The expression of both domains of the 69/71 kDa 2',5' oligoadenylate synthetase generates a catalytically active enzyme and mediates an anti-viral response.

The 2',5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which, when activated by double-stranded (ds) RNA, polymerizes ATP into 2',5'-linked oligomers with the general formula pppA(2'p5'A)n, where n >/= 1. The 69-kDa form of human OAS has two isoforms (p69 and p71) that are identical for their first 683 amino acids and consist of two homologous and adjacent domains, each homologous to the small 40-kDa OAS. Here, we demonstrate that mRNA species specific for the isoforms p69 and p71 are enhanced in interferon-treated cells, with the p69 mRNA being more abundant than that of p71. In transfected cells, both isoforms could be expressed independently to generate enzymes with similar catalytic activity, typical of the natural 69-kDa OAS from interferon-treated cells. On the other hand, deletion mutants expressing either the N- or C-terminal domain common in p69 and p71 were greatly unstable and were found to be devoid of catalytic activity, in spite of the capacity of the C-terminal domain to bind dsRNA. Finally, we show that murine cell lines stably expressing either p69 or p71 isoforms partially resist infection by the encephalomyocarditis virus. These results indicate that both isoforms of the 69-kDa form of 2',5' OAS are expressed in interferon-treated cells, and that each isoform could be implicated in the mechanism of the anti-viral action of interferon.     

The human 2'-5'oligoadenylate synthetase family: unique interferon-inducible enzymes catalyzing 2'-5' instead of 3'-5' phosphodiester bond formation

The demonstration by Kerr and colleagues that double-stranded (ds) RNA inhibits drastically protein synthesis in cell-free systems prepared from interferon-treated cells, suggested the existence of an interferon-induced enzyme, which is dependent on dsRNA. Consequently, two distinct dsRNA-dependent enzymes were discovered: a serine/threonine protein kinase that nowadays is referred to as PKR and a 2'-5'oligoadenylate synthetase (2'-5'OAS) that polymerizes ATP to 2'-5'-linked oligomers of adenosine with the general formula pppA(2'p5'A)(n), n>or=1. The product is pppG2'p5'G when GTP is used as a substrate. Three distinct forms of 2'-5'OAS exist in human cells, small, medium, and large, which contain one, two, and three OAS units, respectively, and are encoded by distinct genes clustered on the 2'-5'OAS locus on human chromosome 12. OASL is an OAS like IFN-induced protein encoded by a gene located about 8 Mb telomeric from the 2'-5'OAS locus. OASL is composed of one OAS unit fused at its C-terminus with two ubiquitin-like repeats. The human OASL is devoid of the typical 2'-5'OAS catalytic activity. In addition to these structural differences between the various OAS proteins, the three forms of 2'-5'OAS are characterized by different subcellular locations and enzymatic parameters. These findings illustrate the apparent structural and functional complexity of the human 2'-5'OAS family, and suggest that these proteins may have distinct roles in the cell.     

Expansion and molecular evolution of the interferon-induced 2'-5' oligoadenylate synthetase gene family

The mammalian 2'-5' oligoadenylate synthetases (2'-5'OASs) are enzymes that are crucial in the interferon-induced antiviral response. They catalyze the polymerization of ATP into 2'-5'-linked oligoadenylates which activate a constitutively expressed latent endonuclease, RNaseL, to block viral replication at the level of mRNA degradation. A molecular evolutionary analysis of available OAS sequences suggests that the vertebrate genes are members of a multigene family with its roots in the early history of tetrapods. The modern mammalian 2'-5'OAS genes underwent successive gene duplication events resulting in three size classes of enzymes, containing one, two, or three homologous domains. Expansion of the OAS gene family occurred by whole-gene duplications to increase gene content and by domain couplings to produce the multidomain genes. Evolutionary analyses show that the 2'-5'OAS genes in rodents underwent gene duplications as recently as 11 MYA and predict the existence of additional undiscovered OAS genes in mammals.     

Inhibition of 2'-5' oligoadenylate synthetase by divalent metal ions.

OAS1 is the small form and OAS2 is the medium form of the human interferon-induced 2'-5' oligoadenylate synthetases. The p42 isoform of OAS1 and the p69 isoform of OAS2 have been expressed in insect cells and purified to give pure, highly active 2'-5' oligoadenylate synthetase. The catalysis of 2'-5' oligoadenylate synthesis is strictly dependent on double-stranded RNA and magnesium ions. We have examined the effect of a series of divalent metal ions: copper, iron and zinc ions strongly inhibited the enzymatic activity, cobalt and nickel ions were partly inhibitory whereas calcium and manganese ions were without effect. However, manganese ions can replace magnesium ions as activator. The inhibitory effect of zinc ions was characterised in detail. The inhibitory constants of Zn(2+) were estimated to be 0.10 mM for OAS1p42 and to 0.02 mM for OAS2p69. Cross-linking experiments showed that zinc ions can control the oligomerisation by enhancing the formation of tetrameric forms of OAS1p42     

Structure of equine 2'-5'oligoadenylate synthetase (OAS) gene family and FISH mapping of OAS genes to ECA8p15-->p14 and BTA17q24-->q25

Mammalian 2'-5' oligoadenylate (2-5A) synthetases are important mediators of the antiviral activity of interferons. Both human and mouse 2-5A synthetase gene families encode four forms of enzymes: small, medium, large and ubiquitin-like. In this study, the structures of four equine OAS genes were determined using DNA sequences derived from fifteen cDNA and four BAC clones. Composition of the equine OAS gene family is more similar to that of the human OAS family than the mouse Oas family. Two OAS-containing bovine BAC clones were identified in GenBank. Both equine and bovine BAC clones were physically assigned by FISH to horse and cattle chromosomes, ECA8p15-->p14 and BTA17q24--> q25, respectively. The comparative mapping data confirm conservation of synteny between ungulates, humans and rodents.     

Interleukin-6 induces the (2'-5') oligoadenylate synthetase gene in M1 cells through an effect on the interferon-responsive enhancer

Interleukin-6 (IL-6) activates (2'-5') A synthetase (2'-5' AS) gene expression in differentiating myeloleukemic M1 cells. Antibodies to type I interferon (IFN) inhibit 2'-5' AS induction but not differentiation. Analysis of the mechanism of 2'-5' AS induction shows that it does not result from increased IFN formation, but from a synergism between IL-6 and endogenously secreted IFN. IL-6 can activate expression of a CAT construct fused to the interferon response sequence (IRS) of the 2'-5' AS gene. In extracts of IL-6-treated M1 cells, changes in protein binding to IRS DNA can be demonstrated. One of the effects of IL-6 on M1 cells is, therefore, to induce DNA binding factors, some of which act on the same enhancer sequence as IFNs, resulting in a synergistic gene activation. M1 variants resistant to differentiation by IL-6 have lost the ability to induce the 2'-5' AS gene.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Gene structure and expression of the 5'-(CGG)(n)-3'-binding protein (CGGBP1)

The human nuclear 5'-(CGG)(n)-3'-binding protein (CGGBP1) influences the expression of the fragile X mental retardation (FMR1) gene by specifically interacting with the 5'-(CGG)(n>5)-3' repeat in its 5' untranslated region. Here, we show that CGGBP1 binds to 5'-(CGG)(n)-3' repeats with n>or=5 and to interrupted repeats. The genomic and mRNA organization of the human and murine CGGBP1 genes was studied and the human gene was mapped to chromosome 3p. Due to alternative polyadenylation, mRNAs of 1.2 and 4.5 kb are transcribed at varying ratios in human and murine cells and in embryonic, fetal, and adult tissues. The human and the murine genes, including promoters and large parts of the untranslated regions, are highly conserved. A sequence of 235 nucleotides 5' upstream of CGGBP1 is essential for promoter activity in transfection experiments. Complete in vitro methylation inactivates the promoter, which is unmethylated in human cells as shown by bisulfite genomic sequencing.     

Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene

It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine triphosphate in 2'-specific nucleotidyl transfer reactions to synthesize 2',5'-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. We showed elsewhere that constitutive (basal) activity of 2'5'AS is correlated with virus-stimulated activity. In the present study, we asked whether constitutive activity is genetically determined and, if so, by which variants. Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs (P<.0001) and sibling pairs (P=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1 an excellent candidate for a human gene that influences host susceptibility to viral infection.     

Molecular cloning, expression and chromosomal localization of a novel human REG family gene, REG III

Regenerating gene (Reg), first isolated from a regenerating islet cDNA library, encodes a secretory protein with a growth stimulating effect on pancreatic beta cells that ameliorates the diabetes of 90% depancreatized rats and non-obese diabetic mice. Reg and Reg-related genes have been revealed to constitute a multigene family, the Reg family, which consists of four subtypes (types I, II, III, IV) based on the primary structures of the encoded proteins of the genes [Diabetes 51(Suppl. 3) (2002) S462]. Plural type III Reg genes were found in mouse and rat. On the other hand, only one type III REG gene, HIP/PAP (gene expressed in hepatocellular carcinoma-intestine-pancreas/gene encoding pancreatitis-associated protein), was found in human. In the present study, we found a novel human type III REG gene, REG III. This gene is divided into six exons spanning about 3 kilobase pairs (kb), and encodes a 175 amino acid (aa) protein with 85% homology with HIP/PAP. REG III was expressed predominantly in pancreas and testis, but not in small intestine, whereas HIP/PAP was expressed strongly in pancreas and small intestine. IL-6 responsive elements existed in the 5'-upstream region of the human REG III gene indicating that the human REG III gene might be induced during acute pancreatitis. All the human REG family genes identified so far (REG Ialpha, REG Ibeta, HIP/PAP, REG III and REG IV) have a common gene structure with 6 exons and 5 introns, and encode homologous 158-175-aa secretory proteins. By database searching and PCR analysis using a yeast artificial chromosome clone, the human REG family genes on chromosome 2, except for REG IV on chromosome 1, were mapped to a contiguous 140 kb region of the human chromosome 2p12. The gene order from centromere to telomere was 5' HIP/PAP 3'-5' RS 3'-3' REG Ialpha 5'-5' REG Ibeta 3'-3' REG III 5'. These results suggest that the human REG gene family is constituted from an ancestor gene by gene duplication and forms a gene cluster on the region.     

2'-5' Oligoadenylate synthetase plays a critical role in interferon-gamma inhibition of respiratory syncytial virus infection of human epithelial cells

Respiratory syncytial virus (RSV), associated with bronchiolitis and asthma, is resistant to the antiviral effects of type-I interferons (IFN), but not IFN-gamma. However, the antiviral mechanism of IFN-gamma action against RSV infection is unknown. The molecular mechanism of IFN-gamma-induced antiviral activity was examined in this study using human epithelial cell lines HEp-2 and A549. Exposure of these cells to 100-1000 units/ml of IFN-gamma, either before or after RSV infection, results in a significant decrease in RSV infection. After 1 h of exposure, IFN-gamma induces protein expression of IFN regulatory factor-1 (IRF-1) but not IRF-2, double-stranded RNA-activated protein kinase, and inducible nitric-oxide synthase in these cells. The mRNA for IRF-1, p40, and p69 isoforms of 2'-5' oligoadenylate synthetase (2-5 AS) are detectable, respectively, at 1 and 4 h of IFN-gamma exposure. Studies using cycloheximide and antisense oligonucleotides to IRF-1 indicate a direct role of IRF-1 in activating 2-5 AS. Cells transfected with 2-5 AS antisense oligonucleotides inhibit the antiviral effect of IFN-gamma. A stable cell line of HEp-2 overexpressing RNase L inhibitor, RLI-14, which exhibits an IFN-gamma-induced gene expression pattern similar to that of the parent cell line, shows a significant reduction in RNase L activity and IFN-gamma-mediated antiviral effect, compared with HEp-2 cells. These results provide direct evidence of the involvement of 2-5 AS in IFN-gamma-mediated antiviral activity in these cells.