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Background: Antimicrobial resistance (AMR) poses a critical threat to public health and disproportionately affects the health and well-being of persons in low-income and middle-income countries. Our aim was to identify synthetic antimicrobials termed conjugated oligoelectrolytes (COEs) that effectively treated AMR infections and whose structures could be readily modified to address current and anticipated patient needs., Methods: Fifteen chemical variants were synthesized that contain specific alterations to the COE modular structure, and each variant was evaluated for broad-spectrum antibacterial activity and for in vitro cytotoxicity in cultured mammalian cells. Antibiotic efficacy was analyzed in murine models of sepsis; in vivo toxicity was evaluated via a blinded study of mouse clinical signs as an outcome of drug treatment., Findings: We identified a compound, COE2-2hexyl, that displayed broad-spectrum antibacterial activity. This compound cured mice infected with clinical bacterial isolates derived from patients with refractory bacteremia and did not evoke bacterial resistance. COE2-2hexyl has specific effects on multiple membrane-associated functions (e.g., septation, motility, ATP synthesis, respiration, membrane permeability to small molecules) that may act together to negate bacterial cell viability and the evolution of drug-resistance. Disruption of these bacterial properties may occur through alteration of critical protein-protein or protein-lipid membrane interfaces-a mechanism of action distinct from many membrane disrupting antimicrobials or detergents that destabilize membranes to induce bacterial cell lysis., Interpretation: The ease of molecular design, synthesis and modular nature of COEs offer many advantages over conventional antimicrobials, making synthesis simple, scalable and affordable. These COE features enable the construction of a spectrum of compounds with the potential for development as a new versatile therapy for an imminent global health crisis., Funding: U.S. Army Research Office, National Institute of Allergy and Infectious Diseases, and National Heart, Lung, and Blood Institute., Competing Interests: Declaration of interests A UC Santa Barbara patent application describing the composition and use of COEs and derivatives as antibiotics is currently pending (G.C.B., M.J.M., D.M.H., J.L.; US20210017179A1). G.C.B. is a principal of Xiretsa Inc., which had no role in the design, execution, analysis, or funding of the work. The remaining authors made no declarations., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

C6 deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA) is catalyzed by a family of enzymes known as ADARs (adenosine deaminases acting on RNA) encoded by three genes in mammals. Alternative promoters and splicing produce two ADAR1 proteins, an interferon-inducible cytoplasmic p150 and a constitutively expressed p110 that like ADAR2 is a nuclear enzyme. ADAR3 lacks deaminase activity. A-to-I editing occurs with both viral and cellular RNAs. Deamination activity is dependent on dsRNA substrate structure and regulatory RNA-binding proteins and ranges from highly site selective with hepatitis D RNA and glutamate receptor precursor messenger RNA (pre-mRNA) to hyperediting of measles virus and polyomavirus transcripts and cellular inverted Alu elements. Because I base-pairs as guanosine instead of A, editing can alter mRNA decoding, pre-mRNA splicing, and microRNA silencing. Editing also alters dsRNA structure, thereby suppressing innate immune responses including interferon production and action.

Replication of negative-strand RNA viruses occurs in association with discrete cytoplasmic foci called inclusion bodies. Whereas inclusion bodies represent a prominent subcellular structure induced by viral infection, our knowledge of the cellular protein components involved in inclusion body formation and function is limited. Using measles virus-infected HeLa cells, we found that the WD repeat-containing protein 5 (WDR5), a subunit of histone H3 lysine 4 methyltransferases, was selectively recruited to virus-induced inclusion bodies. Furthermore, WDR5 was found in complexes containing viral proteins associated with RNA replication. WDR5 was not detected with mitochondria, stress granules, or other known secretory or endocytic compartments of infected cells. WDR5 deficiency decreased both viral protein production and infectious virus yields. Interferon production was modestly increased in WDR5-deficient cells. Thus, our study identifies WDR5 as a novel viral inclusion body-associated cellular protein and suggests a role for WDR5 in promoting viral replication. IMPORTANCE Measles virus is a human pathogen that remains a global concern, with more than 100,000 measles-related deaths annually despite the availability of an effective vaccine. As measles continues to cause significant morbidity and mortality, understanding the virus-host interactions at the molecular level that affect virus replication efficiency is important for development and optimization of treatment procedures. Measles virus is an RNA virus that encodes six genes and replicates in the cytoplasm of infected cells in discrete cytoplasmic replication bodies, though little is known of the biochemical nature of these structures. Here, we show that the cellular protein WDR5 is enriched in the cytoplasmic viral replication factories and enhances virus growth. WDR5-containing protein complex includes viral proteins responsible for viral RNA replication. Thus, we have identified WDR5 as a host factor that enhances the replication of measles virus., (Copyright © 2018 American Society for Microbiology.)

Adenosine-to-inosine (A-to-I) RNA editing is a conserved post-transcriptional mechanism mediated by ADAR enzymes that diversifies the transcriptome by altering selected nucleotides in RNA molecules. Although many editing sites have recently been discovered, the extent to which most sites are edited and how the editing is regulated in different biological contexts are not fully understood. Here we report dynamic spatiotemporal patterns and new regulators of RNA editing, discovered through an extensive profiling of A-to-I RNA editing in 8,551 human samples (representing 53 body sites from 552 individuals) from the Genotype-Tissue Expression (GTEx) project and in hundreds of other primate and mouse samples. We show that editing levels in non-repetitive coding regions vary more between tissues than editing levels in repetitive regions. Globally, ADAR1 is the primary editor of repetitive sites and ADAR2 is the primary editor of non-repetitive coding sites, whereas the catalytically inactive ADAR3 predominantly acts as an inhibitor of editing. Cross-species analysis of RNA editing in several tissues revealed that species, rather than tissue type, is the primary determinant of editing levels, suggesting stronger cis-directed regulation of RNA editing for most sites, although the small set of conserved coding sites is under stronger trans-regulation. In addition, we curated an extensive set of ADAR1 and ADAR2 targets and showed that many editing sites display distinct tissue-specific regulation by the ADAR enzymes in vivo. Further analysis of the GTEx data revealed several potential regulators of editing, such as AIMP2, which reduces editing in muscles by enhancing the degradation of the ADAR proteins. Collectively, our work provides insights into the complex cis- and trans-regulation of A-to-I editing.

Adenosine deaminases acting on double-stranded RNA (ADARs) catalyze the deamination of adenosine (A) to produce inosine (I) in double-stranded (ds) RNA structures, a process known as A-to-I RNA editing. dsRNA is an important trigger of innate immune responses, including interferon (IFN) production and action. We examined the role of A-to-I RNA editing by two ADARs, ADAR1 and ADAR2, in the sensing of self-RNA in the absence of pathogen infection, leading to activation of IFN-induced, RNA-mediated responses in mouse embryo fibroblasts. IFN treatment of Adar1(-/-) cells lacking both the p110 constitutive and p150 IFN-inducible ADAR1 proteins induced formation of stress granules, whereas neither wild-type (WT) nor Adar2(-/-) cells displayed a comparable stress granule response following IFN treatment. Phosphorylation of protein synthesis initiation factor eIF2α at serine 51 was increased in IFN-treated Adar1(-/-) cells but not in either WT or Adar2(-/-) cells following IFN treatment. Analysis by deep sequencing of mouse exonic loci containing A-to-I-editing sites revealed that the majority of editing in mouse embryo fibroblasts was carried out by ADAR1. IFN treatment increased editing in both WT and Adar2(-/-) cells but not in either Adar1(-/-) or Adar1(-/-) (p150) cells or Stat1(-/-) or Stat2(-/-) cells. Hyper-edited sites found in predicted duplex structures showed strand bias of editing for some RNAs. These results implicate ADAR1 p150 as the major A-to-I editor in mouse embryo fibroblasts, acting as a feedback suppressor of innate immune responses otherwise triggered by self-RNAs possessing regions of double-stranded character., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Expression of adenosine deaminase acting on RNA1 (ADAR1) is driven by alternative promoters. Promoter PA, activated by interferon (IFN), produces transcripts that encode the inducible p150 ADAR1 protein, whereas PB specifies the constitutively expressed p110 protein. We show using Stat1(-/-), Stat2(-/-) and IRF9(-/-) MEFs that induction of ADAR1 p150 occurs by STAT2- and IRF9-dependent signaling that is enhanced by, but not obligatorily dependent upon, STAT1. Chromatin immunoprecipitation analysis demonstrated STAT2 at the PA promoter in IFN-treated Stat1(-/-) cells, whereas IFN-treated wild-type cells showed both STAT1 and STAT2 bound at PA. By contrast, with human 2fTGH cells and mutants U3A or U6A, ADAR1 induction by IFN was dependent upon both STAT1 and STAT2. These results suggest that transcriptional activation of Adar1 by IFN occurs in the absence of STAT1 by a non-canonical STAT2-dependent pathway in mouse but not human cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Adenosine deaminase acting on RNA1 (ADAR1) catalyzes the C6 deamination of adenosine (A) to produce inosine (I) in regions of RNA with double-stranded (ds) character. This process is known as A-to-I RNA editing. Alternative promoters drive the expression of the Adar1 gene and alternative splicing gives rise to transcripts that encode 2 ADAR1 protein size isoforms. ADAR1 p150 is an interferon (IFN)-inducible dsRNA adenosine deaminase found in the cytoplasm and nucleus, whereas ADAR1 p110 is constitutively expressed and nuclear in localization. Dependent on the duplex structure of the dsRNA substrate, deamination of adenosine by ADAR can be either highly site-selective or nonspecific. A-to-I editing can alter the stability of RNA structures and the coding of RNA as I is read as G instead of A by ribosomes during mRNA translation and by polymerases during RNA replication. A-to-I editing is of broad physiologic significance. Both the production and the action of IFNs, and hence the subsequent interaction of viruses with their hosts, are among the processes affected by A-to-I editing.

Double-stranded RNA (dsRNA) plays a centrally important role in antiviral innate immunity, both for the production of interferon (IFN) and also in the actions of IFN. Among the IFN-inducible gene products are the protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA 1 (ADAR1). PKR is an established key player in the antiviral actions of IFN, through dsRNA-dependent activation and subsequent phosphorylation of protein synthesis initiation factor eIF2α thereby altering the translational pattern in cells. In addition, PKR plays an important role as a positive effector that amplifies the production of IFN. ADAR1 catalyzes the deamination of adenosine (A) in RNA with double-stranded (ds) character, leading to the destabilization of RNA duplex structures and genetic recoding. By contrast to the antiviral and proapoptotic functions associated with PKR, the actions of ADAR1 in some instances are proviral and cell protective as ADAR1 functions as a suppressor of dsRNA-mediated antiviral responses including activation of PKR and interferon regulatory factor 3., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Adenosine deaminases acting on RNA (ADARs) catalyze the C-6 deamination of adenosine (A) to produce inosine (I), which behaves as guanine (G), thereby altering base pairing in RNAs with double-stranded character. Two genes, adar1 and adar2, are known to encode enzymatically active ADARs in mammalian cells. Furthermore, two size forms of ADAR1 are expressed by alternative promoter usage, a short (p110) nuclear form that is constitutively made and a long (p150) form that is interferon inducible and present in both the cytoplasm and nucleus. ADAR2 is also a constitutively expressed nuclear protein. Extensive A-to-G substitution has been described in mouse polyomavirus (PyV) RNA isolated late times after infection, suggesting modification by ADAR. To test the role of ADAR in PyV infection, we used genetically null mouse embryo fibroblast cells deficient in either ADAR1 or ADAR2. The single-cycle yields and growth kinetics of PyV were comparable between adar1(-/-) and adar2(-/-) genetic null fibroblast cells. While large T antigen was expressed to higher levels in adar1(-/-) cells than adar2(-/-) cells, less difference was seen in VP1 protein expression levels between the two knockout MEFs. However, virus-induced cell killing was greatly enhanced in PyV-infected adar1(-/-) cells compared to that of adar2(-/-) cells. Complementation with p110 protected cells from PyV-induced cytotoxicity. UV-irradiated PyV did not display any enhanced cytopathic effect in adar1(-/-) cells. Reovirus and vesicular stomatitis virus single-cycle yields were comparable between adar1(-/-) and adar2(-/-) cells, and neither reovirus nor VSV showed enhanced cytotoxicity in adar1(-/-)-infected cells. These results suggest that ADAR1 plays a virus-selective role in the host response to infection.

Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11-E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150(-/-) but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150(-/-) cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150(-/-) cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus, reovirus, or lymphocytic choriomeningitis virus replication but protected against CPE resulting from infection with Newcastle disease virus, Sendai virus, canine distemper virus, and influenza A virus. Thus, ADAR1 is a restriction factor in the replication of paramyxoviruses and orthomyxoviruses.

Adenosine deaminases acting on RNA (ADARs) catalyze adenosine (A) to inosine (I) editing of RNA that possesses double-stranded (ds) structure. A-to-I RNA editing results in nucleotide substitution, because I is recognized as G instead of A both by ribosomes and by RNA polymerases. A-to-I substitution can also cause dsRNA destabilization, as I:U mismatch base pairs are less stable than A:U base pairs. Three mammalian ADAR genes are known, of which two encode active deaminases (ADAR1 and ADAR2). Alternative promoters together with alternative splicing give rise to two protein size forms of ADAR1: an interferon-inducible ADAR1-p150 deaminase that binds dsRNA and Z-DNA, and a constitutively expressed ADAR1-p110 deaminase. ADAR2, like ADAR1-p110, is constitutively expressed and binds dsRNA. A-to-I editing occurs with both viral and cellular RNAs, and affects a broad range of biological processes. These include virus growth and persistence, apoptosis and embryogenesis, neurotransmitter receptor and ion channel function, pancreatic cell function, and post-transcriptional gene regulation by microRNAs. Biochemical processes that provide a framework for understanding the physiologic changes following ADAR-catalyzed A-to-I ( = G) editing events include mRNA translation by changing codons and hence the amino acid sequence of proteins; pre-mRNA splicing by altering splice site recognition sequences; RNA stability by changing sequences involved in nuclease recognition; genetic stability in the case of RNA virus genomes by changing sequences during viral RNA replication; and RNA-structure-dependent activities such as microRNA production or targeting or protein-RNA interactions.

The protein kinase regulated by RNA (PKR) and the adenosine deaminase acting on RNA (ADAR1) are interferon-inducible enzymes that play important roles in biologic processes including the antiviral actions of interferons, signal transduction, and apoptosis. PKR catalyzes the RNA-dependent phosphorylation of protein synthesis initiation factor eIF-2 alpha, thereby leading to altered translational patterns in interferon-treated and virus-infected cells. PKR also modulates signal transduction responses, including the induction of interferon. ADAR1 catalyzes the deamination of adenosine (A) to generate inosine (I) in RNAs with double-stranded character. Because I is recognized as G instead of A, A-to-I editing by ADAR1 can lead to genetic recoding and altered RNA structures. The importance of PKR and ADAR1 in innate antiviral immunity is illustrated by a number of viruses that encode either RNA or protein viral gene products that antagonize PKR and ADAR1 enzymatic activity, localization, or stability.

The p150 form of the RNA-specific adenosine deaminase ADAR1 is interferon-inducible and catalyzes A-to-I editing of viral and cellular RNAs. We have characterized mouse genomic clones containing the promoter regions required for Adar1 gene transcription and analyzed interferon induction of the p150 protein using mutant mouse cell lines. Transient transfection analyses using reporter constructs led to the identification of three promoters, one interferon-inducible (P(A)) and two constitutively active (P(B) and P(C)). The TATA-less P(A) promoter, characterized by the presence of a consensus ISRE element and a PKR kinase KCS-like element, directed interferon-inducible reporter expression in rodent and human cells. Interferon induction of p150 was impaired in mouse cells deficient in IFNAR receptor, JAK1 kinase or STAT2 but not STAT1. Whereas Adar1 gene organization involving multiple promoters and alternative exon 1 structures was highly preserved, sequences of the promoters and exon 1 structures were not well conserved between human and mouse.

Background: Gene carriers based on lipids or polymers-rather than on engineered viruses-constitute the latest technique for delivering genes into cells for gene therapy. Cationic liposome-DNA (CL-DNA) complexes have emerged as leading nonviral vectors in worldwide gene therapy clinical trials. To arrive at therapeutic dosages, however, their efficiency requires substantial further improvement., Methods: Newly synthesized multivalent lipids (MVLs) enable control of headgroup charge and size. Complexes comprised of MVLs and DNA have been characterized by X-ray diffraction and ethidium bromide displacement assays. Their transfection efficiency (TE) in L-cells was measured with a luciferase assay., Results: Plots of TE versus the membrane charge density (sigmaM, average charge/unit area of membrane) for the MVLs and monovalent 2,3-dioleyloxypropyltrimethylammonium chloride (DOTAP) merge onto a universal, bell-shaped curve. This bell curve leads to the identification of three distinct regimes, related to interactions between complexes and cells: at low sigmaM, TE increases with increasing sigmaM; at intermediate sigmaM, TE exhibits saturated behavior; and unexpectedly, at high sigmaM, TE decreases with increasing sigmaM., Conclusions: Complexes with low sigmaM remain trapped in the endosome. In the high sigmaM regime, accessible for the first time with the new MVLs, complexes escape by overcoming a kinetic barrier to fusion with the endosomal membrane (activated fusion), yet they exhibit a reduced level of efficiency, presumably due to the inability of the DNA to dissociate from the highly charged membranes in the cytosol. The intermediate, optimal regime reflects a compromise between the opposing demands on sigmaM for endosomal escape and dissociation in the cytosol., (Copyright (c) 2005 John Wiley & Sons, Ltd.)

ADAR1 (adenosine deaminase acting on RNA) is widely expressed in adult mammals and has a critical role during embryogenesis. Two size forms of ADAR1 are known that possess adenosine-to-inosine editing activity: an interferon (IFN)-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. We defined the structure of the 5'-flanking region of the mouse Adar1 gene, and we show here that mouse Adar1 transcripts possess alternative exon 1 structures (1A, 1B, and 1C) that initiate from unique promoters and are spliced to a common exon 2 junction. Exon 1A-containing transcripts encoding p150 were expressed in all tissues examined from adult mice (brain, cecum, heart, kidney, liver, lung, spleen, and Peyer's patches) and were elevated most significantly in liver but remained lowest in brain following oral infection with Salmonella. Exon 1B-containing RNA was most abundant in brain and was not increased in any tissue examined following infection. Exon 1C-containing RNA was very scarce. Exon 1A, but not exon 1B or 1C, expression was increased in fibroblast L cells treated with IFN, and a consensus ISRE element was present in the promoter driving exon 1A expression. Exon 1B, but not 1A, was detectable in embryonic day 10.5 embryos and was abundantly expressed in embryonic day 15 embryos. Furthermore, the ADAR1 p110 protein isoform was detected in embryonic tissue, whereas both p110 and the inducible p150 proteins were found in IFN-treated L cells. Finally, the presence of alternative exon 7a correlated with exon 1B-containing RNA, and alternative exon 7b correlated with exon 1A-containing RNA. These results establish that multiple promoters drive the expression of the Adar1 gene in adult mice, that the IFN inducible promoter and exon 1A-containing RNA are primarily responsible for the increased ADAR1 observed in Salmonella-infected mice, and that the constitutive exon 1B-containing transcript and encoded p110 protein product are abundantly expressed both in adult brain and during embryogenesis.

Cationic liposomes (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L(alpha)(C) and inverted hexagonal H(II)(C) nanostructures, and mouse fibroblast cells. Confocal images of L(alpha)(C) complexes in cells identified two regimes. For low membrane charge density (sigma(M)), DNA remained trapped in CL-vectors. By contrast, for high sigma(M), released DNA was observed in the cytoplasm, indicative of escape from endosomes through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L(alpha)(C)-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids merged into a single curve as a function of sigma(M), identifying it as a key universal parameter. The universal curve for transfection by L(alpha)(C) complexes climbs exponentially over approximately four decades with increasing sigma(M) below an optimal charge density (sigma(M)(*)), and saturates for at a value rivaling the high transfection efficiency of H(II)(C) complexes. In contrast, the transfection efficiency of H(II)(C) complexes is independent of sigma(M). The exponential dependence of TE on sigma(M) for L(alpha)(C) complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sigma(M) lowers the barrier. In the saturated TE regime, for both L(alpha)(C) complexes and H(II)(C), confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.

Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear lambda-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled L(alpha)C with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled H(II)C) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, phiDOPE = 0.72) and a low transfectant complex (DOTAP/DOPC, (phiDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.

The double-stranded RNA-dependent protein kinase PKR is an interferon-inducible enzyme that possesses antiviral and antiproliferative activities. We examined expression of PKR transcripts in human placenta tissue and cultured human amnion U cells. Alternative exon 2 structures were identified and characterized that possess different functional activities. Cloning and sequence analyses of 5'-RACE cDNAs from human placenta established a linkage between exon 1 and three alternative exon 2 structures that constitute, together with part of exon 3, the 5'-untranslated region of the PKR mRNA. The alternative splice variants of exon 2 were designated Ex2alpha (83 nucleotides), Ex2beta (167 nucleotides), and Ex2gamma (401 nucleotides). All three exon 2 variants were present in placenta tissue. However, only the Ex2alpha and Ex2beta forms were detectable in the amnion U cell line. Nuclease protection analysis revealed that the Ex2beta form was slightly more abundant than the Ex2alpha form, in both placenta tissue and U cells. Interferon treatment of U cells increased the level of both Ex2alpha and Ex2beta RNA by approximately 5-fold. The translational activities, measured in a luciferase reporter assay, of RNA transcripts possessing the Ex2alpha and Ex2beta forms of the PKR 5'-UTR were comparable to each other and more efficient than those with the Ex2gamma form., (Copyright 1999 Academic Press.)

RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. We have now identified alternative exon 1 structures of human ADAR1 transcripts that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5'-rapid amplification of cDNA ends (RACE) cDNAs from human placenta established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrated that exon 1B-exon 2 transcripts were synthesized in the absence of IFN and were not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts were IFN inducible. Transient transfection analysis with reporter constructs led to the identification of two functional promoters, designated PC and PI. Exon 1B transcripts were initiated from the PC promoter whose activity in transient transfection reporter assays was not increased by IFN treatment. The 107-nt exon 1B mapped 14.5 kb upstream of exon 2. The 201-nt exon 1A that mapped 5.4 kb upstream of exon 2 was initiated from the interferon-inducible PI promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A).

The double-stranded RNA-specific adenosine deaminase (ADAR1) is inducible by interferon (IFN) and is implicated in the editing of viral RNAs during lytic and persistent infection. We have now isolated and characterized human genomic clones that contain the promoter region required for transcription of the ADAR1 gene. Rapid amplification of cDNA 5'-ends (5'-RACE) identified additional upstream exon 1 sequence that was localized on P1-phage and lambda-phage genomic clones by Southern gel-blot analysis and sequence analysis. A Northern gel-blot analysis using a probe corresponding to the 5'-RACE exon 1 sequence and adjacent exon 2 sequence detected a major RNA transcript of approximately 6.7kb that was IFN-inducible in human amnion U cells. Transient transfection assays, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the ADAR1 gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed a consensus copy of the IFN-Stimulated Response Element (ISRE) involved in IFN inducibility that was flanked by a Kinase Conserved Sequence (KCS)-like element previously found to be unique to the human and mouse PKR gene promoters. A 63-bp minimal promoter fragment possessing the KCS-like and ISRE elements was sufficient to drive IFN-inducible transcription.

The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.

The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes, HIS3 and IacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of the HIS3 and lacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathione S-transferase (GST) fusions in Escherichia coli were purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1-184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR.

Post-translational modifications such as protein phosphorylation provide an important mechanism by which the functional activity of proteins can be controlled and, hence, biological processes regulated. Interferons (IFN) are a multigene family of cytokines that can profoundly affect a wide variety of functions in animal cells including virus replication, cell growth and differentiation, and the immune response. Changes in protein phosphorylation mediated by the IFN-inducible, RNA-dependent protein kinase (PKR) are implicated in the control of cell proliferation mediated by IFNs. Our knowledge of the structure, regulation and function of PKR will be summarized in this brief review, with focus on those aspects of protein phosphorylation and interferon action involving PKR that are central to the roles of the enzyme in the control of cell growth and proliferation.

The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. We have isolated and characterized human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein. Southern blot and sequence analyses revealed that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) was exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differed in exons 6 and 7. ADAR-b was a 5'-splice site variant that possessed a 26-amino acid deletion within exon 7; ADAR-c was a 3'-splice site variant that possessed an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possessed comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c variants altered the functional importance of each of the three R-motifs.

Heparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent elF-2 alpha protein kinase (PKR). We have used heparin oligosaccharides of defined lengths to examine the heparin-mediated activation of human PKR. Heparin oligosaccharide with 8 sugar residues was nearly as efficient as 16-residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas 6-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistically in activating PKR autophosphorylation. The RNA-binding activity of recombinant PKR, measured with adenovirus VA RNA, was competed by poly(rl):poly(rC) but not by Hep-16. When the catalytically inactive, histidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a substrate for purified wild-type PKR, the intermolecular phosphorylation of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR but not by heparin-activated PKR. However, elF-2 alpha phosphorylation was catalyzed by both heparin-and dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosphorylation mediated either by Hep-16 or dsRNA, whereas preincubation with dsRNA either alone or in combination with Hep-16 did not impair subsequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detectable degradation of PKR during preincubation or subsequent autophosphorylation of PKR. These results suggest that, while both dsRNA and heparin are capable of activating PKR autophosphorylation, the structural and functional basis of PKR activation differs for these two classes of polyanionic biomolecules.

The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations viral RNAs and the site-selective editing of mammalian mRNAs of neural origin. The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2. Simultaneous multicolor FISH including lambda clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705.

Seven new inorganic heteropolyanions were tested for their antiviral activity. One of these, sodium 12-tungstoborate, was found to protect mice from Semliki forest virus and mouse embryo fibroblast monolayers from vaccinia virus infections. In mice these heteropolyanions exhibited no synergistic antiviral activity with the interferon inducing mycoviral double-stranded RNA.

Hexagonal virus-like particles (VLPs) measuring 45 nm across were detected in mycelial extracts from Trichothecium roseum Himachal strain, the source fungus for the production of T-poly (Trichothecium polysaccharide), a known inhibitor of plant viruses. VLPs were found to contain double-stranded RNA (ds RNA) and the purified ds RNA was capable of inhibiting tobacco mosaic virus infection in Nicotiana glutinosa plants. Active preparations of T-poly were found to contain traces of ds RNA, probably of mycoviral origin.

Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons. Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level. The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level. Of 147 silent nt differences in the translated region, 136 were in the third base position of codons.

Antiviral activity of an interferon-inducing mycoviral ds RNA against Semliki Forest virus infection was considerably enhanced by N-palmitoylmuramyl-L-alanyl-D-isoglutamine (PMDP), a new muramyl dipeptide. This enhancement in activity was not due to increased production of interferon, but resulted probably from a PMDP-induced increase in nonspecific resistance to infection. These results indicate that a combined treatment with an interferon inducer and muramyl dipeptide may prove highly useful to control effectively viral infections.

The effect of interferon on the expression of the reovirus serotype 1 Lang strain S3 gene was examined in simian COS cells transfected with the expression vector pSVS3 containing the S3 gene under the control of the SV40 late promoter. When COS cells were treated with type I interferon-alpha 24 hr after transfection, the synthesis of the reovirus S3-encoded sigma NS polypeptide was inhibited about 10-fold as compared to that in untreated control cells. By contrast, under the same conditions, neither the plasmid DNA copy number nor the S3 gene mRNA levels were significantly affected by interferon treatment. Type II interferon-gamma, unlike the type I interferons-alpha, did not affect the rate of synthesis of polypeptide sigma NS in pSVS3-transfected cells.

Human reovirus serotype 1 Lang strain s3 mRNA, which encodes the nonstructural RNA-binding polypeptide sigma NS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s3 mRNA is 1198 nucleotides in length and possesses an open reading frame with a coding capacity of 366 amino acids, sufficient to account for a sigma NS polypeptide of 41,179 daltons. Comparison of the serotype 1 (Lang) s3 sequence with the serotype 3 (Dearing) s3 sequence reveals 86.8 percent homology at the nucleotide level. The predicted sigma NS polypeptides of the Lang and Dearing strains display 97 percent homology at the amino acid level.