The Oligoadenylate Synthetase Family: An Ancient ProteinFamily with Multiple Antiviral Activities

Helle Kristiansen,1 Hans Henrik Gad,2 Signe Eskildsen-Larsen,1,3 Philippe Despres,2 and Rune Hartmann1

The 20-50 oligoadenylate synthetases (OAS) are interferon-induced antiviral enzymes that recognize virallyproduced dsRNA and initiate RNA destabilization through activation of RNase L within infected cells. How-ever, recent evidence points toward several RNase L–independent pathways, through which members of theOAS family can exert antiviral activity. The crystal structure of OAS led to a novel insight into the catalyticmechanism, and revealed a remarkable similarity between OAS, Polyadenosine polymerase, and the class ICCA-adding enzyme from Archeoglobus fulgidus. This, combined with a variety of bioinformatic data, leads tothe definition of a superfamily of template independent polymerases and proved that the OAS family are ancientproteins, which probably arose as early as the beginning of metazoan evolution.

Introduction

The 20-50 oligoadenylate synthetase (OAS) family ofproteins are members of an ancient class of template-

independent RNA polymerases, along with the eukaryoticpolyadenosine polymerase (PAP) and the class I CCA add-ing enzyme (CCA) from Archaea (Torralba and others 2008).We presume that the OAS proteins diverged from PAP andCCA at the beginning of metazoan (multicellular animals)evolution. This view is supported by the presence of OASproteins in some of the oldest metazoans, sea sponges (Por-ifera) (Wiens and others 1999). The important role played bythe OAS proteins in the defense against viral infections inmammalians is well documented, whereas the biological roleof recently identified OAS proteins in lower animals, par-ticularly invertebrates, is less clear.

The OAS proteins identify viral infection by binding to(viral derived) dsRNA. This results in an allosteric activationof OAS followed by synthesis of 20-50 oligoadenylates (2-5A),which in turn bind and activate the latent RNase, RNase L.Upon 2-5A binding, RNase L dimerizes and degrades RNApresent in the cell, cellular as well as viral, leading to theinhibition of protein synthesis (Clemens and Williams 1978;Dong and Silverman 1997). This is the classical OAS/RNaseL pathway. Recently, additional exciting information regard-ing RNase L–independent antiviral pathways has becomeavailable. Here, we review both the RNase L–dependent andRNase L–independent functions of the OAS family, as wellas providing an overview of the structure, function, andevolution of this interesting family of proteins.

The Oligoadenylate Synthetase Family:Genome Organization, Protein Structure,and Catalytic Mechanism

The OAS genes

The human OAS family is a multigene family that consistsof 4 genes, OAS1, OAS2, OAS3, and OASL. All 4 genes areactively induced by interferon. In addition, OASL, but notOAS1, is induced directly in response to viral infection(interferon-regulatory factor [IRF]-3 dependent) (Melchjor-sen and others 2009). Further, traces of an OASL2 pseudo-gene is found next to the OASL gene (Eskildsen and others2003). The OAS genes are located on the human chromosome12 with OAS1 to -3 clustered in the 12q24.1 region and OASLat 12q24.2 (Hovnanian and others 1998; Justesen and others2000). All mammalian OAS genes share a general exon/in-tron structure, consisting of a basal OAS unit, encoded by 5exons. This unit is repeated once in OAS1 and OASL, twice inOAS2, and thrice in OAS3. All the OAS genes contain 1(OAS3 and OASL) or 2 (OAS1 and OAS2) 30 exons, and thesegive rise to splice variants of OAS1 (designated p42, p44,p46, and p48) and OAS2 (designated p69 and p71) (Benechand others 1985; Ghosh and others 1991; Marie and Hova-nessian 1992). Only 1 splice variant of OAS3 has beenidentified (Rebouillat and others 1999). Two groups simul-taneously identified the fourth member of the OAS family,OASL (Hartmann and others 1998b; Rebouillat and others1998). The OASL gene shares high homology with the otherOAS family members; however, it is devoid of enzymaticactivity (Marques and others 2008). Further, it contains a 30

1Centre for Structural Biology, Department of Molecular Biology, Aarhus University, Aarhus, Denmark.2Unite Interactions Moleculaires Flavivirus-Hotes, Institut Pasteur, Paris, France.3Novozymes A/S, Copenhagen, Denmark.

JOURNAL OF INTERFERON & CYTOKINE RESEARCHVolume 31, Number 1, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/jir.2010.0107

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exon that shares homology with 2 consecutive ubiquitinrepeats (Hartmann and others 1998b).

Oas gene organization in mice. The rodent Oas gene locushas undergone a recent series of duplications event, leadingto the presence of 8 mOas1 genes in the murine genome(Eskildsen and others 2002; Mashimo and others 2002; Per-elygin and others 2002). In addition to the 8 complete mOas1genes, we found a group of 3 exons (exon C, D, and E) and asingle exon E within the Oas loci (Eskildsen and others 2002).These duplications appear to have occurred after the sepa-ration of the rodent order, and are thus specific to rodents.For a comprehensive study of the mammalian OAS1 geneloci, see Perelygin and others (2006). We also observed thatthe mOas1a and mOas1g genes, which are almost identical inamino acid composition, are located within 2 18 kb DNArepeat sequences, suggesting that they originate from a re-cent DNA duplication. Further, 4 of the 8 mOas1 genes aretranscribed in the centromere to telomere direction (mOas1a,mOas1c, mOas1e, and mOas1g) and 4 in the opposite direction

(mOas1b, mOas1d, mOas1f, and mOas1h) as indicated by theirposition either above or below the chromosome in Fig. 1.

Sequence analysis of the active site of the mOas1 proteinsencoded by the various genes revealed that they could bedivided into 2 groups. The sequence of mOas1a and mOas1gproteins suggests that they are active enzymes, which alsoagree with experiential information. On the contrary, theremaining mOas1 proteins contain changes within the activesite, which are considered to be incompatible with activity.Experiential evidence from Kakuta and others (2002) sup-ports this division of mOas1 proteins into a group of active(mOas1a and -g) and a group of catalytic inactive proteins(mOas1b, -c, -d, -e, -f, and -h). However, real-time polymerasechain reaction analysis revealed that all 8 mOas1 genes areactively transcribed and is induced by interferon (Eskildsenand others 2002).

In a pioneering effort using positional cloning, 2 groupssimultaneously identified the murine Oas1b gene as respon-sible for restricting West Nile virus (WNV) replication in

FIG. 1. Genomic structure of the human and murine OAS genes. (A) The human OAS genomic structure. (B) The murineOas genomic structure. Squares above the line indicate genes transcribed in the centromere-to-telomere direction; squaresbelow the line indicate genes transcribed in the telomere-to-centromere direction. Genes are not drawn to scale. OAS, 20-50

oligoadenylate synthetase; TEL, telomere; CEN, centromere; CDE, Exon duplications, see text for further explanation.

FIG. 2. Structure and active site of OAS. (A) Crystal structure of the porcine OAS1. (B) Close-up of the active site of OAS1.Residues demonstrated to be involved in catalysis by site-directed mutagenesis are shown in ball and stick, yellow indicatesimpaired activity if mutated to alanine, and red indicates no activity if mutated to alanine. A model, based on mutagenesisdata, of the assumed position of the donor ATP is shown.

42 KRISTIANSEN ET AL.

wild mice (Mashimo and others 2002; Perelygin and others2002). In the majority of laboratory mouse strains, this genecontains a premature stop codon, leading to the productionof a truncated protein. The antiviral activity of the mOas1bprotein is discussed later.

Structure of the OAS proteins

The determination of the porcine OAS1 crystal structureprovided valuable insight into the enzymatic mechanism ofthe OAS family. One OAS unit forms 1 defined globularstructure, containing 2 domains: an N-terminal b-sheet do-main, containing 5 anti-parallel b-strands, and a C-terminal, a-helical domain (Hartmann and others 2003). The 2 domainsare connected by a linker region and a short N-terminal ex-tension. The N-terminal extends across to the C-terminal do-main, and secures the 2 domains together (Fig. 2A).

In the following, we discuss the structure of OAS and theimplications for the catalytic function. Before doing so, wewould like to clarify the nomenclature used by us. OASbinds 2 molecules of ATP: the donor and acceptor ATP. Thedonor ATP is the molecule of ATP added to the growingchain of 2-5As, whereas the acceptor ATP provides the free 20

hydroxyl to which the AMP moiety of the donor ATP isadded. The acceptor ATP can be replaced by a growing chainof 2-5As. In terms of catalysis, the acceptor ATP is equivalentof the primer in other polymerases.

The active site of OAS is located on the interface betweenthe N-terminal and C-terminal domains, and consists ofresidues from both domains (Fig. 2B). The N-terminal do-main contains 3 aspartic acids, D74, D76, and D147 (num-bered according to the porcine OAS1), which coordinate the2 Mg2þ ions critical for catalysis (Hartmann and others 2003).The coordination of 2 catalytic Mg2þ ions is a feature con-served among all nucleotidyl transferase enzymes (see alsoThe unique 20 polymerase activity and the origin of the OASproteins section) (Holm and Sander 1995). The N-terminaldomain also contains a b–turn between strand 1 and 2 (alsoknown as a phosphate binding loop or the Walker A motif),responsible for binding the triphosphate of the incomingdonor ATP. The C-terminal domain contains 2 basic residues(R209 and K212 in the porcine OAS1), which are presumedto neutralize the positive charge on the phosphates of thedonor ATP. These residues are conserved in all catalytic ac-tive members of the OAS family, as well as in both PAP andCCA. Mutation of these residues leads to a strong impair-ment of catalytic activity in the OAS enzymes (Sarkar andothers 1999b; Hartmann and others 2003), as well as PAPand CCA (Martin and others 1999; Cho and others 2005).

The C-terminal domain also contains several residues,which, based upon mutational studies, are believed to interactwith the base moiety of the donor ATP (Torralba and others2008) (see Fig. 2 for details). These residues are found withinthe second helix and a small b-loop-b motif, located betweenhelix 3 and 4, and are conserved in all OAS family members, aswell as in CCA, but not in PAP. Interestingly, the active siteresidues in the C-terminal 4-helix bundle domain are all con-served in the inactive OASL protein, whereas the N-terminalb-sheet domain in OASL has diverged substantially from theother members of the OAS family; in particular, the last of theessential aspartic acids is absent from OASL.

At present, we know little about the interaction with theacceptor nucleotide. Obviously, the 20 hydroxyl has to be in a

position to react with the a-phosphate of the donor, and thusthe ribose of the acceptor must be positioned in a differentway from that seen in 30 specific polymerases.

The unique 2 0 polymerase activity and the originof the OAS proteins

The formation of 20 linked oligonucleotides is a uniqueproperty of the OAS proteins. Nevertheless, the fundamentalchemical reaction between the formation of 20 and 30 linkedoligonucleotides is similar. A unified mechanism utilizing 2metal ions was proposed for the phosphoryl transfer reaction ofall polymerases (Delarue and others 1990; Beese and Steitz 1991;Steitz and others 1994; Steitz 1999). This naturally raised thequestion if the OAS family would adhere to this mechanism.

First part of the answer came from bioinformatics. Usingstructure-guided alignments, the OAS family was classifiedinto the nucleotidyl transferase superfamily based on theconservation of a specific sequence motif in the active site(Holm and Sander 1995). Martin and Keller worked exten-sively on the characterization of PAP, and noted a conserva-tion of several specific motifs between the OAS family and thefamily of eukaryotic PAPs (Martin and others 1999; Martinand Keller 2007). PAP and OAS are both template-indepen-dent, RNA-specific ribonucleotidyl transferases using ATP assubstrate. The enzymatic activity of OAS differs from PAP in 2important ways: they add nucleotides to the 20 hydroxyl in-stead of the 30, and the OAS enzymes are primer independent,whereas PAP adds the polyA tail to the 30 end of mRNA.

The crystal structure of porcine OAS1 confirmed the re-lation between OAS and other nucleotidyl transferases, andhelped define a new family of structurally related template-independent polymerases. It revealed a remarkable similar-ity to the structure of the catalytic core of both PAP and classI CCA adding enzymes. These 3 enzymes have a common 2domain catalytic core, equivalent to 1 OAS unit as describedabove. The classical 5-stranded b-sheet domain is found in allnucleotidyl transferases (the palm domain in the handanalogy used by Steitz and others) and the C-terminal do-main is a 4-helix bundle domain, which replaces the thumbsand finger domains found in template dependent polymer-ases. Together, these 3 families are proposed to constitute asuperfamily of template independent polymerases (Torralbaand others 2008), which, we believe, shares a common evo-lutionary origin. Structure-guided mutagenesis confirmedthat catalysis is performed in a highly similar way in these 3enzymes, despite OAS being 20 specific. This leads to theconclusion that the 20 specificity of the OAS enzymes arisesfrom a different orientation of the ribose of the acceptorsubstrate in such a way that the 20 hydroxyl, not the 30 hy-droxyl, is posed for catalysis (Torralba and others 2008).

A search for the 2-5A binding protein, RNase L, only gavepositive results in tetrapods, leading to the assumption thatthe OAS system only exists in tetrapods (Cayley and others1982). This might still hold true for the classical OAS/RNaseL pathway. However, it is now clear that the OAS proteinsare part of an ancient family of template-independent poly-merases. With the progress in genomic sequencing, homo-logs of OAS have been identified throughout a number ofanimal phyla (Torralba and others 2008; Kjaer and others2009), including sponges, which are some of the oldestmetazoans (Wiens and others 1999). At the same time, it ap-pears that OAS has been secondarily lost in certain animals, as

THE OLIGOADENYLATE SYNTHETASE FAMILY 43

it is absent from the genomes of Caenorhabditis elegans andDrosophila melanogaster. In vertebrates, members of the OASfamily have been identified in all classes, except from bonyfishes (Osteichthyes). On the basis of the evidence presentedabove, we presume that the 20-specific OAS arose at the be-ginning of metazoan evolution (Torralba and others 2008).

Detection of Viral Infection: Binding of dsRNAand Activation of the OAS Enzymatic Activity

The activation of OAS1 by dsRNA and certain single-stranded RNAs has been characterized extensively in vitro.There is a tendency toward better activation of OAS1 bylonger dsRNAs (Desai and Sen 1997). This requirement forlonger dsRNA is somewhat enigmatic, as the RNA bindinggroove on OAS only covers 18 to 20 base pairs. It was sug-gested that the active form of OAS1 is tetrameric (Marie andothers 1990; Ghosh and others 1997), which could explain thepreference of OAS1 for longer dsRNAs. However, evidencefrom both our own work and that of others clearly demon-strated that monomeric OAS1 is fully active (Wathelet andothers 1986; Hartmann and others 2001, 2003). Thus, at leastfor OAS1, multimerization cannot explain the preference forlonger dsRNA. Contrary to this, relatively strong data havebeen generated to support a dimeric model for activatedOAS2 (Sarkar and others 1999a, 2002).

The mechanistic details of RNA activation have primarilybeen studied using OAS1, and the discussion in this para-graph refers to data obtained using OAS1. At present,we believe that the basic mechanism is similar for bothOAS2 and OAS3. The RNA binding site of OAS has beenidentified as a positively charged groove, found at theinterface of the N- and C- terminal domains (Hartmann andothers 2003). Single charge-swap mutations (Lys/Arg toGlu) within this area prevent the protein from entering theactivated state but have limited effect on RNA binding.However, multiple mutations within the basic RNA bindinggrove have an additive effect upon RNA binding. Thesedata lead to the conclusion that the activation of OAS is a2-step process: OAS binds RNA, and if the RNA is a suit-able activator, the protein will undergo a conformationalchange, involving, but not limited to, a rotation of theN- and C-terminal domains relative to each other, assem-bling the active site (Hartmann and others 1998a, 2003).Binding of dsRNA is not equal to activation, as the OASprotein can bind to a number of single-stranded RNAs,which does not lead to activation. Our current under-standing of the molecular details involved in OAS recog-nition of, and activation by, dsRNA is poor, in particulardue to the lack of structural information on the activatedOAS-dsRNA complex.

It is presumed that, in vivo, OAS recognizes viral dsRNA,which will activate OAS and initiate RNA degradation withinthe infected cell via the RNase L pathway. Thus, OAS servesas a combined pattern recognition receptor and antiviralprotein. However, few in vivo data exist to aid in the identi-fication of what kind of viral RNA is recognized by OAS.

RNase L–Dependent and RNase L–IndependentAntiviral Effects of the OAS Family Members

A number of studies have been performed over the years,documenting the important role played by the members of

the OAS family in controlling viral replication within mam-malian cells. Unfortunately, space does not permit us to gothrough all these studies systematically, but a recent reviewcan be found in (Silverman 2007). Here, we will focus uponthe different fundamental mechanisms by which the OASproteins can exert antiviral activity.

The first direct demonstration of an antiviral effect by amember of the OAS family was performed by Judith Che-bath and others (1987). They measured the replication ofEncephalomyocarditis virus (EMCV, a picornavirus) in cellstransfected with a plasmid overexpressing human OAS1,and compared it to cells transfected with the empty vector.Expression of OAS1 led to 10- to 100-fold reduction of viraltiters. Similar experiments were carried out for human OAS2with similar results (Marie and others 1999; Ghosh andothers 2000). In both cases, the authors also tested a differentvirus, Vesicular Stomatitis virus, (VSV a rhabdovirus) andfound no effect of OAS expression on the replication of thisvirus. Recent experiments with cells overexpressing OAS3reduced the viral titers of alphaviruses such as Chikungunyavirus, Sindbis virus, and Semliki Forest virus (Brehin andothers 2009).

Beside the demonstrated antiviral effects, polymorphismsin the human OAS gene have been also shown to be asso-ciated with susceptibility to infection with West Nile Virus(WNV, a flavivirus) (Yakub and others 2005; Lim and others2009) and Hepatitis C virus (a flavivirus) (Knapp and others2003) as well as SARS coronavirus (a coronavirus) (Hamanoand others 2005; He and others 2006).

RNase L–dependent antiviral effectof the OAS family

The classical OAS/RNase L pathway was described al-ready in the late 1970s (Roberts and others 1976; Hova-nessian and others 1977; Kerr and others 1977; Baglioni andothers 1978; Zilberstein and others 1978). Activation of thispathway results in RNA degradation within infected cells,both viral and cellular, including ribosomal RNA. Thus, ineffect, protein translation is inhibited in cells where theOAS/RNase L pathway is active, which effectively preventsviral replication. The activity of the OAS/RNase L pathwaycan be monitored by following degradation of ribosomalRNA in virus-infected cells or cells transfected with 2-5As.Generation of RNase L–deficient mice (RNase L�/�) con-firmed the importance of RNase L in the replication of pi-cornaviruses as these mice displayed an increased sensitivitytoward EMCV infection (Zhou and others 1997).

A recent study investigated the role of the OAS familyagainst dengue virus (a flavivirus) and found that both OAS1and OAS3 possess antiviral activity against dengue virus inan RNase L–dependent manner (Lin and others 2009).

RNase L–independent antiviral effectof the OAS family

Recently, evidence has been accumulating about at least 1,and probably several RNase L–independent pathways, bywhich members of the OAS family can prevent viral repli-cation. The human OASL and murine Oas1b are both devoidof enzymatic activity and thus incapable of activating RNaseL. Nevertheless, both proteins display antiviral activity ifexpressed in mammalian cells.

44 KRISTIANSEN ET AL.

The murine Oas1b gene. Mice have multiple duplicationsof their Oas1 gene, leading to 8 Oas1 genes in mice. In 2002, 2groups simultaneously identified 1 of the 8 genes, Oas1b, asthe gene responsible for the relative resistance of wild micetoward infection with WNV (Mashimo and others 2002;Perelygin and others 2002). All inbred mouse strains, except1, were susceptible to infection with WNV, whereas all of thestrains derived from wild mice were resistant to infection.Contrary to wild mice, inbred mice contained an Oas1b genewith a premature stop codon, hence encoding a truncatedversion of Oas1b, lacking *30% of its C-terminal domain.This truncated protein is not expected to fold properly, and isexpected to be unstable within the cell. The resistant mice allharbored a full-length version of Oas1b. Additional experi-ments confirmed the importance of the Oas1b gene in resis-tance to WNV. Knock-in of the full-length Oas1b from aresistant strain results in the reversal of the flavivirus-susceptible phenotype (Scherbik and others 2007). Further,expression of Oas1b protein suppresses WNV replication incell culture (Kajaste-Rudnitski and others 2006).

So why is this listed under the RNase L–independent ef-fect? First, the inability of any of the other 7 genes to com-pensate suggests a different mechanism. Second, mutationswithin the active site of the Oas1b protein are incompatiblewith enzymatic activity (Hartmann and others 2003). Usingcell lines with inducible expression of Oas1b, we demon-strated that the suppression of WNV was restricted tothe early phases of infection, and knock-out of RNase L bysiRNA does not affect the inhibition of WNV replicationby Oas1b, clearly indicating an RNase L–independentmechanism (Kajaste-Rudnitski and others 2006).

The OASL gene. OASL is a unique member of the OASfamily that lacks the enzymatic activity but has a C-terminaldomain, which displays homology to ubiquitin. OASL dis-plays antiviral activity against EMCV in a manner requiring

the ubiquitine-like domain, but is independent of RNase L(Marques and others 2008). The ubiqutin-like domain ofOASL does not appear to function as an ubiqutin molecule,as we were unable to detect any conjugation. Further, theubiqutin-like domain does not have any effect on the stabilityof the OASL protein (Hartmann, unpublished). The OASLgene appears to be conserved in mammals, with some spe-cies harboring 2 genes of the OASL type. This is best studiedin mice, where both genes have been characterized. ThemOasl1 gene is the ortholog of the human OASL gene andenzymatically inactive (Eskildsen and others 2003). ThemOasl2 gene is enzymatically active, which is reflected in thesequence of their active sites. Further, the mOasl1 and mOasl2genes differ substantially in the sequence of their ubiqutin-like domains: the mOasl1 gene is closer to its human orthologthan to its murine paralog. In humans, the OASL2 gene is apseudogene. We believe that the mOasl2 gene represents anevolutionary intermediate between the active OAS1/mOas1genes and the inactive OASL/mOasl1 genes.

Concluding Remarks

On the basis of the evidence listed above, we suggest theexistence of at least 2 RNase L–independent pathways bywhich members of the OAS family can exert antiviral activity(Fig. 3). We believe that the OASL and mOas1b proteinsrepresent 2 different molecular pathways, as the OASLprotein requires its unique ubiquitin-like domain for activity,and no such domain is found in the mOas1b protein.Recent work by us has provided evidence for an RNaseL–independent antiviral activity of both the porcine andhuman OAS1 proteins (Kristiansen and others, under re-view). It is possible that this pathway is overlapping with thepathway represented by the mOas1b gene. We speculate thatthe OAS family (excluding OASL), in general, has 2 antiviral

FIG. 3. Alternative pathways of the OAS family. Representation of potential pathways utilized by proteins of the OASfamily. IFN, interferon; ?, mechanism unknown.

THE OLIGOADENYLATE SYNTHETASE FAMILY 45

pathways, the classical RNase L–dependent pathway and ayet uncharacterized RNase L–independent pathway, whichcan act independently of each other. The mOas1b gene is theresult of a recent duplication event that has so far only beenidentified in the muroidea (mice and rats), and it is possi-ble that the mOas1b gene has specialized in the RNase L–independent pathway. However, this is purely speculativeand experiential evidence, in particular a clarification of themolecular details of the novel RNase L–independent path-way, is required.

Acknowledgments

Research in the author’s laboratory was supported bygrants from the Danish Cancer Society, The Danish MedicalResearch Council (Grant no. 22-04-0704), Novo NordiskFoundation, and the Carlsberg Foundation.

Author Disclosure Statement

No financial competing interests exist.

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Address correspondence to:Dr. Rune Hartmann

Centre for Structural BiologyDepartment of Molecular Biology

Aarhus UniversityGustav Wieds vej 10C

Aarhus 8000Denmark

E-mail: rh@mb.au.dk

Received 10 August 2010/Accepted 10 August 2010

THE OLIGOADENYLATE SYNTHETASE FAMILY 47