RNA editing enzyme adenosine deaminase is
a restriction factor for controlling measles virus
replication that also is required for embryogenesis
Simone V. Warda,1, Cyril X. Georgeb,1, Megan J. Welcha, Li-Ying Lioua, Bumsuk Hahma,c, Hanna Lewickia, Juan C. de la
Torrea, Charles E. Samuelb,d, and Michael B. Oldstonea,2
aDepartment of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037;bDepartment of Molecular, Cellular, and
Developmental Biology anddBiomolecular Sciences and Engineering Program, University of California, Santa Barbara, CA 92106; andcDepartments of Surgery
and Molecular Microbiology and Immunology, University of Missouri, Columbia, MO 65212
Contributed by Michael B. Oldstone, November 19, 2010 (sent for review October 13, 2010)
exclusively human pathogen, is among the most infectious viruses.
A progressive fatal neurodegenerative complication, subacute
sclerosing panencephalitis (SSPE), occurs during persistent MV
infection of the CNS and is associated with biased hypermutations
of the viral genome. The observed hypermutations of A-to-G are
consistent with conversions catalyzed by the adenosine deaminase
acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV
infection, we selectively disrupted expression of the IFN-inducible
p150 ADAR1 isoform and found it caused embryonic lethality at
embryoday(E) 11–E12. We thereforegeneratedp150-deficient and
WT mouse embryo fibroblast (MEF) cells stably expressing the MV
receptor signaling lymphocyte activation molecule (SLAM or
CD150). The p150−/−but not WT MEF cells displayed extensive syn-
cytium formation and cytopathic effect (CPE) following infection
with MV, consistent with an anti-MV role of the p150 isoform of
ADAR1. MV titers were 3 to 4 log higher in p150−/−cells compared
with WT cells at 21 h postinfection, and restoration of ADAR1 in
p150−/−cells prevented MV cytopathology. In contrast to infection
reovirus, or lymphocytic choriomeningitis virus replication but pro-
tected against CPE resulting from infection with Newcastle disease
virus, Sendai virus, canine distemper virus, and influenza A virus.
Thus, ADAR1 is a restriction factor in the replication of paramyxovi-
ruses and orthomyxoviruses.
myxoviridae, infects more than 10 million persons world-
wide each year, resulting in several hundred thousand deaths (1,
2). A serious complication is the persistent infection of the CNS
known as subacute sclerosing panencephalitis (SSPE) that occurs
at a frequency of 4–11 cases per 100,000 cases of MV infection.
SSPE is a progressive fatal neurodegenerative disease with
characteristic features of replication of MV in neurons in the
presence of high titers of MV antibodies, modest infiltration of T
and B cells into the CNS, and replication of defective MV in the
CNS with biased mutation of U-to-C and A-to-G in the viral
genome (3–5). These hypermutations occur primarily in the
matrix (M) gene but are also observed to a lesser extent in the
fusion (F) and hemagglutinin (H) genes (3, 4). A transgenic
mouse model that expresses the human MV receptor CD46
recapitulates all the features of SSPE on infection with MV, with
biased hypermutations of U-to-C and A-to-G accounting for
more than 95% of point mutations in the M gene (3, 4, 6). In-
terestingly, these biased hypermutations play a direct role in the
pathogenesis of SSPE by facilitating a significant prolongation of
MV persistence within the CNS, as opposed to mere accumu-
lation as a result of persistent infection (7). In support of a direct
role of M gene hypermutations in the establishment of SSPE,
MV generated by reverse genetics and containing a hyper-
mutated M gene caused a significant prolongation of the viral
persistent state when used to infect the CD46 transgenic mice
compared with MV containing a normal M gene (7). Although
easles virus (MV), a member of the family Para-
this provided evidence for a biological importance of biased
hypermutations of the M gene in the establishment of persistent
infection, neither the mechanism by which mutations in the MV
genome arise nor the effect of adenosine deaminase acting on
RNA (ADAR1) on MV replication was known. Because of the
uniformity of mutations observed (4), it has been postulated that
the biased hypermutations are likely the result of an enzymatic
activity, with an attractive candidate enzyme being ADAR1.
ADAR1 catalyzes the conversion of adenosine (A) to inosine
(I) on double-stranded RNA substrates, thereby introducing A-
to-G mutations because I is recognized as G by the translation
and viral RNA-dependent RNA transcription and replication
machineries (8–10). Thus, A-to-I editing of both cellular and
viral RNA substrates has the capacity to alter coding capacity
and RNA structure (9). ADAR1 expression is driven by three
promoters, one of which is inducible by IFN. The IFN-inducible
form of ADAR1 is a 150-kDa protein referred to as p150 that
localizes to both the cytoplasm and the nucleus (11), whereas
a smaller constitutively expressed form of ADAR1 referred to as
p110 is localized predominantly in the nucleus. Several features
of the p150 protein make it a likely candidate enzyme re-
sponsible for generating biased hypermutations of the M gene
associated with SSPE, including its presence in the cytoplasm,
the site of MV replication, and its induction by IFN, a cytokine
induced in response to viral infection (12). In addition, induction
of ADAR1 in the CNS as a result of viral infection likely con-
tributes to neuronal cell dysfunction, because ADAR1 is essen-
tial for glutamate and serotonin receptor editing (3, 9, 13).
Nevertheless, whether the p150 isoform of ADAR1 had a di-
rect antiviral function remained unknown. Earlier gene dis-
ruptions of ADAR1 eliminated expression of both the p110 and
p150 isoforms, resulting in embryonic lethality (14, 15). Here,
we report that selective disruption of expression of the IFN-
inducible p150 isoform of ADAR1 was embryonic-lethal. Fur-
ther, we recovered mouse embryo fibroblast (MEF) cells ho-
mozygous and heterozygous for deletion of p150 and show that
the p150 isoform of ADAR1 inhibits MV replication as well as
protecting against infection with representative viruses of the
paramyxovirus and orthomyxovirus families.
Disruption of the Gene Encoding the p150 Isoform of ADAR1 Results
in Embryonic Lethality. We generated a targeted gene disruption
that selectively abolishes expression of only p150 and leaves ex-
Author contributions: S.V.W., C.E.S., and M.B.O. designed research; S.V.W., C.X.G., M.J.W.,
L.-Y.L., B.H., H.L., and J.C.d.l.T. performed research; S.V.W., C.X.G., L.-Y.L., B.H., J.C.d.l.T.,
C.E.S., and M.B.O. analyzed data; and S.V.W., C.E.S., and M.B.O. wrote the paper.
The authors declare no conflict of interest.
1S.V.W. and C.X.G. contributed equally to this work.
2To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| January 4, 2011
| vol. 108
| no. 1
pression of p110 intact. A targeting vector was constructed con-
taining Adar1 genomic sequences to disrupt the IFN-inducible PA
promoter and its associated IFN-inducible exon 1A region spe-
cifically (16, 17) (Fig. 1A). Successful targeting of Adar1p150was
confirmed by PCR, with the disrupted and WT alleles yielding
the predicted bands of 1,129 and 384 bp, respectively (Fig. 1B).
Although live embryos that were either WT or heterozygous for
the disrupted Adar1p150allele were readily observed, no live
embryos homozygous for the Adar1p150gene disruption were
recovered. Consistent with the conclusion that specific targeting
of the p150 isoform of ADAR1 resulted in embryonic lethality,
no live animals that possessed both copies of the disrupted allele
were born on intercrossing Adar1p150+/−heterozygotes (Fig. 1 B
and C). By contrast, WT Adar1p150+/+
Adar1p150+/−animals were born at the approximate expected
Mendelian frequencies. Moreover, Adar1p150−/−embryos dis-
played abnormal morphology compared with their WT and
heterozygous counterparts (Fig. 1D). Thus, embryonic lethality
occurred only with deletion of both copies of the Adar1p150gene.
Lethality at embryo day (E) 11 to E12 of development was
similar to previously described Adar1 gene disruptions that tar-
geted expression of both p110 and p150 (14, 15).
Deletion of the p150 Isoform of ADAR1 Increases Susceptibility of
MEF Cells to MV Infection. To study the effect of selective dis-
ruption of the p150 isoform of ADAR1 on MV replication, we
generated immortalized MEF cells from WT, Adar1p150+/−, and
Adar1p150−/−embryos at E12. Expression of the constitutive
transcripts containing exon 1B and encoding the p110 isoform of
ADAR1 was readily detectable in WT, p150+/−, and p150−/−
MEF cells (Fig. 1E). By contrast, the exon 1A-containing tran-
script that encodes the p150 protein was only detectable in WT
and p150+/−MEF cells but not in p150−/−MEF cells even on
treatment with IFN-α. These results confirm selective disruption
of p150 expression while leaving expression of p110 intact.
Because rodent cells are not permissive to MV infection as a
result of the lack of a virus receptor, we generated WT, p150+/−,
and p150−/−MEF cells expressing the MV receptor (CD150)
fused to GFP at its C terminus (hSLAM-GFP). Functionality of
the fusion protein as evidenced by syncytium formation following
infection with MV was confirmed by introduction of hSLAM-
GFP into nonpermissive BHK-21 cells (Fig. S1A). We next
transiently transfected hSLAM-GFP into WT, p150+/−, or
p150−/−MEF cells. On infection with MV, only p150−/−MEF
cells that expressed hSLAM-GFP but not vector control-trans-
fected cells displayed significant cytopathic effect (CPE) (Fig.
S1B, data not shown). By contrast, neither WT nor p150+/−MEF
cells that expressed the hSLAM-GFP fusion protein displayed
CPE (Fig. S1B). Thus, development of CPE was dependent on
both expression of human SLAM and infection with MV.
To evaluate the effect of MV infection in p150−/−MEF cells
quantitatively, we used a lentiviral vector system to generate
MEF cells that stably expressed hSLAM-GFP. WT and p150−/−
MEF cells showed similar GFP expression and equivalent high
transduction efficiencies (98% or more) when transduced with
GFP control vectors (Fig. S2 A and B). Similar to results
obtained with GFP control vectors, comparable expression of the
hSLAM-GFP fusion protein was seen in both WT and p150−/−
MEF cells following lentiviral vector transduction (Fig. S2C).
Importantly, although fluorescence intensity of the hSLAM-GFP
fusion protein was lower than that seen for cytoplasmic GFP,
equivalent expression of hSLAM-GFP was seen by flow cytom-
etry in WT and p150−/−MEF cells (Fig. S2D).
Having established MEF cells stably expressing hSLAM-GFP
or GFP as a control, WT and p150−/−MEF cells were infected
with MV and observed for development of CPE at various time
points postinfection, including at 21, 31, and 45 h. As shown in
Fig. 2A, at 21 h postinfection, extensive CPE, including forma-
tion of syncytia, developed only in p150−/−MEF cells that stably
expressed hSLAM-GFP but not GFP as a control. Likewise, no
CPE was detectable in infected WT cells that expressed either
GFP or the hSLAM-GFP fusion protein at 21 h postinfection.
The differences observed in development of CPE between WT
and p150−/−MEF cells following infection with MV correlated
with viral titers obtained at this time point, with 3- to 4-log higher
titers seen in p150−/−MEF cells compared with WT cells (Table
1). Indeed, for WT cells, no infectious progeny was detected at
21 h postinfection. Taken together, these results indicate that the
p150 isoform of ADAR1 possesses antiviral functions in the
context of MV replication.
WT cells that expressed the hSLAM-GFP fusion protein. CPE
seen in infected p150−/−MEF cells that expressed hSLAM-GFP
was significantly more extensive, however. Furthermore, in con-
trast to complete destruction of the monolayer seen for infected
WT cells was not destroyed at this time point. Titers were not
statistically different for WT and p150−/−MEF cells at the later
time points of 31 and 45 h postinfection, indicating that both WT
and p150−/−hSLAM-GFP-positive MEF cells had been rendered
permissive to MV (Table 1). No CPE was observed in uninfected
4.14 kb 4.5 kb
Adar1p150 +/- X Adar1p150 +/-
Adar1p150+/+ (25%), +/- (50%), -/- (25%)
: : :1
: :: :115 0
Day 11 Day 12 Day 14
tion. (A) Schematic illustrating the gene targeting
strategy to disrupt expression of the p150 isoform of
ADAR1 specifically. Genomic sequences flanking the
Neo cassette and encompassing the IFN-inducible PA
promoter and associated exon 1A regions were
inserted into the targeting vector to yield pKO-
Adar1p150-floxP, thus resulting in specific disruption
of the corresponding region of the Adar1 locus on
genotyping results of embryos obtained at E14 and
homozygous for the disrupted Adar1p150allele
(lanes 2 and 3) and heterozygous for the Adar1p150
disruption (lanes 4 and 5) or WT (lanes 6 and 7). The
of offspring and associated genotypes resulting
from interbreeding of mice heterozygous for dis-
ruption of the p150 isoform of ADAR1. (D) Repre-
embryos or embryos heterozygous for the Adar1p150
RNA isolated from WT, p150+/−, and p150−/−MEF cells detecting IFN-inducible exon 1A-containing and constitutive exon 1B-containing transcripts encoding the
p150 and p110 isoforms of ADAR1, respectively. GAPDH is shown as an internal control.
Generation of the Adar1p150gene disrup-
seen for WT
| www.pnas.org/cgi/doi/10.1073/pnas.1017241108Ward et al.
control cells expressing GFP did not display CPE and were com-
parable in appearance to uninfected cells (Fig. 2). Similar results
infection (MOI) of 0.8 pfu/cell as shown in Fig. 2A or at MOIs of
0.1 and 3.0 pfu/cell (data not shown). Infection of p150−/−MEF
cellswithMVrecoveredfrom WTMEFcells at 45hpostinfection
CPE seen with the parental MV grown in Vero cells (data not
shown), thus ruling out the possibility of an intrinsic viral defect in
infected WT MEF cells, such as selection of a variant unable to
To determine definitively that deficiency of p150 was re-
sponsible for the apparent heightened susceptibility of p150−/−
cells to MV infection, p150 expression was restored in p150−/−
MEF cells with a lentiviral vector expressing murine ADAR1. As
shown in Fig. 2B, p150−/−MEF cells reconstituted with murine
ADAR1 were protected from development of complete CPE
following infection with MV. By contrast, p150−/−MEF cells that
were transduced with an empty lentiviral vector possessing no
transgene displayed complete CPE on infection with MV similar
to the parental p150−/−MEF cells. Taken together, these results
firmly establish an important role for the p150 isoform of
ADAR1 in MV replication.
p150 Isoform of ADAR1 Protects Against Infection with Members of
the Paramyxoviridae Family. Because our results indicated that the
p150 isoform of ADAR1 restricted MV replication, we next
determined the generality of the effect by studying the replica-
tion of additional members of the Paramyxoviridae family of
viruses, including Newcastle disease virus (NDV), Sendai virus
(SeV), and the wild 5804P strain of canine distemper virus
(CDV) in hSLAM-GFP-positive WT and p150−/−MEF cells
(Fig. 3). For all these viruses, infection of p150−/−MEF cells
resulted in development of complete CPE as indicated by de-
struction of the cell monolayer. By contrast, no CPE was ob-
served in uninfected p150−/−MEF cells or when WT MEF cells
were infected. Furthermore, reconstitution of p150−/−MEF cells
with a lentiviral vector delivering murine ADAR1 but not with
an empty control lentiviral vector completely protected from
development of CPE. Similar results were seen when infections
were carried out with the Onderstepoort vaccine strain of CDV,
although protection from CPE in p150-reconstituted cells was
partial and more variable than that seen for the wild strain of
CDV. Taken together, these results indicate a general protective
role of the p150 isoform of ADAR1 against infection with
p150 Isoform of ADAR1 Protects Against Influenza Virus but Not
Lymphocytic Choriomeningitis Virus, Vesicular Stomatitis Virus, or
Reovirus. To extend the role of p150 in virus infection further, we
infected WT and p150−/−MEF cells with the mouse-adapted
WSN strain of influenza A virus, lymphocytic choriomeningitis
virus (LCMV), vesicular stomatitis virus (VSV), and reovirus.
Infection of p150−/−MEF cells with influenza A virus resulted in
complete CPE by 48 h postinfection (Fig. 4A), with minimal CPE
observed on infection of WT cells. Similar to results seen for
members of the Paramyxoviridae family, reconstitution of p150−/−
MEF cells with murine ADAR1 provided protection from de-
velopment of complete CPE. By contrast, p150−/−MEF cells
transduced with an empty lentiviral vector displayed CPE similar
to the parental p150−/−MEF cells, indicating that p150 plays an
important protective role in infection with influenza A virus,
a representative member of the family Orthomyxoviridae.
In contrast to infection with MV, NDV, SeV, CDV, and in-
fluenza A virus, no significant differences were seen at 8, 12, and
24 h postinfection when WT, p150+/−, and p150−/−MEF cells
were infected with the clone 13 strain of LCMV, as indicated by
the comparable presence of viral antigen (Fig. 4B). In support of
this finding, LCMV produced comparable amounts of virus in
supernatants from infected WT, p150+/−, and p150−/−MEF cells
(data not shown). We next performed single-cycle yield analyses
of VSV (Fig. 4C) and reovirus (Fig. 4D) in both untreated and
IFN-treated WT, p150+/−, and p150−/−MEF cells. Although
single-cycle yields of VSV and reovirus were reduced, as ex-
pected, by treatment with IFN, no significant differences were
seen in virus yields from WT, p150+/−, and p150−/−MEF cells.
In this paper, we make three observations. First, disruption of
the p150 isoform of ADAR1 itself, although maintaining ex-
pression of the p110 isoform, is sufficient to cause lethality in
WT p150 -/-
MV. (A) WT and p150−/−MEF cells expressing either GFP as a control or
hSLAM-GFP as indicated were infected with MV and observed for de-
velopment of CPE by bright-field microscopy at the indicated times post-
infection. (B) Restoration of mouse ADAR1 expression in p150−/−MEF cells
protects from development of full CPE as a result of infection with MV. WT
and p150−/−MEF cells expressing hSLAM-GFP and reconstituted with lenti-
viral vectors delivering mouse ADAR1 (p150−/−Lenti-mADAR1) or with
empty control lentiviral vectors (p150−/−Lenti-empty) were left uninfected
or infected with MV. Cells were observed for development of CPE by bright-
field microscopy at 48 h postinfection.
p150 isoform of ADAR1 plays an antiviral role in the replication of
cells stably expressing hSLAM-GFP
Titers obtained from MV-infected WT or p150−/−MEF
0 h pi
21 h pi
31 h pi
45 h pi
1.2 × 104pfu
1.9 × 104pfu
7.4 × 103pfu
8.4 × 103pfu
4.6 × 103pfu
Infections were carried out at an MOI of 0.8 pfu/cell, and titers were
determined in combined cells and supernatants at the indicated times post-
infection (pi). Results are from a representative experiment. With the excep-
tion of 0 h postinfection, titers were obtained from duplicate samples.
Ward et al.PNAS
| January 4, 2011
| vol. 108
| no. 1
embryos at E11 to E12. Second, the p150 isoform of ADAR1 is
a restriction factor that inhibits MV as well as NDV, SeV, CDV,
and WSN influenza virus replication and virus-induced cytotox-
icity. Third, the p150 isoform of ADAR1 does not inhibit the
replication of LCMV, VSV, or reovirus.
Previous targeted disruptions of Adar1 that involved deletions
within the exon 2–15 region and eliminated expression of both
the p110 and p150 isoforms of ADAR1 resulted in embryonic
lethality (14, 15, 18). To circumvent the embryonic lethality as-
sociated with simultaneous disruption of p110 and p150, we
specifically targeted the promoter and exon 1A region of the
p150 isoform of ADAR1 while leaving expression of p110 intact.
Selective disruption of p150 alone resulted in embryonic lethality
at E11 to E12, similar to the time point of embryonic lethality
seen previously with disruption of p110 and p150 (14, 15). These
results indicate that the p150 isoform of ADAR1 plays a criti-
cally important but not previously recognized role in embryonic
development. Furthermore, our results raise the possibility that
embryonic lethality seen in previously described Adar1 gene
disruptions may have resulted primarily from ablation of
Our ability to generate MEF cells selectively deficient in the
IFN-inducible p150 isoform of ADAR1 allowed us to examine
resulted in higher replication of MV by 21 h postinfection and
extensive CPEat alltimespostinfection. CPEwaseither absentor
minimal in WT MEF cells infected with MV, highlighting the
susceptibility of p150−/−MEF cells to MV infection. Re-
constitution of p150−/−MEF cells with murine ADAR1 firmly
asseen by protection from CPE in p150-reconstituted cells. These
results demonstrate an antiviral function of the p150 isoform of
ADAR1. The delay in production of infectious progeny in WT
MEF cells compared with p150−/−MEF cells may reflect viral
adaptation to overcome an intact host antiviral response or in-
complete inhibition of viral replication by p150 during a single
however, in SSPE, it allows the infection to be prolonged, likely as
current inability to generate ADAR1 p150 KO mice precludes us
from directly testing the role of ADAR1 in vivo in generating bi-
ased hypermutations in MV infection.
Our results of increased MV-induced CPE obtained with
p150−/−MEF cells are similar to those of previous studies that
examined the role of ADAR1 in MV replication using a shRNA
knockdown strategy that stably diminished expression of both the
p110 and p150 isoforms (19). In contrast to our results with MEF
cells genetically deficient in p150, however, the shRNA knock-
down did not restrict MV replication (19). The differences be-
tween these observations are likely explained by only a partial
knockdown of p150 obtained with shRNA (19) compared with
complete absence of p150 obtained by genetic ablation or by the
different virus strains used (Edmonston, recombinant Moraten).
Neither the overexpression nor the knockdown of ADAR1 af-
fected VSV replication in the absence of IFN treatment (20),
similar to our findings with p150−/−MEFs described herein.
Our studies importantly show that the p150 isoform of
ADAR1 protects against infection with members of the Para-
myxoviridae family in addition to MV, including NDV, SeV, and
CDV. For each of these viruses, complete protection from virus-
induced CPE was seen in p150−/−MEF cells reconstituted with
murine p150 compared with p150−/−control cells. Thus, p150
likely plays an important role in the replication of members of
the Paramyxoviridae family in general. ADAR1 p150 is the only
known ADAR that is found in the cytoplasm, where members of
the Paramyxoviridae family of viruses replicate; the p110 isoform
of ADAR1 is an exclusively nuclear protein (1, 9). The role of
p150 in virus infection was independent of the level of virus at-
tenuation, as exemplified by the Edmonston vaccine strain of
MV and the wild 5804P strain of CDV that both belong to the
Morbillivirus genus. Likewise, the role of p150 was independent
of a viral requirement for human SLAM, because similar results
were obtained for viruses capable of using hSLAM for cell entry
(MV and CDV) and viruses independent of hSLAM (SeV,
NDV, and influenza A virus) (21, 22).
In addition to several genera of the Paramyxoviridae family of
viruses, our findings with influenza A virus, a representative
member of the Orthomyxoviridae family, show an antiviral role
for ADAR1. Not all viruses are inhibited by the p150 isoform of
ADAR1, however. We found that p150 deletion did not alter the
replication of LCMV, VSV, or reovirus. That replication of some
described in the legend for Fig. 2 were left uninfected or infected with NDV or SeV. Cells were observed for development of CPE by bright-field microscopy at
48 h postinfection. (B) Infection of MEF cells as described above with wild CDV, followed by bright-field microscopy at 67 h postinfection. The following MOIs
were used for infections: NDV and SeV, MOI of 0.1 pfu/cell; CDV, MOI of 0.5 50% tissue culture infectious dose per cell.
p150 isoform of ADAR1 protects from infection with members of the Paramyxoviridae. (A) WT, parental p150−/−, or reconstituted p150−/−MEF cells as
| www.pnas.org/cgi/doi/10.1073/pnas.1017241108Ward et al.
viruses was not changed in the p150−/−MEFs is not unexpected,
given the pleiotropic nature of the IFN response and redundancy
of host factors that can affect virus replication (8, 23). Others
have implicated an antiviral function of ADAR1 in infection with
the WE strain of LCMV based on mutations observed in the
LCMV glycoprotein over time (24); however, the respective
contributions of p110 and p150 were not examined.
Thus, the IFN-inducible p150 isoform of ADAR1 provides
protection against the replication of paramyxoviruses and
orthomyxoviruses. It is interesting to note that the representative
paramyxovirus and orthomyxovirus family members share a re-
spiratory component for in vivo infection. It is tempting to
speculate that the p150 isoform of ADAR1 functions as a host
restriction factor of respiratory RNA viruses analogous to the
role of the cytidine deaminase APOBEC3G, which exerts its
antiviral function by generating hypermutations in the proviral
DNA of retroviruses, including HIV (25–28). It is possible that
extensive hypermutations of the M gene of MV seen in vivo are
the result of the known dispensability of the M protein for viral
replication (29), with M gene sequences representing a viral
decoy as targets for hypermutation. Significant A-to-G sub-
stitutions have also been seen in the viral M gene sequences of
influenza A virus recovered from WT animals with intact innate
immune responses (30). A-to-G substitutions in the M gene were
reduced in viruses recovered from IKKε-deficient animals with
impaired innate immunity (30). In this animal model, homozy-
gous deletion of the gene encoding the IKKε kinase resulted in
hypersusceptibility to influenza A virus infection that correlated
with reduced transcriptional up-regulation of several IFN-
stimulated genes, including ADAR1 in lungs of infected animals
(30). We plan studies to address the function of ADAR1 in vivo
as well as the mechanism of p150 action at the molecular level in
vitro, including an examination of MV proteins for their ability
to antagonize ADAR1 function analogous to the antagonism of
ABOBEC3G by the HIV protein Vif (25–28).
Materials and Methods
Targeted Disruption of the Gene Encoding the p150 Isoform of ADAR1 and
Generation of p150−/−MEF Cells. To generate a targeting vector for specific
disruption of the p150 isoform of ADAR1, genomic sequences encompassing
the IFN-inducible PApromoter and associated exon 1A regions were cloned
into the pKO-loxP vector (Stratagene). The left arm of the targeting vector
contained Adar1 genomic sequence from nucleotides 2,060–6,203, whereas
the right arm contained Adar1 genomic sequence from nucleotides 8,192–
12,653. Following electroporation of ES cells of strain 129Sv/Ev and micro-
injection, chimeric male mice were bred to female C57BL/6J mice. Animals
heterozygous for the targeted Adar1 allele were interbred to obtain em-
bryos with both copies of the gene region encoding the p150 isoform of
Adar1 deleted. Embryos were collected at E11, E12, and E14, and genomic
DNA was isolated for genotyping. MEF cells were derived from embryos at
E12, immortalized, and maintained as described below. The following pri-
mers were used for genotyping: pADAR1-5902 5′ ATCAGGTACCCAGGGA-
TATGGAA 3′, exon 1A PR 5′ AGCAGGGCACTATATACGTTCTCT 3′, and neo-
7030 5′ GAACTGTTCGCCAGGCTCAA 3′, with primer pairs pADAR1-5902/
exon1A PR and pADAR1-5902/Neo-7030 detecting the WT and targeted
Adar1 alleles, respectively.
Cells and Viruses. MEF cells, either WT, heterozygous, or deficient in the p150
isoform of ADAR1, were maintained in high-glucose DMEM supplemented
with 10% (vol/vol) FBS, 2 mM glutamine, 50 μM β-mercaptoethanol, 1 μg/mL
doxycycline, penicillin, and streptomycin. The Edmonston vaccine strain of
MV, the clone 13 strain of LCMV, the Indiana strain of VSV, and the T3
Dearing strain of reovirus were grown and titered as previously described
(31–34). Immunofluorescence detection was carried out using an antibody
against the LCMV nucleoprotein (35). NDV and SeV were generously pro-
vided by Juan C. de la Torre (The Scripps Research Institute). The 5804P wild
and Onderstepoort vaccine strains of CDV were generously provided by
Veronika von Messling [INRS Institute Armand-Frappier, Université du
Québec, Laval, QC, Canada (22)]. The WSN strain of influenza A virus was
p150 +/- p150 -/-WT
++ - +
Yield log10 (PFU/ml)
reconstituted p150−/−MEF cells as described in the legend for Fig. 2 were left uninfected or infected with the WSN strain of influenza A virus at an MOI of 1.0 pfu/
cell and observed by bright-field microscopy at 48 h postinfection. (B) WT MEF or MEF cells heterozygous or homozygous for the p150 disruption were infected
with the clone 13 strain of LCMV. Cells were fixed at 12 h postinfection, stained with antibody to LCMV nucleoprotein to detect the presence of viral antigen, and
observed by fluorescence microscopy. Single-cycle yields of VSV (C) and reovirus (D) in untreated and IFN-treated WT, p150+/−, and p150−/−MEF cells are shown.
p150 isoform of ADAR1 protects from infection with influenza A virus but not from infection with LCMV, VSV, or reovirus. (A) WT, parental p150−/−, or
Ward et al.PNAS
| January 4, 2011
| vol. 108
| no. 1
generously provided by John Teijaro (The Scripps Research Institute, La Jolla, Download full-text
CA) of this laboratory.
RNA Isolation and RT-PCR Analysis. RT-PCR analyses of Adar1 transcripts were
carried out essentially as described (36). Total RNA was isolated by the TRIzol
method (Invitrogen) following the manufacturer’s recommendations. PCR
for mouse Adar1 (36). The forward primer used for the p150 transcript was
exon 1A plus 19 corresponding to nucleotides 19–40, and the reverse primer
was exon 2 minus 842 corresponding to nucleotides 842–822; for the p110
transcript, the primer pairs were forward exon 1B plus 72 corresponding to
nucleotides 72–94 and minus primer 842. Amplification with GAPDH-specific
primers as an internal control was as described (37). IFN treatment was carried
out with 1,000 U/mL IFN-αA/D (Pestka Biomedical Laboratories) for 24 h.
Reconstitution of p150 Expression in MEF Cells. The full-length mouse ADAR1
cDNA (accession no. BC042505) in pCMV-SPORT6 (Thermo Fisher Scientific,
Inc.) was amplified using the forward primer 5′ CAGTCGGATCCCCACCATG-
TCTCAAGGGTTCAGG 3′ with the reverse primer 5′ CATGCTCTCGAGTCA-
AGCGTAATCTGGAACATCGTATGGGTAGTCATTGGGTACTGGACA 3′, with the
reverse primer designed to introduce an HA epitope tag sequence (under-
lined) to the 3′ end of the ORF. The ADAR1-HA cDNA was introduced into
the lentiviral vector pCSC-SP-PW (38), followed by generation of high-titer
lentiviral vector preparations as described (39). Western blotting of purified
vectors using anti–HIV-1 p24 monoclonal antibody 183-H12-5C obtained
through the National Institutes of Health AIDS Research and Reference Re-
agent Program confirmed p24 incorporation equivalent to control lentiviral
vectors with a known biological titer of 1 × 1010TU/mL. p150−/−MEF cells
stably expressing hSLAM-GFP were transduced twice using 15 and 30 μL of
concentrated vector preparations.
Infection of hSLAM-GFP Stable MEF Cells and Plaque Assay. A total of 5 × 106
MEF cells stably expressing hSLAM-GFP or GFP were infected in suspension
with MV for 2 h at 37 °C at an MOI of 0.8 pfu/cell unless otherwise noted.
Cells were plated in 24-well plates at 5 × 105cells per well and observed by
bright-field microscopy for development of CPE. For plaque assays, both cells
and supernatants were harvested, followed by a single freeze-thaw cycle
and titration on Vero cells as described (7). Infections of 1.2 × 105MEF cells
with NDV and SeV were carried out at an MOI of 0.1 pfu/cell. Infections with
the WSN strain of influenza virus were performed at an MOI of 1.0 pfu/cell,
and infections with the wild 5804P strain of CDV were carried out at an MOI
of 0.5 50% tissue culture infectious dose per cell.
ACKNOWLEDGMENTS. This is Publication 20928 from the Department of
Immunology and Microbial Science, The Scripps Research Institute. We thank
Drs. Inder Verma (Salk Institute for Biological Studies, La Jolla, CA) and Didier
Trono (Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland) for
Chesebro and Kathy Wehrly (National Institutes of Health AIDS Research and
and Dr. Young-Jin Seo for technical assistance. We also thank Drs. Andrew Lee,
John Teijaro, and Brian Sullivan for critical reading of the manuscript. This work
was supported by National Institutes of Health Research Grants AI-09484, AI-
45927, and AI-70967 (to M.B.O.) and National Institutes of Health Research
Grants AI-12250 and AI-20611 (to C.E.S.).
1. Griffin DE (2007) Measles virus. Fields Virology, eds Knipe DM, Howley PM (Lippincott
Williams & Wilkins, Philadelphia), 5th Ed, pp 1551–1585.
2. Moss WJ, Griffin DE (2006) Global measles elimination. Nat Rev Microbiol 4:900–908.
3. Oldstone MB (2009) Modeling subacute sclerosing panencephalitis in a transgenic
mouse system: Uncoding pathogenesis of disease and illuminating components of
immune control. Curr Top Microbiol Immunol 330:31–54.
4. Oldstone MB, Dales S, Tishon A, Lewicki H, Martin L (2005) A role for dual viral hits in
causation of subacute sclerosing panencephalitis. J Exp Med 202:1185–1190.
5. Cattaneo R, et al. (1988) Biased hypermutation and other genetic changes in defective
measles viruses in human brain infections. Cell 55:255–265.
6. Oldstone MB, et al. (1999) Measles virus infection in a transgenic model: Virus-induced
immunosuppression and central nervous system disease. Cell 98:629–640.
7. Patterson JB, et al. (2001) Evidence that the hypermutated M protein of a subacute
sclerosing panencephalitis measles virus
progressive CNS disease. Virology 291:215–225.
8. Samuel CE (2001) Antiviral actions of interferons. Clin Microbiol Rev 14:778–809.
9. Toth AM, Zhang P, Das S, George CX, Samuel CE (2006) Interferon action and the
double-stranded RNA-dependent enzymes ADAR1 adenosine deaminase and PKR
protein kinase. Prog Nucleic Acid Res Mol Biol 81:369–434.
10. Patterson JB, Thomis DC, Hans SL, Samuel CE (1995) Mechanism of interferon action:
Double-stranded RNA-specific adenosine deaminase from human cells is inducible by
alpha and gamma interferons. Virology 210:508–511.
11. Patterson JB, Samuel CE (1995) Expression and regulation by interferon of a double-
stranded-RNA-specific adenosine deaminase from human cells: Evidence for two
forms of the deaminase. Mol Cell Biol 15:5376–5388.
12. Isaacs A, Lindenmann J (1957) Virus interference. I. The interferon. Proc R Soc Lond B
Biol Sci 147:258–267.
13. Seeburg PH, Hartner J (2003) Regulation of ion channel/neurotransmitter receptor
function by RNA editing. Curr Opin Neurobiol 13:279–283.
14. Hartner JC, et al. (2004) Liver disintegration in the mouse embryo caused by
deficiency in the RNA-editing enzyme ADAR1. J Biol Chem 279:4894–4902.
15. Wang Q, et al. (2004) Stress-induced apoptosis associated with null mutation of
ADAR1 RNA editing deaminase gene. J Biol Chem 279:4952–4961.
16. George CX, DasS,SamuelCE (2008)Organization ofthe mouse RNA-specific adenosine
deaminase Adar1 gene 5′-region and demonstration of STAT1-independent,
STAT2-dependent transcriptional activation by interferon. Virology 380:338–343.
17. George CX, Samuel CE (1999) Human RNA-specific adenosine deaminase ADAR1
transcripts possess alternative exon 1 structures that initiate from different
promoters, one constitutively active and the other interferon inducible. Proc Natl
Acad Sci USA 96:4621–4626.
18. Hartner JC, Walkley CR, Lu J, Orkin SH (2009) ADAR1 is essential for the maintenance
of hematopoiesis and suppression of interferon signaling. Nat Immunol 10:109–115.
19. Toth AM, Li Z, Cattaneo R, Samuel CE (2009) RNA-specific adenosine deaminase
ADAR1 suppresses measles virus-induced apoptosis and activation of protein kinase
PKR. J Biol Chem 284:29350–29356.
20. Li Z, Wolff KC, Samuel CE (2010) RNA adenosine deaminase ADAR1 deficiency leads to
increased activation of protein kinase PKR and reduced vesicular stomatitis virus
growth following interferon treatment. Virology 396:316–322.
actively contributes to the chronic
21. Tatsuo H, Ono N, Tanaka K, Yanagi Y (2000) SLAM (CDw150) is a cellular receptor for
measles virus. Nature 406:893–897.
22. von Messling V, Zimmer G, Herrler G, Haas L, Cattaneo R (2001) The hemagglutinin of
canine distemper virus determines tropism and cytopathogenicity. J Virol 75:
23. Randall RE, Goodbourn S (2008) Interferons and viruses: An interplay between
induction, signalling, antiviral responses and virus countermeasures. J Gen Virol 89:
24. Zahn RC, Schelp I, Utermöhlen O, von Laer D (2007) A-to-G hypermutation in the
genome of lymphocytic choriomeningitis virus. J Virol 81:457–464.
25. Sheehy AM, Gaddis NC, Choi JD, Malim MH (2002) Isolation of a human gene that
inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature 418:646–650.
26. Harris RS, et al. (2003) DNA deamination mediates innate immunity to retroviral
infection. Cell 113:803–809.
27. Mangeat B, et al. (2003) Broad antiretroviral defence by human APOBEC3G through
lethal editing of nascent reverse transcripts. Nature 424:99–103.
28. Zhang H, et al. (2003) The cytidine deaminase CEM15 induces hypermutation in newly
synthesized HIV-1 DNA. Nature 424:94–98.
29. Young VA, Rall GF (2009) Making it to the synapse: Measles virus spread in and
among neurons. Curr Top Microbiol Immunol 330:3–30.
30. Tenoever BR, et al. (2007) Multiple functions of the IKK-related kinase IKKepsilon in
interferon-mediated antiviral immunity. Science 315:1274–1278.
31. Patterson JB, Thomas D, Lewicki H, Billeter MA, Oldstone MB (2000) V and C proteins
of measles virus function as virulence factors in vivo. Virology 267:80–89.
32. Borrow P, Evans CF, Oldstone MB (1995) Virus-induced immunosuppression: Immune
system-mediated destruction of virus-infected dendritic cells results in generalized
immune suppression. J Virol 69:1059–1070.
33. Samuel CE, Knutson GS (1982) Mechanism of interferon action. Kinetics of induction
of the antiviral state and protein phosphorylation in mouse fibroblasts treated with
natural and cloned interferons. J Biol Chem 257:11791–11795.
34. Samuel CE, Knutson GS (1981) Mechanism of interferon action: Cloned human
leukocyte interferons induce protein kinase and inhibit vesicular stomatitis virus but
not reovirus replication in human amnion cells. Virology 114:302–306.
35. Buchmeier MJ, Lewicki HA, Tomori O, Oldstone MB (1981) Monoclonal antibodies to
lymphocytic choriomeningitis and pichinde viruses: Generation, characterization, and
cross-reactivity with other arenaviruses. Virology 113:73–85.
36. George CX, Wagner MV, Samuel CE (2005) Expression of interferon-inducible RNA
adenosine deaminase ADAR1 during pathogen infection and mouse embryo
development involves tissue-selective promoter utilization and alternative splicing. J
Biol Chem 280:15020–15028.
37. Shtrichman R, Heithoff DM, Mahan MJ, Samuel CE (2002) Tissue selectivity of
interferon-stimulated gene expression in mice infected with Dam(+) versus Dam(−)
Salmonella enterica serovar Typhimurium strains. Infect Immun 70:5579–5588.
38. Marr RA, et al. (2004) Neprilysin regulates amyloid Beta peptide levels. J Mol Neurosci
39. Tiscornia G, Singer O, Verma IM (2006) Production and purification of lentiviral
vectors. Nat Protoc 1:241–245.
| www.pnas.org/cgi/doi/10.1073/pnas.1017241108Ward et al.