2-5A-dependent RNAase, an interferon-induced enzyme that is activated by 5'-phosphorylated, 2',5'-linked oligoadenylates (2-5A), is implicated in both the molecular mechanisms of interferon action and the fundamental control of RNA stability in mammalian cells. Here we report the expression cloning and analysis of murine and human 2-5A-dependent RNAases. The 2-5A binding properties and RNAse activities of recombinant and naturally occurring forms of 2-5A-dependent RNAase were identical. Interferon induction of 2-5A-dependent RNAse expression was demonstrated by measuring the mRNA levels in cells treated with interferon and cycloheximide. Analysis of aligned murine and human 2-5A-dependent RNAse sequences revealed several intriguing features, including similarity to RNAase E, which is implicated in the control of mRNA stability in E. coli. Interestingly, a duplicated phosphate-binding loop motif was determined by deletion analysis and site-directed mutagenesis to function in the binding of 2-5A.

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The 2–5A system contributes to the antiviral effect of interferons through the synthesis of 2–5A and its activation of the ribonuclease, RNase L. RNase L degrades viral and cellular RNA after activation by unique, 2′–5′ phosphodiester-linked, oligoadenylates [2–5A, (pp)p5′ A2′(P5′A2′)]n, n ⩾2. Because both the 2–5A system and apoptosis can serve as viral defense mechanisms and RNA degradation occurs during both processes, we investigated the potential role of RNase L in apoptosis. Overexpression of human RNase L by an inducible promoter in NIH3T3 fibroblasts decreased cell viability and triggered apoptosis. Activation of endogenous RNase L, specifically with 2–5A or with dsRNA, induced apoptosis. Inhibition of RNase L with a dominant negative mutant suppressed poly (I)·poly (C)–induced apoptosis in interferon-primed fibroblasts. Moreover, inhibition of RNase L suppressed apoptosis induced by poliovirus. Thus, increased RNase L levels induced apoptosis and inhibition of RNase L activity blocked viral-induced apoptosis. Apoptosis may be one of the antiviral mechanisms regulated by the 2–5A system.