3166±3173 Nucleic Acids Research, 2003, Vol. 31, No.

12
DOI: 10.1093/nar/gkg427

Characterization of the 2¢±5¢-oligoadenylate

synthetase ubiquitin-like family
Signe Eskildsen1, Just Justesen1, Mikkel Heide Schierup2 and Rune Hartmann1,3,*
1
Department of Molecular Biology, University of Aarhus, CF Moellers Alle 130, DK-8000 Aarhus C, Denmark,
2
Bioinformatics Research Center, University of Aarhus, Ny Munkegade 116, DK-8000 Aarhus C, Denmark and
3
Department of Molecular Cardiology/NB20, Lerner Research Institute, The Cleveland Clinic Foundation, OH, USA

Received February 14, 2003; Revised April 5, 2003; Accepted April 22, 2003

ABSTRACT activation of the OAS enzymes results in the synthesis of

The interferon-induced 2¢±5¢-oligoadenylate synthet-
ases (OAS) are important for the antiviral activity
of interferons. The human and murine OAS gene
families each contain four genes: OAS1, OAS2,
OAS3 and OASL, all having one or more conserved
OAS units composed of ®ve translated exons. The
OASL gene has both an OAS unit and a C-terminus
of two ubiquitin-like repeats. In this study, we
demonstrate that murine Oasl1 protein is inactive
while murine Oasl2 is active as an OAS. Further-
more, murine Oasl2 requires double-stranded RNA
as co-factor. The af®nity of murine Oasl2 for the
double-stranded RNA activator is higher than that of
human OAS1 (p42 isoform). We propose a model for
the evolutionary origin of the murine Oasl1 and
Oasl2 genes. The identi®cation of a human ortho-
logue (hOASL2) to the murine Oasl2 gene estab-
lishes that the OASL gene was duplicated prior to
the radiation of the rodent and primate groups.
We suggest that murine Oasl2, which has both
enzymatic activity and a ubiquitin-like domain, is a
functional intermediate between the active OAS
species and the inactive human OASL1/murine
Oasl1 proteins. In addition, we propose that murine
Oasl1 appears to have gained a hitherto unchar-
acterized function independent of 2¢±5¢-linked
oligoadenylate synthesis.
INTRODUCTION
Interferons (IFNs) constitute an important part of the mam-
malian innate immune system (1). They confer resistance to
viral infections by regulating the transcription of a large
number of genes (2). The 2¢±5¢-oligoadenylate synthetases
(OAS) were among the ®rst characterised IFN-induced
antiviral proteins (3±5). The OAS gene family is induced by
both type I and type II IFNs. The OAS proteins are expressed
as latent enzymes, which require double-stranded RNA
(dsRNA) for activation; however, they do not harbour any
of the characteristic dsRNA-binding motifs (6). This
2¢±5¢-linked oligoadenylates (2±5As) from ATP (6). In turn,
these 2±5As can bind to the latent RNase L which subse-
quently dimerises into the active form. The activated RNase L
then degrades viral and cellular RNAs, suppressing protein
synthesis and viral growth (7). Most viruses produce dsRNA at
some stage in their life cycle, and it is generally believed that
the activating dsRNA in infected cells is of viral origin (8).
Furthermore, the OAS are involved in the induction of
apoptosis and control of cell growth (9,10), and studies show
that RNase L and the 2±5A system play a role in tumorigenesis
(11).
In humans, the OAS gene family is composed of four genes
located on chromosome 12 (6,12,13) The hOAS1, hOAS2 and
hOAS3 genes are encoded by a tightly coupled locus on
12q24.1. The additional member of the OAS family in humans
is the hOASL gene which is located on 12q24.2. Each OAS
gene consists of a conserved OAS unit composed of ®ve
translated exons (exons A±E). OAS1 has one unit, whereas
OAS2 and OAS3 have two and three units, respectively, and all
three genes encode active 2±5A synthetases. The hOASL gene
encodes a two-domain protein composed of an OAS unit fused
to a 164 amino acid C-terminal domain, homologous to a
tandem repeat of ubiquitin. In contrast to the different
enzymes encoded by the hOAS1, hOAS2 and hOAS3 genes,
no enzymatic activity has been detected for the hOASL
protein (14,15).
Another member of the OASL class proteins was identi®ed
in chicken (16). The ChOASL protein is active upon induction
by poly(I)´poly(C), and the identi®cation of this protein
divided the OASL proteins into two classes: the active and the
inactive. We have recently described the structure of the
murine Oas gene family which included two mOasl genes
(mOasl1 and mOasl2) that exist head to tail on chromosome
5F (17,18).
Exons A and B of the OAS genes encode a ®ve-stranded
antiparallel b-sheet domain (19, R.Hartmann, unpublished).
This domain is structurally homologous to the `palm' domain
found in other polymerases and contains a number of amino
acids crucial for nucleotide transferase activity (20). The exon
B of the OAS family contains a P-loop, which is a short
¯exible stretch of amino acids forming a helical turn; this
motif is present in many nucleoside triphosphate-binding
proteins such as kanamycin nucleotidyltransferase and

*To whom correspondence should be addressed. Tel: +1 216 445 6909; Fax: +1 216 445 1466; Email: rh@mb.au.dk
Correspondence may also be addressed to Just Justesen. Tel: +45 89422682; Fax: +45 89422637; Email: jj@mb.au.dk

Nucleic Acids Research, Vol. 31 No. 12 ã Oxford University Press 2003; all rights reserved

different polymerases (20). The P-loop is involved in the
binding of the triphosphate of the nucleotide. Mutational
studies have demonstrated the importance of the P-loop in
hOAS1 protein for catalytic activity (21). The second motif
present in exon B is composed of three conserved aspartic
acids in b-strands 2 and 5; these three aspartic acids coordinate
the catalytically active magnesium ions (19,21). When
considering the amino acid sequence of exon B encoded by
mOasl1 and mOasl2 genes, notable differences exist. In
mOasl1 protein, two of the three aspartic acids are replaced,
and in hOASL1 all three are substituted. Furthermore, in both
mOasl1 and hOASL1, several mutations are introduced into
the P-loop motif, explaining why the hOASL1 protein is
inactive as expected for mOasl1. In contrast, mOasl2 and
ChOASL proteins have both motifs conserved, suggesting that
the mOasl2 protein belongs to the class of active OASL
proteins.
In this study, we have investigated the biochemical
properties of mOasl1 and mOasl2 proteins. We puri®ed
recombinant protein for examination of the 2±5A synthetase
activity and demonstrated that while mOasl1 has no activity,
mOasl2 is an active 2±5A synthetase that requires dsRNA.
Furthermore, we have determined the af®nity of mOasl2 for
poly(I)´poly(C) in comparison with hOAS1 (p42 isoform). We
propose a model for the evolutionary origin of the mOasl1 and
mOasl2 genes. Our data suggest that mOasl2 protein is a
functional intermediate between the active hOAS and the
inactive hOASL1/mOasl1 proteins. In addition, mOasl1
protein has gained several substitutions in exon B, resulting
in loss of 2±5A synthetase activity, suggesting that mOasl1
may have obtained an novel function independent of 2±5A
synthesis.
MATERIALS AND METHODS
RT±PCR for determination of IFN induction
Total RNA was puri®ed from L929 (DSMZ: accession 2)
cells using the Midi RNEasy puri®cation kit (Qiagen,
Valencia, CA) according to the manufacturer's instructions.
The cells were either untreated, or treated with murine IFN-a
(500 U/ml) or IFN-g (100 U/ml) for 24 h. A 5 mg aliquot of
total RNA was reverse-transcribed using the First strand
cDNA synthesis kit (Amersham Biosciences, Piscataway, NJ).
The PCR was carried out with 30 cycles of 96°C for 30 s, 60°C
for 30 s, and 72°C for 2 min, resulting in a 1535 bp PCR
product for mOasl1 and a 1526 bp product for mOasl2. The
murine b-actin gene (GenBank accession no. M12481) was
included as control. The mOasl and control reactions were mixed
in equal amounts before gel electrophoresis (1% agarose).
Cloning, expression and puri®cation of recombinant
His-tagged protein
The IMAGE clone 1546490 (GenBank accession no.
BE13926) was used as a template for mOasl1, and cDNA
from IFN-a-induced L929 cells was used as template for
mOasl2. The expression vectors (pET30b vector, Novagen,
Madison, WI) were introduced into BL21 pRI cells by
electroporation using standard conditions. Protein synthesis
was induced by addition of 0.5 mM isopropyl-b-D-
thiogalactoside (IPTG) overnight at room temperature. Cells
Nucleic Acids Research, 2003, Vol. 31, No. 12 3167
were lysed in 25 ml lysis buffer [50 mM Na2HPO4 pH 8.0,
500 mM NaCl, 10% glycerol, 20 mM imidazole, 0.1% (v/v)
NP-40, 5 mM b-mercaptoethanol and protease inhibitor
cocktail without EDTA (CompleteÔ, Roche Molecular
Biochemicals, Indianapolis, IN)] using a French press. The
lysate was clari®ed by centrifugation, and the supernatant was
mixed with Ni2+-NTA±agarose beads (Qiagen) and rotated
for 1 h. The beads were washed (pH 8.0, 50 mM Na2HPO4,
500 mM NaCl, 10% glycerol and 50 mM imidazole), applied
to a column (Bio-Rad, Hercules, CA) and eluted with 15 ml of
elution buffer (pH 8.0, 50 mM Na2HPO4, 500 mM NaCl, 10%
glycerol, 250 mM imidazole) in 2 ml fractions. The fractions
were analysed by 10% SDS±PAGE. The eluted His-tagged
protein was loaded on a Heparin HiTrap (5 ml) column
(Amersham Biosciences) (buffer A: 20 mM Tris±HCl pH 7.0,
5% glycerol). Bound protein was eluted in a linear gradient of
an increasing concentration of NaCl (20±100%) (buffer B:
20 mM Tris±HCl pH 7.0, 5% glycerol, 1 M NaCl). The
volume of the gradient was 10.054 ml. The fractions were
collected in 0.5 ml and analysed by SDS±PAGE (10%). The
eluted protein was dialysed for 2 h (600 mM NaCl, 25 mM
HEPES pH 6.8, 5% glycerol), diluted 1:2 with cold 100%
glycerol and stored at ±20°C.
2¢±5¢-Oligoadenylate synthetase activity assay by PEI
thin-layer chromatography
The activity of the recombinant proteins was determined as
previously described (22) with the following overall reaction
mixture: 4 ml of 53 OAS buffer, 4 ml of water, 4 ml of
recombinant protein, 4 ml of ATP pH 7.5 including 0.025 mCi
of [a-32P]ATP/ml, 4 ml of poly(I)´poly(C) (Amersham
Biosciences) or water. The 53 OAS buffer contains 20 mM
Tris±HCl pH 7.5, 75 mM Mg(OAc)2, 1 mM dithiothreitol,
0.2 mM EDTA, 0.5 mg/ml bovine serum albumin, 10% (v/v)
glycerol, 30 mM creatine phosphate (Boehringer Mannheim
GmbH) and 0.5 mg/ml creatine kinase (Boehringer Mannheim
GmbH). The reactions were stopped with 10 ml of 50 mM
EDTA after either 1 or 2 h. Separation of the ATP and the
2¢±5¢-oligomers was performed by thin-layer chromatography
(TLC) as described previously (22). The TLC plate was
visualised using a PhosphoImagerÔ (Molecular Dynamics,
Sunnyvale, CA).
Activity assay using Mono Q to detect 2±5As
The incubations to make 2±5As were prepared in a similar
way as the activity assay for TLC; however, the total volume
was 100 ml. After incubation for 4 h, the OAS enzymes
were heat-inactivated, treated with alkaline phosphatase
(Boehringer Mannheim GmbH) and applied to a Mono Q
HR5/5 column (Amersham Biosciences). The different 2¢±5¢-
oligomers were eluted by a linear gradient of NaCl (0±70%)
(buffer B: Tris±HCl pH 7.5, 700 mM NaCl) of 19.64 ml. The
2±5As were detected at 254 nm.
Determination of kinetic parameters
In order to estimate the kinetic parameters of both mOasl2
and hOAS1 (p42 isoform) enzymes, we used the
ANEMONA.XLT which is an Excel template (23). This
template implements among others the Michaelis±Menten
model, and the KM and Vmax parameters are calculated by non-
linear regression performed by the least-squares method. The
3168 Nucleic Acids Research, 2003, Vol. 31, No. 12

Figure 1. (A) Schematic drawing of the hOASL and mOasl genes on human chromosome 12 and mouse chromosome 5. Genes with reading frames in the
same direction as the annotated nucleotide sequence are indicated by squares above the lines; genes below are in the reverse direction. The genes are not
drawn to scale. CEN, centromere; TEL, telomere. (B) Amino acid alignments of hOAS1 p42 (D00068), ChOASL (AB037592), hOASL1 (AJ22089), hOASL2
(NT_028327), mOasl1 (AY089728) and mOasl2 (AK010034) (numbers in parentheses indicate the GenBank accession no. for each protein). Black boxes
indicate conserved residues, grey boxes related sequences. Gaps are indicated by ±. The middle part of exon B has been cut out as indicated by black dots.
The three catalytic aspartate residues are indicated by asterisks. (C) Transcription of mOasl2 (lanes 1±3) and mOasl1 (lanes 4±6) genes. Products of PCRs
(30 cycles) with cDNA from untreated (lanes 1 and 4), IFN-a-induced (lanes 2 and 5) and IFN-g-induced (lanes 3 and 6) mouse L929 cells, analysed by gel
electrophoresis (1% agarose). The mOasl reaction and the control reaction (murine b-actin) were mixed in equal amounts before gel electrophoresis.

kcat constant de®nes the number of substrate molecules (ATP)
oligomerised per molecule of enzyme per second.
Western blotting
The His-tagged and heparin-puri®ed protein was subjected to
10% SDS±PAGE and blotted onto polyvinylidene di¯uoride
(Immobilon-P, Millipore) membranes. The puri®ed,
recombinant proteins were detected using the monoclonal
mouse anti-His6 antibody (Boehringer Mannheim GmbH);
monoclonal goat anti-mouse horseradish peroxidase (HRP)-
conjugated secondary antibody (Dako) was used. The blots
were visualised using the enhanced chemiluminescence
method (ECL; Amersham Biosciences) according to the
manufacturer's instructions.
Alignment and phylogenetic analysis
The alignment of sequences was done at the amino acid level
using default settings of the ClustalX 1.82 program, followed
by manual adjustments. The following sequences were
used: hOAS1 (D00068), hOASL1 (AJ225089), mOasl2
(AK010034), mOasl1 (AY089728), ChOASL (AB037592),
Geodia cydonium OAS1 (Y18497) and a pseudogene
sequence (hOASL2) from the human genome (NT_028327).
Phylogenetic trees were reconstructed with and without the
partial hOASL2 sequence, using the Geodia sequence as an
outgroup, and the minimum evolution criterion for choosing
the tree topology. Pairwise distances between sequences were
calculated assuming rate heterogeneity in evolutionary rate
modelled by a gamma distribution with shape parameter
a = 1.5. Only alignment columns without gaps (complete
deletion) were used. Standard bootstrap was done using 1000
replicates. For testing for differences in evolutionary rates
between human and mouse OASL genes (24), a one-parameter
version of the relative rate test was used employing either the
hOAS1 sequence or ChOAS1 sequence as outgroup. All
analyses were done using MEGA 2.1 (25).
RESULTS
A pseudogene corresponding to mOasl2 in humans
To date, only a single OASL gene has been identi®ed in
humans, in contrast to the two genes (mOasl1 and mOasl2)
characterised in mice. Therefore, we searched the human
genome for a counterpart of the mOasl2 gene. This was done
by performing a BLAST search of the translated human
genome with the amino acid sequence of mOasl2. Using this
approach, we identi®ed two exons (exons A and B) located
downstream of the hOASL gene on chromosome 12 position
127.482 kb (exon A) and 127.476 kb (exon B) (Fig. 1A).
These exons exhibit 54 and 53% identity to exons A and B in
mOasl2, respectively, and their positions correspond to the
homologous position of the mOasl2 gene. The N- and
C-termini of exon B from human, mouse and chicken OASL
proteins are aligned in Figure 1B, showing the important
amino acids in the active site. As expected for a pseudogene,
several stop codons are present in hOASL2 exon A, but no
traces of the remaining sequences of the hOASL2 gene were
found. We believe that the identi®ed exons represent an
hOASL2 pseudogene, which has not previously been
described.
Interferon induction of mOasl1 and mOasl2
In order to investigate whether the mOasl genes are induced by
IFNs, we extracted total RNA from IFN-a- and IFN-g-induced
and untreated mouse L929 cells. By RT±PCR studies, we

Figure 2. Puri®cation of recombinant mOasl2 protein. (A) The elution pro-
®le of mOasl2 using the heparin±Sepharose column for puri®cation. The
graphs show the absorption at 280 nm and the NaCl gradient from 0 to 1 M.
(B) A 10% SDS±polyacrylamide gel with 5 mM of pooled His tag fractions
(lane 1) and 5 mM of pooled fractions from the heparin column (lane 2) was
stained with Coomassie brilliant blue. The size of the molecular markers is
indicated. (C) Western blot analysis of the samples loaded in (B). They
were visualised using an anti-His tag antibody and a secondary goat
anti-mouse HRP-conjugated antibody.
show that mOasl1 and mOasl2 are induced by IFN-a in
murine L929 cells; additionally, mOasl2 is induced by IFN-g
although to a lower extent (Fig. 1C). No transcript was
detected in untreated L929 cells with the PCR conditions used
here. We have no evidence of the p54/mOasl2 gene identi®ed
by Tiefenthaler et al. (26) (GenBank accession no. AF068835)
in IFN-induced cells. This p54/mOasl2 variant has a shorter
ubiquitin-like domain (1.5 ubiquitin-like repeat). A poly-
morphism giving allelic variation in the different mouse cells
may explain the two forms of mOasl2. The following analysis
is based solely on the mOasl2 gene with two ubiquitin-like
repeats (GenBank accession no. AK010034).
Puri®cation of active murine Oasl2 protein
To explore the biochemical properties of mOasl2, we cloned
the cDNA from IFN-a-induced L929 cells. N-terminal His-
tagged protein was expressed in Escherichia coli at room
temperature to avoid storage of protein in inclusion bodies.
The ®rst puri®cation step was His tag puri®cation using Ni2+-
NTA beads. mOasl2 was puri®ed further by af®nity
chromatography using a heparin column to remove several
putative degradation products. The elution pro®le of the
heparin column is shown in Figure 2A. A sample of the pooled
His tag fractions is shown in Figure 2B (lane 1). This was
Nucleic Acids Research, 2003, Vol. 31, No. 12 3169
loaded on the heparin column and then the eluted fractions
containing the highest amount of protein were pooled (lane 2).
The identity of the protein was veri®ed by western blotting
using an anti-His tag antibody (Fig. 2C). mOasl1 protein did
not bind to the heparin; therefore, the puri®cation was limited
to nickel af®nity, followed by dialysis removing the imidazole
(data not shown).
mOasl2 possesses true 2¢±5¢-oligoadenylate synthetase
activity
The putative OAS activities of mOasl1 and mOasl2 were
tested by assays using synthetic dsRNA [poly(I)´poly(C)] as
activator. In the following experiments, the total amount of
2±5A production was measured by TLC. As shown in
Figure 3A, mOasl2 is highly active and the protein is
enzymatically active after 22 h at 37°C (Fig. 3A and data
not shown). As expected for an enzyme, a linear correlation
exists between the activity and concentration of mOasl2
enzyme (Fig. 3C). In contrast, mOasl1 was unable to
synthesize 2±5A (Fig. 3B) even after longer periods of
incubation (22 h at 37°C), hence this protein possesses no
2±5A synthetase activity.
In order to verify that the products synthesised by mOasl2
indeed are 2±5A oligomers, we employed a second method.
The reaction products were treated with alkaline phosphatase
and the oligonucleotides were separated by an anion exchange
column (Mono Q). The migration of the mOasl2 products was
compared with standard 2±5As produced by hOAS1. The
superimposed elution pro®les of products synthesised by
mOasl2 and hOAS1 were identical (Fig. 3D). mOasl2 is able
to synthesise dimeric (pppApA), trimeric (pppApApA) and
longer oligomers after an extended incubation time (22 h)
(data not shown).
The activity of mOasl2 is dependent on dsRNA
In the previously described experiments, we have used the
synthetic dsRNA poly(I)´poly(C) as an activator. Next, we
investigated whether mOasl2, like all the human 2±5A
synthetases, needs dsRNA as co-factor. Dose±response curves
using poly(I)´poly(C) as activator of mOasl2 and hOAS1 are
presented in Figure 4A and B, respectively. These curves
clearly indicate that the 2±5A synthetase activity of
mOasl2 is activated by increasing amounts of dsRNA and
that without dsRNA, no 2±5A was produced. hOAS1 (p42
isoform) was included for comparison. Hence mOasl2 needs
dsRNA/poly(I)´poly(C) as a cofactor for 2±5A synthetase
activity.
From the dose±response curves, we have estimated the
maximal velocity (Vmax) and the apparent dissociation
constant (Kapp) by non-linear curve ®t. The Kapp de®nes the
concentration of dsRNA activator, which induces 1/2 Vmax
activity. The estimated parameters using either poly(I)´
poly(C), poly(A)´poly(U) or poly(GU) are presented in
Table 1. mOasl2 has a lower Kapp for poly(I)´poly(C) than
hOAS1 (p42 isoform); consequently, our data suggest that
mOasl2 has higher af®nity for poly(I)´poly(C) than hOAS1.
We continued the investigation of the dsRNA binding of
mOasl2 by using poly(A)´poly(U) which has been shown to
activate hOAS1, although to a lower extent than poly(I)´
poly(C) (27). Our experiments show that mOasl2, on the
contrary, is not activated by poly(A)´poly(U) to any extent.
3170 Nucleic Acids Research, 2003, Vol. 31, No. 12

Figure 4. mOasl2 is induced by poly(I)´poly(C). (A) Dose±response curve

for poly(I)´poly(C) induction of mOasl2 (2.5 ng/ml). (B) Dose±response
curve for poly(I)´poly(C) induction of hOAS1 (2.5 ng/ml).

Table 1. Kapp for induction by different dsRNA

Kappa mOasl2 p42

Poly(I)´poly(C) 0.13 mg/ml 1.40 mg/ml

Poly(A)´poly(U) No activation 25.00 mg/ml
Poly(GU) No activation No activation
Figure 3. mOasl2 is active whereas mOasl1 is not. (A) ATP was separated aThe
from the produced 2±5A products (pppApA and pppApApA) synthesised by apparent dissociation constant (Kapp) was estimated by a non-linear
mOasl2 using TLC. Increasing amounts of mOasl2 protein were added (0, curve ®t.
0.0007, 0.0014, 0.0028, 0.0056, 0.0112 and 0.0224 mg/ml) with a ®nal con-
centration of 100 mg/ml poly(I)´poly(C). Cont., contaminations present in all
reactions. (B) 2±5A synthetase activity assay using mOasl1 protein, and Table 2. Kinetic parameters
nucleotides separated by TLC. The same experimental conditions as in (A). mOasl2 p42
(C) The amount of 2±5A produced by mOasl2 in (A) was quanti®ed and
normalised to the ATP spot. A linear correlation exists between the activity KM(ATP) 0.66 mM 0.31 mM
(nmol ATP/min) and the concentration of mOasl2 (mg). The experiment was kcat 0.39 s±1 7.20 s±1
repeated twice. (D) The heat-inactivated 2±5A synthetase reaction [®nal
concentration of 100 mg/ml poly(I)´poly(C)] was treated with alkaline phos-
phatase and separated using a Mono Q column. The elution pro®les of the
products produced by mOasl2 (2.5 ng/ml) (blue) and p42 (2.5 ng/ml) (red)
are superimposed. 1, adenylate; 2, pppApA; 3, pppA(pA)2; 4, pppA(pA)3; 5,
pppA(pA)4; 6, pppA(pA)5; and 7, pppA(pA)6. enzyme. By ®tting our data to this equation, we were able to
estimate the KM values as shown in Table 2. The KM for
mOasl2 with ATP as substrate is 0.66 mM and is 0.31 mM for
hOAS1.
We also investigated whether poly(GU) can activate mOasl2
even though it does not activate hOAS1. Our studies show no Phylogenetic analysis
activation of mOasl2 with poly(GU). The identi®cation of the hOASL2 pseudogene establishes that
the duplication of the OASL gene has occurred prior to the
Determination of kinetic parameters [KM(ATP) and kcat] radiation of the rodent and primate groups. However, subse-
We continued our biochemical analyses of mOasl2 by quently during evolution, the hOASL2 gene has evolved into a
determination of the kinetic parameters. We plotted the pseudogene and, during this process, exons C±E and T have
amount of synthesized 2±5A as a function of the ATP been lost and several stop codons have arisen in exon A. We
substrate concentration. The Michaelis±Menten equation performed a phylogenetic analysis by aligning mOasl1,
proposes a model for estimation of substrate af®nity of an mOasl2, human p42 (OAS1), hOASL1, hOASL2 (exon B

Figure 5. Phylogenetic analyses. (A) Phylogenetic tree of exons A±E.
Bootstrap values are based on 1000 bootstrap replications. Sequences from
mOasl2 (AK010034), mOasl1 (AY089725), hOAS1 p42 (D00068), hOASL
(AJ22089), hOASL2 (NT_028327), ChOASL (AB037592) and Geodia
OAS1 (Y18497) are aligned (numbers in parentheses indicate the GenBank
accession no. for each protein). (B) Phylogenetic tree of exon B.
only), ChOASL and the OAS gene from the marine sponge
G.cydonium (28). In the subsequent phylogenetic analysis, the
Geodia OAS sequence was used as an outgroup to root the
trees.
Phylogenetic trees were constructed for the complete gene
(Fig. 5A) and for exon B (Fig. 5B) only, using the minimum
evolution criterion on a gamma distance matrix (a = 1.5).
Analysis on exon B alone allowed us to include the hOASL2
psudogene. Figure 5B shows that mOasl1/hOASL1 and
mOasl2/hOASL2 group together with high bootstrap support,
suggesting that they indeed represent orthologous pairs of
genes and that the duplication occurred prior to the diversi-
®cation of human and mouse but before the diversi®cation of
mammals and birds. Bootstrap support of the shown topology
was robust to other choices of a (between 1 and 5) and to other
principles of phylogenetic reconstruction (including likeli-
hood methods). The non-parametric relative rates test (24)
revealed that mOasl1 has evolved signi®cantly faster than
mOasl2 since their divergence (45 versus 28 changes, P <
0.05). This agrees well with the fact that mOasl2 has retained
activity and therefore has more common constraints with the
ChOASL and hOAS1 sequences. Previous analysis of the OAS
family made by Kumar et al. (29) showed that the evolution-
ary distance between hOASL1 and mOasl2 is longer than
expected for two orthologous genes, which led them to suggest
that the two genes were paralogues. This was later proven to
be true by the identi®cation of the mOasl1 gene. For exon B,
the relative rate test suggests not surprisingly that the hOASL2
pseudogene has evolved faster than the mOASL2 sequence
since their divergence, though not signi®cantly so (15 versus 7
changes, P = 0.08).
Nucleic Acids Research, 2003, Vol. 31, No. 12 3171
DISCUSSION
This study was performed to understand the biochemical
properties of the two mOasl proteins. We have cloned and
puri®ed protein encoded by the mOasl1 and mOasl2 genes and
characterised their 2±5A synthetase activity. Our results
demonstrate that the mOasl2 protein is an active 2±5A
synthetase requiring dsRNA as cofactor for activity. As
expected from sequence comparisons, mOasl1 is inactive like
its human orthologue, the hOASL1 protein (15). Thus it is
tempting to speculate that mOasl1 is the functional
counterpart of hOASL1.
The OASL proteins consist of an OAS domain and a
C-terminal domain of two ubiquitin-like repeats. They have
been identi®ed in human, mouse and chicken, and thus we
expect this family to exist throughout the mammalian and
avian classes. The function of the ubiquitin-like domain of the
OASL proteins is unknown. Mice have two mOasl genes,
mOasl1 and mOasl2, with striking differences observed in the
sequences of exon B of these genes. As described in the
Introduction, the P-loop motif and a triad of aspartic acids
coordinating the catalytic active magnesium ions are present
in exon B. Both these motifs have been altered in the mOasl1
protein, whereas they are retained in the mOasl2 protein (see
also Fig. 1B).
In comparison with hOAS1, the speci®c activity of mOasl2
is ~20-fold lower; this could at least partly be due to evolution
of the mOasl2 protein towards a 2±5A-independent function.
The KM we have determined for mOasl2 is marginally higher
than for human p42; however, it is still signi®cantly lower than
the KM for both human p69 OAS2 and p100 OAS3 proteins
(30).
Previous workers have shown that mOasl2 protein only
synthesises dimeric 2±5As (pppApA) (18) whereas we report
that mOasl2 is able to synthesise up to pentamers, although the
major product is dimeric 2±5As. We use creatine kinase and
creatine phosphate in our OAS assays to regenerate ATP from
ADP and AMP. Since ADP and AMP may be inhibitory to
mOasl2, this can explain the lower activity observed in the
experiments of Kakuta et al. (18). The activity of mOasl2 is
more sensitive towards ADP and AMP inhibition than human
p42 under the same experimental conditions (data not shown).
Thus it is crucial to avoid degradation of ATP in the reaction
when investigating the biochemistry of mOasl2. The assay
system used here has the advantage that an eventual degrad-
ation of ATP is visualised directly on the chromatogram.
We have measured a higher af®nity of mOasl2 for
poly(I)´poly(C) compared with hOAS1. This supports pub-
lished results from other groups showing that the hOAS1
protein in general requires the highest concentrations of
dsRNA for activation among the OAS family members (30).
The hOAS2 protein requires intermediate concentrations of
dsRNA for activation, and the OAS3 proteins are the most
sensitive to dsRNA (31). It should be noted that large batch to
batch variations of commercially available poly(I)´poly(C)
exist, making comparison of the absolute values for
poly(I)´poly(C) dif®cult. In addition, mOasl2 was not activ-
ated by poly(A)´poly(U), in contrast to hOAS1 which was
activated by this dsRNA although higher concentrations were
required. This suggests that the dsRNA recognition mechan-
ism might differ between the mOasl2 and hOAS1 proteins.
3172 Nucleic Acids Research, 2003, Vol. 31, No. 12
Figure 6. Model for the evolutionary origin of the OASL1 and OASL2
genes.
Previously published results by Tiefenthaler et al. claimed
that the activity of mOasl2 was independent of dsRNA (26).
We were unable to detect any activity of the mOasl2 protein in
the absence of dsRNA. Furthermore, Tiefenthaler et al. did not
observe any increase in the 2±5A synthetase activity upon
stimulation with poly(I)´poly(C), whereas we observed a
strong and dose-dependent activation of the mOasl2 enzyme
by poly(I)´poly(C). The speci®c activity of the enzyme used
by Tiefenthaler et al., although not stated in the paper, appears
low, since they use 0.5 mg of protein in contrast to the 0.05 mg
used here. The method we utilised for puri®cation of
recombinant mOasl2 protein is optimised for expressing the
protein at low temperatures, which is essential for recovering
active protein. However, at present, we are unable to explain
the dsRNA-independent activity observed by Tiefenthaler
et al. (26)
Based on our phylogenetic analyses, we propose a model
for the evolutionary origin of the mOasl1 and mOasl2 genes. A
duplication of the ancestral OAS gene followed by fusion to
the ubiquitin-like domain by exon shuf¯ing resulted in the
formation of the original OASL gene (Fig. 6). This gene
possesses both the ubiquitin-like domain and a catalytically
active OAS domain, as is the case for both mOasl2 and
ChOASL genes. Duplication of the ancestral OASL gene led to
the precursors of mOasl1 and mOasl2. This duplication
occurred either prior to the establishment of the mammalian
class or early during mammalian evolution prior to the
radiation of the rodent and primate groups, as demonstrated by
the identi®cation of an hOASL2 pseudogene. After the
duplication of the ancestral OASL gene, mOasl2 has main-
tained the 2±5A synthetase activity, which is the putative
original function of the OASL protein. This is manifested in
the conservation of the catalytically important amino acids in
the sequence of exon B. Some substitutions of amino acids
have occurred outside exon B, explaining why mOasl2 is more
closely related to the mOasl1/hOASL1 proteins when con-
sidering the entire OAS unit. The hOASL2 gene is not
functional in humans, and only exons A and B remain as a
pseudogene. Hence, at least in humans, the hOASL2 protein is
non-essential. Thus we suggest that mOasl2 having both
activity and an ubiquitin-like domain is a functional inter-
mediate between the active hOAS proteins and the inactive
hOASL1/mOasl1 proteins.
In contrast, accumulations of amino acid substitutions in the
mOasl1 gene led to loss of 2±5A synthetase activity. Since
both hOASL1 and mOasl1 proteins are inactive, the loss of
activity occurred in the common ancestor of man and mouse.
Sequence comparisons and functional data demonstrate that
the hOASL1 gene is an orthologue to the mOasl1 gene. The
most common evolutionary fate of gene duplicates is silencing
(32); since this does not seem to be the case for the mOasl1
gene, it appears that mOasl1 has acquired a putative novel
function which is independent of 2±5A synthetase activity.
Preliminary data suggest that this function requires the
ubiquitin-like domain and that hOASL1 has an antiviral
activity conferred by the ubiquitin-like domain (33).
ACKNOWLEDGEMENTS
The authors thank Lisbet Kjeldberg for excellent assistance
with enzymatic measurements, Niels Ole Kjeldgaard for
stimulating and strong interest, and Robert H. Silverman for
critical reading of this manuscript. This work was supported
by the Danish Natural Science Research Council, The Novo-
Nordisk Foundation and the Danish Cancer Society.
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