polymorphisms (rs1293762, rs15895, and rs1732778) and 2 OAS3 single nucleotide polymorphisms (rs2285932 and rs2072136) were detected between patients with central nervous system disease and both those with fever and/or meningitis and the control group. The data suggest a possible association between these 5 OAS single nucleotide polymorphisms and the outcome of tickborne encephalitis virus infection in a Russian population. [ABSTRACT FROM AUTHOR]

2′-5′-oligoadenylate synthetases are a family of interferon-induced enzymes playing an important role in antiviral defense in mammals. In the human genome, three genes encoding functional synthetases ( OAS1, OAS2 and OAS3) form a cluster. Previously, we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) of OAS2 and OAS3 are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russians. In the current study, we investigated the distribution of three of the above SNPs, OAS3 rs2285932 (C/T, Ile438Ile), OAS3 rs2072136 (G/A, Ser567Ser), and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform), in seven populations of North Eurasia: Caucasians (Russians, Germans from Altai region), Central Asian Mongoloids (Altaians, Khakass, Tuvinians, and Shorians), and Arctic Mongoloids (Chukchi). Interpopulational differences in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. These frequencies correlated with the ethnicity of the populations and with their supposed differential exposure to the TBE virus. In particular, the lowest frequencies of G/G genotype for OAS3 SNP rs2072136 (which, according to our earlier results, is associated with predisposition to severe forms of TBE) were found in Altaians, Khakass, Tuvinians, and Shorians, who commonly contact with the TBE virus in their habitation regions. Thus, the data obtained suggest that the TBE virus might act as a selection factor for particular OAS variants in Central Asian Mongoloids. [ABSTRACT FROM AUTHOR]

The 2′-5′ oligoadenylate synthetase (OAS) proteins are traditionally considered intracellular antiviral proteins. However, several studies demonstrate a correlation between the concentration of freely circulating OAS protein in sera from hepatitis C patients and their clinical prognosis. Here we demonstrate that extracellular OAS1 enters into cells and possesses a strong antiviral activity, both in vitro and in vivo, which is independent of RNase L. The OAS protein directly inhibits viral proliferation and does not require the activation of known antiviral signaling pathways. We propose that OAS produced by cells infected with viruses is released to the extracellular space, where it acts as a paracrine antiviral agent. Thus, the OAS protein represents the first direct antiviral compound released by virus-infected cells [ABSTRACT FROM AUTHOR]

Abstract: Upon viral infection, double-stranded viral RNA is detected very early in the host cell by several cellular 2′–5′ oligoadenylate synthetases, which synthesize 2′–5′ adenylate oligonucleotides that activate the cellular RNase L, firing an early primary antiviral response through self and non-self RNA cleavage. Transfecting cells with synthetic 2′–5′ adenylate oligonucleotides activate RNase L, and thus provide a useful shortcut to study the early steps of cellular and viral commitments into this pathway. Defined 2′–5′ adenylate oligonucleotides can be produced in vitro, but their controlled synthesis, purification, and characterisation have not been reported in detail. Here, we report a method suitable to produce large amounts of 2–5As of defined lengths in vitro using porcine OAS1 (pOAS) and human OAS2 (hOAS). We have synthesized a broad spectrum of 2–5As at the milligram scale and report an HPLC-purification and characterisation protocol with quantified yield for 2–5A of various lengths. [ABSTRACT FROM AUTHOR]