Induction and activation of antiviral enzyme 2',5'-oligoadenylate synthetase by in vitro transcribed insulin mRNA and other cellular RNAs.

Double-stranded RNA (dsRNA) can induce antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) expression and activate latent 2'5'AS. Our previous data have shown pancreatic ß cells are sensitive to dsRNA-induced 2'5'AS expression, and constitutive high basal 2'5'AS expression is associated with susceptibility to developing type 1 diabetes (T1D), a disease due to pancreatic ß cell loss. Here we report that in vitro transcribed human insulin mRNA induces the activation of human OAS gene promoter sequences, and specifically and dose-dependently induces 2'5'AS expression in murine pancreatic ßTC3 cells. Over-expression of dsRNA receptor retinoic acid-inducible gene-1 enhances insulin mRNA-induced 2'5'AS expression. In vitro transcribed insulin and other mRNAs, as well as total cellular RNAs, activate latent 2'5'AS in vitro with activation ability likely associated with the sequence and length of individual mRNAs or the sample source of total cellular RNA. Insulin mRNA does not show any specificity to activate 2'5'AS, but total cellular RNA from ßTC3 cells has high activation ability. Constitutive 2'5'AS expression in ßTC3 cells leads to cell proliferation inhibition and apoptosis. Our study suggests the possibility of cellular RNA-regulated 2'5'AS expression and activation, and the potential risk of high insulin gene transcription in pancreatic ß cells, and may help explain genetic predisposition to T1D associated with INS VNTR class I alleles.

OAS1 splice site polymorphism controlling antiviral enzyme activity influences susceptibility to type 1 diabetes.

Both genetic and nongenetic factors contribute to the development of type 1 diabetes. Many investigations, including prospective studies of high-risk children, have implicated virus infections as predisposing environmental agents. We previously reported that basal activity of the key antiviral enzyme 2'5'-oligoadenylate synthetase (2'5'AS) was significantly elevated in type 1 diabetic patients compared with healthy control subjects. Recently, we showed that an A/G splice site single nucleotide polymorphism (SNP) in the OAS1 gene encoding 2'5'AS is strongly associated with basal 2'5'AS activity. Basal enzyme activity was highest in individuals with GG genotype and lowest in those with AA genotype. In the present study, we genotyped 835 type 1 diabetic and 401 healthy siblings at the OAS1 splice site polymorphism and (for comparison) at an A/C SNP of the insulin (IDDM2) locus. Results showed that OAS1 GG and GA were significantly increased in diabetic compared with healthy siblings (P = 0.0023). The strength of association was similar to that at IDDM2, where, as expected, the C/C (variable number tandem repeat class I homozygote) genotype was increased in affected compared with healthy siblings (P = 0.0025). The results suggest that host genetic response to virus infection could influence susceptibility to type 1 diabetes.

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.

Evidence from twins for acquired cellular immune hyperactivity in type 1 diabetes.

Type 1 diabetes has been associated with an increased frequency of activated T cells and T-cell hyperactivity to non-specific and disease-specific stimuli including the islet autoantigen glutamic acid decarboxylase 65 (GAD). To address whether T-cell hyperactivity is genetic or acquired we measured whole blood cytokines in vitro in response to GAD or tetanus in 18 identical twin pairs, nine discordant for type 1 diabetes. In addition, the activity of 2', 5' oligoadenylate synthetase (OAS) in blood mononuclear cells was measured as a marker of viral infection. Interleukin-2 (IL-2) basally and IL-2 and interferon-gamma (IFN-gamma) in response to GAD, were detected more frequently and at higher levels in diabetic compared to non-diabetic twins. IL-10 was not different between groups. OAS activity was increased in diabetic compared to non-diabetic twins and showed a correlation with basal IL-2 and GAD-stimulated IFN-gamma and IL-10. These findings suggest that T-cell hyperactivity in type 1 diabetes is an acquired trait and could reflect persisting virus expression.