Extracellular 2'-5' oligoadenylate synthetase stimulates RNase L-independent antiviral activity: a novel mechanism of virus-induced innate immunity

The innate immune system limits viral replication during early stages of infections and primes the development of adaptive immunity. Recently, substantial progress has been made in understanding the mechanism by which viral infections are recognized by the innate immune system (1, 40, 41) and how this recognition leads to initiation of an antiviral immune response. Production of antiviral cytokines, like interferon (IFN), by virus-infected cells is an essential part of the antiviral response. IFN-mediated cellular signaling leads to the induction of several hundred antiviral genes within cells (29). Extensive research has provided insight into the molecular details of IFN signaling. However, our current understanding of the specific mechanism by which the IFN-induced proteins inhibit viral replication is limited. The 2′-5′ oligoadenylate synthetases (OAS) are a family of antiviral proteins. In humans, this family consists of four genes, OAS1, OAS2, OAS3, and OASL. As is typical for antiviral genes, the transcription of the OAS genes is induced by both virus infection and IFN stimulation (5, 25, 39). None of the OAS genes encode proteins with signal peptides known to mediate secretion to the extracellular environment. Nevertheless, OAS is found in the sera of hepatitis C virus (HCV)-infected individuals. Furthermore, patients undergoing alpha IFN (IFN-α) therapy display elevated levels of OAS activity in serum, and the levels correlate with the success of treating HCV infection by pegylated IFN-α (19, 26, 35). The OAS1 to -3 genes encode proteins with a characteristic polymerase activity, the 2′-5′ oligoadenylate (2-5A) synthetase activity (16), whereas the OASL protein has significant sequence similarity to the other OAS proteins but is devoid of this enzymatic activity (23). The OAS1 to -3 proteins contain one to three repeats of the basal “OAS unit,” and OASL contains one (4, 13, 15, 22, 30, 31). The OAS1 to -3 proteins are all produced as latent enzymes, which are activated to synthesize 2-5As upon binding to double-stranded RNA (dsRNA) (3). The 2-5As subsequently bind and activate the latent RNase, RNase L (11). Activation of RNase L leads to degradation of cellular as well as viral RNA, resulting in the inhibition of protein synthesis, thus terminating viral replication (7). The generation of small RNAs by RNase L cleavage can further enhance the innate immune response, as they are recognized by the pattern recognition receptors, RIG-I and MDA-5 (21). The crystal structure of porcine p40 OAS1 revealed an enzyme, which is related to the Pol β family despite being 2′ specific (14). Three aspartic acid residues, D74, D76, and D147, form the catalytic triad of the enzyme. These residues coordinate two Mg2+ ions, which are critical for catalysis. Residues R209 and R212, which are located in a helix opposite the catalytic triad, neutralize the negative charge of the triphosphates of the incoming ATP and facilitates catalysis (14, 37). The RNA binding site of OAS is comprised of a positively charged groove, which interacts with the dsRNA through arginine and lysine residues (R38, K41, K59, R194, R198). A strain containing a simultaneous mutation of these five residues to glutamic acid (referred to as the C5 mutant in this paper) yielded a protein with minimal affinity toward dsRNA and no enzymatic activity (14). Overexpression of OAS1 in cell culture has demonstrated antiviral activity against encephalomyocarditis virus (EMCV) (8, 33) and mengovirus (6). A recent study used retroviral vectors to overexpress all known isoforms of OAS in human A549 cells and showed that expression of both OAS1 (the p42 and p46 splice isoforms) and OAS3 resulted in inhibition of West Nile virus (WNV) replication in an RNase L-dependent fashion (20). Interestingly, rodents have multiple duplications of the Oas1 gene, and the murine Oas1b gene was shown to be important for resistance toward WNV infection (24, 28). This activity did not appear to require RNase L, as expression of Oas1b in RNase L-negative cells still resulted in antiviral activity (18). The observation of OAS in the sera of patients suffering from various viral infections, or undergoing IFN treatment, prompted us to investigate whether exogenous OAS could protect cells from infection with virus. We found that exogenous recombinant porcine OAS1 enters into cells and exhibits broad-spectrum antiviral activity. This protection is not mediated through the activation of the IFN system and is independent of RNase L. Moreover, the injection of recombinant OAS1 into mice strongly limited viral replication compared to that in relevant controls, demonstrating this mechanism to be operative in vivo and hence to have a therapeutic potential.