JOURNAL OF VIROLOGY, May 2009, p. 4412–4422
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 9
Proteinase 2AproIs Essential for Enterovirus Replication in Type I
Juliet M. Morrison and Vincent R. Racaniello*
Department of Microbiology, College of Physicians & Surgeons of Columbia University, 701 W. 168th St., New York, New York 10032
Received 14 October 2008/Accepted 3 February 2009
The Picornaviridae family comprises a diverse group of small RNA viruses that cause a variety of human and
animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune
system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has
been no study of the effect of picornaviral infection on the events that occur after interferons have been
produced. To determine whether members of the Enterovirus genus can antagonize the antiviral activity of
interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-?) and then infected the
cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these
viruses were able to replicate in IFN-?-pretreated cells but that replication of vesicular stomatitis virus, a
Rhabdovirus, and encephalomyocarditis virus (EMCV), a picornavirus of the Cardiovirus genus, was completely
inhibited. Although EMCV is sensitive to IFN-?, coinfection of cells with poliovirus and EMCV leads to EMCV
replication in IFN-?-pretreated cells. The enteroviral 2A proteinase (2Apro) is essential for replication in cells
pretreated with interferon, because amino acid changes in this protein render poliovirus sensitive to IFN-?.
The addition of the poliovirus 2Aprogene to the EMCV genome allowed EMCV to replicate in IFN-?-pretreated
cells. These results support an inhibitory role for 2Aproin the most downstream event in interferon signaling,
the antiviral activities of ISGs.
Members of the Picornaviridae family are a diverse group of
viruses that cause a variety of human and animal diseases. The
family comprises eight genera: Aphthovirus, Cardiovirus, En-
terovirus, Erbovirus, Hepatovirus, Kobuvirus, Parechovirus, and
Teschovirus. The best-studied member of the family is poliovi-
rus (PV), the etiologic agent of the paralytic disease poliomy-
elitis (39). The three PV serotypes are classified in the Entero-
virus genus along with members such as enterovirus type 70
(EV70), which causes acute hemorrhagic conjunctivitis (52).
The Enterovirus genus has now been expanded to include the
previously separate Rhinovirus genus, which consists of more
than 100 serotypes of rhinoviruses, causative agents of the
common cold (52). A well-studied member of the Cardiovirus
genus is encephalomyocarditis virus (EMCV), which causes
disease in several mammalian species (52).
The genome of picornaviruses is a positive-strand RNA of
7,000 to 9,000 nucleotides that is translated into a polyprotein
which yields structural and nonstructural proteins upon cleav-
age by viral proteinases. The genomes of enteroviruses encode
a 2A proteinase (2Apro), which is structurally and functionally
distinct from the 2A proteins encoded in the genomes of mem-
bers of other genera. For example, the 2A protein of cardio-
viruses has no sequence homology to the enteroviral 2Aproand
lacks protease activity (54). Enterovirus 2Aproprocesses the
viral polyprotein (62), and it also cleaves a variety of host
proteins, including the translation proteins eIF4GI (36),
eIF4GII (43), and poly(A)-binding protein (27). Proteinase
2Aprohas been linked to the inhibition of host translation that
occurs during enterovirus infection (1, 63) and is also involved
in processes as varied as viral RNA replication (42), viral
translation (33), and viral RNA stability (30).
Interferons (IFNs) are pleiotropic cytokines that are well
known for their role in the cellular antiviral response (4, 35,
57). Type I IFNs, which include alpha and beta IFNs (IFN-?
and -?), are induced in response to viral infection (26). The
replication of RNA viruses leads to the production of double-
stranded RNA (dsRNA) intermediates that are recognized by
Toll-like receptors or by the cytoplasmic retinoic acid-induc-
ible gene I (RIG-I)-like helicases, RIG-I and MDA-5 (61).
Upon activation, MDA-5 and RIG-I bind to mitochondrial
antiviral signaling protein (MAVS), which then coordinates
the activation of the transcription factors IFN regulatory factor
3 (IRF3) and NF-?B to induce the production of type I IFN
(61). RIG-I-like helicase signaling has been shown to inhibit
picornavirus replication (32). Type I IFNs bind to the type I
IFN receptor and elicit a cascade of signaling events that lead
to the formation of the IFN-stimulated gene factor 3 transcrip-
tional complex, which binds consensus DNA sites to stimulate
the transcription of IFN-stimulated genes (ISGs) (20). The
importance of IFNs is underscored by the observation that
mice that lack the type I IFN receptor or proteins of the
Jak-Stat signaling pathway have an increased sensitivity to viral
infections (16, 31, 45).
There are more than 300 ISGs, most of which have not been
characterized (10). The antiviral properties of the better-
known ISGs have been documented. Protein kinase R or
dsRNA-dependent protein kinase (PKR) is activated upon
binding to dsRNA and phosphorylates eukaryotic initiation
factor 2? to inhibit both host and viral translation (46). The
2?-5? oligoadenylate synthetase/RNase L pathway is also acti-
vated by dsRNA and leads to the destruction of viral RNA
* Corresponding author. Mailing address: Department of Microbi-
ology, Columbia University College of Physicians, 701 W. 168th St.,
Room 1310B, New York, NY 10032. Phone: (212) 305-5707. Fax: (212)
305-5106. E-mail: email@example.com.
?Published ahead of print on 11 February 2009.
(14). ISG56 suppresses both host and viral translation by bind-
ing to eukaryotic initiation factor 3 (25).
The IFN system evolved to inhibit viral replication, but vi-
ruses have coevolved with the system to block this response.
This antagonism can occur at three levels: (i) viral recognition
and IFN production, (ii) IFN signaling and ISG production,
and (iii) ISG activity. Examples of viral antagonism at the IFN
production step include hepatitis C virus inhibition of IRF3
and NF-?B activation via the NS3/4A protease (19), vesicular
stomatitis virus (VSV) inhibition of IFN-? transcription by the
matrix protein (18), and the binding of human papillomavirus
16 E6 oncoprotein to IRF3 (58). IFN signaling is disrupted
during many viral infections, including those with human cy-
tomegalovirus (47), dengue virus (28), and human papilloma-
virus (2). Examples of viruses with direct antagonism of ISGs
include herpes simplex virus, which targets RNaseL (6) and
PKR (41), and hepatitis C virus, which blocks activation of
The IFN response is altered by a variety of mechanisms in
cells infected with picornaviruses. The production of type I
IFN is attenuated in cells infected with mengovirus (23), while
the hepatitis A virus 3Cprocleaves MAVS to inhibit type I IFN
production (65). PV infection leads to inhibition of the cellular
secretory pathway, leading to reduced secretion of cytokines,
including IFN-? (13). MDA-5 and RIG-I are cleaved during
PV infection (3; P. Barral, P. Fisher, and V. R. Racaniello,
unpublished data), and the induction of IFN-? mRNA is in-
hibited by the L proteinase of foot-and-mouth disease virus
(8). Type I IFN has been shown to regulate PV tropism (50, 66)
and is known to regulate replication of EMCV replication
Here we investigate viral inhibition of the IFN response at
the most downstream step of the pathway, when ISGs have
already been produced. We show that picornaviruses that en-
code a 2Apro, i.e., members of the Enterovirus genus such as
PV, human rhinovirus 16 (HRV16), and EV70, are able to
replicate in cells that have been pretreated with IFN-?. Picor-
naviruses that do not encode this proteinase, such as EMCV,
are exquisitely sensitive to IFN and are unable to replicate in
IFN-pretreated cells. The synthesis of 2Aprorescues the repli-
cation of EMCV in IFN-pretreated cells, indicating that
2Aprofunctions as an ISG antagonist.
MATERIALS AND METHODS
Cells, viruses, and plasmids. HeLa S3 (human cervical carcinoma), Vero
(African green monkey kidney), and Huh7 (human hepatoma) lines were main-
tained in Dulbecco’s modified essential medium (DMEM; Invitrogen, Carlsbad,
CA) supplemented with a 1% penicillin-streptomycin solution (Invitrogen) and
10% bovine calf serum (HyClone, Logan, UT). The K562 (human myeloid
leukemia) line was maintained in Iscove’s modified medium (Invitrogen) sup-
plemented with a 1% penicillin-streptomycin solution and 10% fetal bovine
serum (Atlanta Biologicals, Atlanta, GA). Mrc5 cells were maintained in DMEM
supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum.
Wild-type and mutant PVs were isolated from HeLa cells transfected with in
vitro-synthesized RNA using DEAE-dextran (51). RNA transcripts were pro-
duced from a PV type 1 Mahoney P1M infectious clone, pT7M (56); one of its
derivatives, pT7M-2A-Y88S, pT7M-2A-Y88L, pT7M-3C-V54A; or a PV type 2
Lansing (P2L) infectious clone, pT7L (55), or the mutant R2-5NC-14 (P2L-
5?UTR; a small-plaque mutant of P2L with a single nucleotide substitution in the
5? untranslated region) (53). EMCV was isolated from HeLa cells transfected
with in vitro-synthesized RNA from an EMCV infectious clone, pEC4 (15). All
in vitro-synthesized RNAs were transcribed from linearized templates using T7
RNA polymerase (Promega, Madison, WI). The EMCV/P1M chimeric viruses
and HRV16 were produced from the infectious clones pEC4-EP1, pEC4-EP3,
pEC4-EP4, pEC4-EP5, and pRV16.11 (40). VSV was obtained from the Amer-
ican Type Culture Collection (Manassas, VA) and propagated in HeLa cells.
EV70 was produced from an infectious clone (34) and propagated in HeLa cells.
Virus titers were determined by plaque assay on HeLa cells.
Plaque assays. To determine the titer of P1M and EV70, HeLa cells were
covered with an overlay consisting of DMEM (Specialty Media, Philipsburg, NJ),
0.2% NaHCO3(Sigma, St. Louis, MO), 5% bovine calf serum, 1% penicillin-
streptomycin, and 0.8% Noble agar (Sigma). To determine the titer of HRV16,
HeLa cells were covered with an overlay of DMEM, 0.2% NaHCO3, 1% bovine
calf serum, 1% penicillin-streptomycin, 0.1 M MgCl2(Sigma), and 0.8% Noble
agar. After the overlay solidified, it was covered with liquid medium consisting of
DMEM (Invitrogen), 0.04 M MgCl2(Sigma), 0.002% glucose (Sigma), 0.1%
bovine serum albumin (Sigma), 2 mM sodium pyruvate (Invitrogen), 4 mM
glutamine (Invitrogen), 4 mM oxaloacetic acid (Sigma), 0.2% NaHCO3(Sigma),
and 1% penicillin-streptomycin. Plaque assays of EMCV were performed using
3T3 or HeLa cells in the same medium that was used for determining the titer of
Construction of PVs with altered 2Aproor 3Cpro. Mutations were introduced
into cloned PV type 1 DNA by mutagenic PCR. All PCR amplifications were
done with PfuUltra or PicoMaxx enzymes (Stratagene, La Jolla, CA). To change
amino acid 88 of 2Aprofrom Y to either S or L, the BstEII fragment of PV DNA
from nucleotide (nt) 3240 to 3930 was replaced with a PCR product containing
the appropriate mutations. To change amino acid 54 of 3Cprofrom V to A, a
BseRI fragment (nt 4742 to 6148) was transferred from Se1-3C-02 DNA (11) to
Construction of EMCV clones expressing PV 2Apro. Sequences encoding 2Apro
of P1M, with or without an amino-terminal FLAG epitope, were inserted be-
tween the coding region for P1 and 2A of EMCV in pEC4. The clone with the
untagged version of 2Aprowas named EP4, and the clone containing a FLAG
epitope at the amino terminus of 2Aprowas named EP1. A cleavage site for
EMCV 3C proteinase, VLMLESPNAL, was placed at the N and C termini of PV
2Apro. The two versions of the 2Aprofragment were constructed by overlap or
mutagenic PCR. The final PCR fragment was cloned into pCR2.1 using the
TOPO TA cloning kit and cut using SpeI and SacI (New England BioLabs)
enzymes. This fragment was used to replace the corresponding SpeI-SacI frag-
ment in pEC4 between nt 2293 and 3609.
To construct EP5 and EP3, two versions of 2Aproof P1M, with or without an
N-terminal FLAG epitope, were inserted before the L coding region in the
genome of EMCV in pEC4. The clone with the untagged version of 2Aprowas
named EP5, and the clone containing a FLAG epitope at the amino terminus of
2Aprowas named EP3. An AUG site was placed at the N terminus of 2Apro, and
a cleavage site for EMCV 3Cpro, DEQEQGPYN, was placed at the C terminus
of PV 2Apro. The two versions of the 2Aprofragment were constructed by overlap
or mutagenic PCR. The final PCR fragment was cloned into pCR2.1 using the
TOPO TA cloning kit and cut using AvrII and BssHII (New England BioLabs)
enzymes. This fragment was used to replace the corresponding AvrII-BssHII
fragment in pEC4 between nt 283 and 734.
Single-step growth analysis. Confluent monolayers of cells in 35-mm plates
that had been mock treated or pretreated with IFN-? for 24 h were infected at
37°C at the desired multiplicity of infection (MOI). After a 1-hour adsorption
step, cells were rinsed once with phosphate-buffered saline (PBS; pH 7.5), fol-
lowed by the addition of DMEM supplemented with 1% penicillin-streptomycin
and 1% bovine calf serum and incubation at 37°C. Cells and supernatants were
collected at each time point and frozen and then thawed to release the virus.
Virus was clarified by centrifugation, and titrated on HeLa cells in the case of
single-virus infections or on both HeLa cells and 3T3 cells in the case of coin-
fections. Because 3T3 cells do not allow entry of PV, these cells can be used to
distinguish this virus from EMCV and its derivatives in the yield of mixed
Protein labeling and Western blot analysis. To visualize proteins in infected
cells, confluent monolayers of HeLa cells in 35-mm plates were infected at an
MOI of 10 with wild-type P1M or with the PV mutants P1M-2A-Y88L (a
small-plaque mutant of P1M with a tyrosine-to-leucine substitution at amino acid
88 in 2Apro), P1M-2A-Y88S (a small-plaque mutant of P1M with a tyrosine-to-
serine substitution at amino acid 88 in 2Apro), or P2L-5?UTR at 37°C. After a
1-hour adsorption step, cells were rinsed once with PBS, followed by the addition
of serum-free DMEM. At each time point, the medium was removed from a
batch of cells and the cells were rinsed with serum-free DMEM lacking methi-
onine and cysteine. Serum-free DMEM containing 25 ?Ci35S-labeled methio-
nine and cysteine (Amersham, Piscataway, NJ) per ml was added to the cells for
15 min. The cells were then washed with PBS and lysed with radioimmunopre-
cipitation buffer (PBS containing 1% NP-40, 0.5% sodium deoxycholate, and
VOL. 83, 2009 ENTEROVIRUS INTERFERON RESISTANCE4413
0.1% sodium dodecyl sulfate [SDS]). Cytoplasmic extracts were denatured in
SDS buffer (250 mM Tris Cl [pH 6.8; Sigma], 50% glycerol [Sigma], 10% SDS
[Sigma], 0.5% bromophenol blue [Sigma], ?-mercaptoethanol [Bio-Rad, Hercu-
les, CA]), and equal amounts of lysate were resolved on a 10% SDS-polyacryl-
amide gel. Gels were stained, fixed, and dried by standard protocols and then
exposed to X-ray film overnight.
For Western blot analysis, confluent monolayers of HeLa cells in 35-mm plates
were infected with virus at an MOI of 10 at 37°C. After a 1-hour adsorption step,
cells were rinsed once with PBS (pH 7.5), followed by the addition of DMEM
supplemented with 1% penicillin-streptomycin and 1% bovine calf serum and
incubation at 37°C. At each time point, cells were washed with PBS and lysed in
radioimmunoprecipitation buffer. Cytoplasmic extracts were denatured in SDS
buffer, and equal amounts of lysate were resolved on a 6%, 10%, or 18%
SDS-polyacrylamide gel, depending on the size of protein being visualized. For
detection of eIF4GI, the proteins were transferred to a polyvinylidene fluoride
membrane and subjected to Western blot analysis with rabbit anti-eIF4GI anti-
body (Abcam, Cambridge, MA) at a 1/1,000 dilution and horseradish peroxidase-
conjugated anti-rabbit immunoglobulin G (Sigma) at a 1/10,000 dilution. For
detection of the FLAG epitope, Western blot analysis was carried out with
mouse M2 anti-FLAG antibody (Sigma) at a 1/4,000 dilution and horseradish
peroxidase-conjugated anti-mouse immunoglobulin G (Promega) at a 1/3,000
Efficiency of plaque formation. Subconfluent 100-mm plates of HeLa cells
were pretreated for 24 h with 2,000 or 100 units/ml IFN-?. Cells were then
infected with a known quantity of virus and overlaid with plaquing medium for
3 days to allow plaque formation. Plaques were picked from each plate and
expanded in HeLa cells, and RNA from these plaques was extracted using the
RNeasy kit (Qiagen, Valencia, CA). The RNA was reverse transcribed to cDNA
using Superscript III (Invitrogen) and amplified via PCR using PicoMaxx enzyme
and primers flanking the 2Aprogene. The nucleotide sequence of the PCR
products was determined to identify changes in the 2Aprosequence. Plaques on
each plate were counted.
Replication of VSV, EMCV, and P1M in IFN-?-treated
HeLa cells. To determine the effect of IFN-? pretreatment on
PV replication, HeLa cells were treated with 1,000 units/ml
IFN-? for 24 h and then infected with PV. IFN-? was not
added to the cells postinfection. Yields of PV were 1,000 PFU/
cell in untreated cells and 100 PFU/cell in IFN-?-treated cells
(Fig. 1A). P2L and PV type 3 Leon (P3L) also replicated to
high titers in IFN-?-treated cells (see Fig. 1B for P2L data;
data not shown for P3L). The replication of these serotypes
was inhibited 5- to 10-fold by IFN-? pretreatment. In contrast,
replication of VSV, which is known to be exquisitely sensitive
to IFN (59), was blocked in cells that had been treated with
IFN-? (Fig. 1C). Replication of EMCV was also blocked in
IFN-?-treated cells (Fig. 1D).
The experiments described above were done at an MOI of
10, which would result in infection of ?99.99% of the cells. To
determine the outcome of infection at a lower MOI, untreated
or IFN-?-treated HeLa cells were infected with P1M at an
MOI of 100, 10, 1, or 0.1, and viral titers were determined 24 h
postinfection. P1M was relatively resistant to IFN pretreat-
ment at an MOI of 10 or 1, but IFN resistance declined at an
MOI of 0.1 (Fig. 1E). These results indicate that the IFN
resistance of PV is dependent on the MOI.
Replication of EMCV and P1M in other cell lines treated
with IFN-?. Since HeLa cells are transformed and aneuploid,
the ability of PV to replicate in IFN-?-treated cells may not be
representative of the results in other cell types. To determine
if IFN resistance is cell type specific, Huh7, Vero, K562, and
Mrc5 cells were treated with 1,000 units/ml IFN-? for 24 h and
infected with P1M or EMCV. In all the cell lines tested, P1M
titers were approximately 1,000 PFU/cell in untreated cells and
FIG. 1. Effect of IFN-? pretreatment on viral replication. Mono-
layers of HeLa cells were pretreated with 1,000 units/ml IFN-? for 24 h
or were left untreated and then were infected with P1M (A), P2L (B),
VSV (C), or EMCV (D) at an MOI of 10. Total virus was harvested at
the indicated times postinfection, and viral titers were determined by
plaque assay on HeLa cells. (E) Monolayers of HeLa cells were pre-
treated with 1,000 units/ml IFN-? for 24 h and then infected with P1M
at an MOI of 0.1, 1, 10, or 100. Total virus was harvested at 24 h
postinfection, and viral titers were determined by plaque assay on
4414MORRISON AND RACANIELLOJ. VIROL.
100 PFU/cell in cells treated with IFN (Fig. 2). EMCV titers
were ?100 PFU/cell in untreated Huh7, K562, and Vero cells,
while replication was completely inhibited in cells that had
been treated with IFN-? (Fig. 2). EMCV was completely in-
hibited in Mrc5 cells that had been treated with IFN-?, but
yields were ?500 PFU/cell in untreated Mrc5 cells. Because
Mrc5 cells are not transformed, the results indicate that the
transformed phenotypes of HeLa cells and other cell types do
not contribute to PV IFN resistance and EMCV IFN sensitiv-
ity. The effects of IFN-? on EMCV and P1M replication are
therefore independent of cell type.
Because Vero cells (17, 48) and K562 cells (12) do not
produce IFN, these cells can be used to dissociate type I IFN
induction from downstream events, such as ISG production
and activity. Treatment of both cell lines with IFN-? results in
partial inhibition of P1M and complete inhibition of EMCV.
Since the extent of inhibition of viral replication in these cells
is the same as in cell types that produce IFN, the phenomenon
being studied here is not antagonism of the IFN induction that
normally occurs upon viral infection and during the type I IFN
feedback loop (44) but antagonism at the level of ISG produc-
tion or activity.
PV 2Aprois essential for replication in IFN-?-treated cells.
To identify viral proteins essential for PV replication in IFN-
?-treated cells, viruses with amino acid changes in one of the
two virus-encoded proteinases were studied. A single amino
acid change in 3Cprowhich leads to defects in trans but not cis
cleavage has been described previously (11). A virus carrying
this change, P1M-3C-V54A (a small-plaque mutant of P1M
with a valine-to-alanine substitution at amino acid 54 in the
3Cproproteinase), replicated to approximately 500 PFU/cell in
untreated cells and to over 100 PFU/cell in IFN-treated cells
(Fig. 3A). Amino acid changes at position 88 of 2Aprothat
affect cleavage of cellular but not viral substrates have been
described previously (64, 67). The growth of PV mutants P1M-
2A-Y88L and P1M-2A-Y88S was similar to that of wild-type
virus in untreated cells (?1,000 PFU/cell) but was completely
inhibited in IFN-?-treated cells (Fig. 3B and C). These results
suggest that 2Aprois essential for PV replication in IFN-?-
Other PV mutants were examined to rule out the possibility
that any replication-defective virus would be sensitive to
IFN-?. A small-plaque mutant of P2L with a nucleotide sub-
stitution in the 5? UTR, P2L-5?UTR (53), replicated as well in
the presence of IFN (Fig. 3D) as did the wild-type parental
virus (Fig. 1B). The Sabin type 3 vaccine strain, whose genome
has 10 mutations that contribute to a small-plaque phenotype
(60), also replicated to high titers in mock-treated and IFN-?-
treated cells (data not shown).
To determine whether changes in other PV proteins could
confer the ability to replicate in IFN-treated cells, we at-
tempted to isolate viruses that could form plaques on mono-
layers of IFN-?-treated HeLa cells. HeLa cell monolayers were
treated with 2,000 units/ml of IFN-? for 24 h, infected with a
known quantity of wild-type P1M, P1M-2A-Y88S, P1M-2A-
Y88L, or EMCV, and covered with an agar overlay containing
IFN-?. The efficiencies of plaque formation differed among the
viruses (Table 1). The plaquing efficiency was highest for wild-
type PV, at 50%. All viruses isolated from plaques arising on
IFN-treated plates encoded wild-type 2Apro. The frequency of
plaque formation was much lower for P1M-2A-Y88S and
P1M-2A-Y88L than for wild-type PV and was zero for EMCV.
The frequency of plaque formation for P1M-2A-Y88S was
approximately 1/120,000 on IFN-treated cells, and all viruses
within the plaques encoded the wild-type amino acid at residue
88. P1M-2A-Y88L had a higher efficiency of plaquing on IFN-
treated cells (?1/3,000), though it was still more than 1,000-
fold lower than that observed for wild-type virus. The viruses
within the P1M-2A-Y88L plaques all encoded a tyrosine or a
FIG. 2. Replication of EMCV and P1M in different cell lines
treated with IFN-?. Monolayers of Huh7 (A), Vero (C), and Mrc5
(D) cells and suspensions of K562 (B) cells were pretreated with 1,000
units/ml IFN-? for 24 h or were left untreated before they were in-
fected with P1M or EMCV at an MOI of 10. Total virus was harvested
at 0 and 24 h postinfection, and viral titers were determined by plaque
assay on HeLa cells.
VOL. 83, 2009ENTEROVIRUS INTERFERON RESISTANCE 4415
phenylalanine at amino acid 88. Only one nucleotide change is
required to convert leucine to phenylalanine, explaining why
revertants arose with greater frequency with P1M-2A-Y88L
than with P1M-2A-Y88S. A P1M-2A-Y88F mutant virus is
able to replicate to wild-type titers in IFN-treated cells (data
Effect of IFN treatment time on virus replication. In all of
the experiments described in this paper, cells were pretreated
with IFN for 24 h. It has been shown that different sets of ISGs
are induced at various times after treatment with IFN (24). To
investigate the effect of IFN treatment times on viral replica-
tion, HeLa cells were treated for different lengths of time with
IFN-? and then infected with virus at an MOI of 10. As in
previous experiments, IFN was not added back to the cells
after infection. Virus production was determined at 0 and 24 h
postinfection. Complete inhibition of EMCV and P1M-2A-
Y88L replication was observed after treatment of cells for 3 h
or longer, indicating that some of the ISGs that are antago-
nized by enteroviruses are induced early after IFN-? treatment
Protein synthesis and eIF4GI levels in cells infected with PV
2Apromutants. An important cellular substrate for 2Aprois
eIF4G (5, 37, 38). Cleavage of this translation protein leads to
inhibition of host protein synthesis during picornavirus infec-
tion (1, 5, 22, 63, 68). To determine whether translation inhi-
bition plays a role in the ability of PV to replicate in cells
treated with IFN-?, cellular protein synthesis was examined
after labeling with [35S]methionine. Host protein synthesis was
inhibited beginning at 3 h after infection with wild-type PV,
and by 5 h postinfection, mainly viral proteins were detected
(Fig. 5A). Inhibition of host protein synthesis coincides with
the complete cleavage of eIF4GI by 3 h postinfection (Fig. 5B).
Infection with the PV 2Apromutant P1M-2A-Y88S, which is
IFN sensitive, also leads to inhibition of cellular protein syn-
thesis (Fig. 5A) and cleavage of eIF4GI (Fig. 5B) with kinetics
similar to those observed during infection with wild-type virus.
Cells infected with the other IFN-sensitive virus, P1M-2A-
Y88L, display a different eIF4GI cleavage and host translation
inhibition phenotype. During P1M-2A-Y88L infection, there is
a lag in eIF4GI cleavage compared to that of the wild type,
with some intact eIF4GI remaining until 7 h postinfection (Fig.
5B). No translation shutoff was observed even at 9 h postin-
fection (Fig. 5A). Protein was not harvested at points past 9 h
postinfection due to the high level of cell death seen at those
times. Infection with the IFN-resistant PV mutant P2L-5?UTR
leads to cleavage of eIF4GI even earlier in infection than with
wild-type virus (Fig. 5B) and inhibition of cellular translation,
though not as effectively as with wild-type virus or P1M-2A-
Y88S (Fig. 5A). These results show that neither PV-induced
host translation inhibition or eIF4GI cleavage correlates with
the ability to replicate in IFN-treated cells. This conclusion is
highlighted by the different properties of the two IFN-sensitive
mutants: infection with P1M-2A-Y88S, but not P1M-2A-Y88L,
leads to inhibition of host translation and eIF4GI cleavage
early in infection.
Effect of PV on EMCV replication in IFN-?-treated HeLa
cells. We hypothesize that replication of P1M in IFN-treated
cells is a consequence of antagonism of the IFN response. To
test this hypothesis, we attempted to rescue an IFN-sensitive
virus by coinfection with P1M. This experiment was compli-
cated by the inhibition of cap-dependent translation that takes
place in cells infected with P1M. Consequently, VSV replica-
tion is completely inhibited in cells that are coinfected with
P1M (data not shown). We used EMCV as the IFN-sensitive
virus, because translation of the genome occurs via an internal
ribosomal entry site and is not affected by cleavage of eIF4G.
TABLE 1. Efficiency of plaque formation on IFN?-pretreated cells
amino acid 88
Nucleotide changes in
codon 88 of 2Apro
L to Y, L to F
CTC to TAC, TAT,
TCG to TAC or
P1M-2A-Y88S1/120,000S to Y
aNumber of plaques on cells treated with 2,000 units/ml IFN? relative to
number of plaques on untreated cells.
bNA, not applicable.
FIG. 3. Replication kinetics of small-plaque PV mutants in IFN-?-pretreated cells. Monolayers of HeLa cells were pretreated with 1,000
units/ml IFN-? for 24 h or were left untreated before they were infected with virus at an MOI of 10. Total virus was harvested at the indicated
times postinfection, and viral titers were determined by plaque assay on HeLa cells. The mutant viruses were P1M-3C-V54A, a small-plaque
mutant of P1M with a valine-to-alanine substitution at amino acid 54 in 3Cpro(A), P1M-2A-Y88L, a small-plaque mutant of P1M with a
tyrosine-to- leucine substitution at amino acid 88 in 2Apro(B), P1M-2A-Y88S, a small-plaque mutant of P1M with a tyrosine-to-serine
substitution at amino acid 88 in 2Apro(C), and P2L-5?UTR, a small-plaque mutant of P2L with a single nucleotide substitution in the 5?
untranslated region (D).
4416 MORRISON AND RACANIELLO J. VIROL.
HeLa cells were coinfected with P1M at an MOI of 1 and
EMCV at an MOI of 10 to minimize PV-mediated inhibition
of EMCV replication at higher MOIs (data not shown). As
expected, EMCV did not replicate in IFN-treated HeLa cells
(Fig. 1 and 6). However, when IFN-?-treated HeLa cells were
coinfected with P1M and EMCV, EMCV replicated to a titer
of about 3 PFU/cell (Fig. 6). These results indicate that infec-
tion with P1M modifies the cells to allow EMCV replication in
the presence of ISGs.
Replication of other enteroviruses in IFN-?-treated HeLa
cells. The picornavirus family includes diverse members in
addition to PV and EMCV. It was therefore of interest to
determine the effect of IFN-? pretreatment on the replication
of other members of this virus family. When untreated cells
were infected with EV70 or HRV16, both viruses replicated to
about 15 PFU/cell (Fig. 7). Neither virus replicated as well in
untreated cells as PV did but nevertheless showed IFN resis-
tance, replicating to about 3 PFU/cell in IFN-treated HeLa
Replication of EMCV-P1M chimeras in IFN-?-treated cells.
Although the genome of EMCV encodes a 2A protein, it is
very different from enterovirus 2Aproand does not have pro-
teolytic activity. We therefore investigated whether insertion of
the coding sequence for PV 2Aprointo the genome of EMCV
would allow viral replication in IFN-?-treated cells. Chimeric
viruses were constructed with 2Aproof P1M (with or without
an N-terminal FLAG epitope) inserted between P1 and P2 of
EMCV (EP1 and EP4) or upstream of L (EP3 and EP5). The
results of Western blot analysis demonstrated that the 2Apro
proteins were produced in HeLa cells infected with chimeric
viruses EP1 and EP3 (data not shown). Cleavage of eIF4GI
was observed in cells infected with all chimeric viruses (Fig. 8D
and data not shown), and shutoff of cellular translation was
observed in cells infected with EP4 and EP5 (Fig. 8C). These
FIG. 4. Effect of IFN treatment time on the replication of EMCV, wild-type P1M (P1M-WT), and P1M-2A-Y88L. Monolayers of HeLa cells
were pretreated with 1,000 units/ml IFN-? for 0, 1, 3, 6, 12, or 24 h and then infected with P1M-WT (A), P1M-2A-Y88L (B), or EMCV (C). Total
virus was harvested at 24 h post infection (p.i.) for all conditions and also at 0 h postinfection for cells that had not been treated with IFN-?. Viral
titers were determined by plaque assay on HeLa cells.
FIG. 5. Protein synthesis and eIF4GI cleavage in HeLa cells infected with different PV mutants. (A) HeLa cell monolayers were infected at
an MOI of 10 with wild-type P1M (P1M-WT) or with the PV mutant P1M-2A-Y88L, P1M-2A-Y88S, or P2L-5?UTR. At the indicated time
postinfection (p.i.), cells were labeled with [35S]methionine for 15 min. Cytoplasmic extracts were analyzed by 10% SDS-polyacrylamide gel
electrophoresis. (B) Monolayers of HeLa cells were infected with virus at an MOI of 10. Cytoplasmic extracts were prepared at each time point
and fractionated on a 6% SDS-polyacrylamide gel. Western blot analysis was carried out with rabbit anti-eIF4GI antibody.
VOL. 83, 2009ENTEROVIRUS INTERFERON RESISTANCE4417
results demonstrate that 2Apro, when produced from the
EMCV genome, is capable of cleaving at least one of its known
host substrates. The chimeric viruses EP4 and EP5 replicated
slightly less well than wild-type EMCV in untreated cells (Fig.
8A and B). However, replication of the chimeric viruses in
HeLa cells was completely inhibited by IFN pretreatment.
Interestingly, the levels of EP4 remained constant in IFN-
treated HeLa cells while the levels of EP5 declined (Fig. 8A
and B). This observation suggests that EP4, unlike EP5, might
have a higher level of IFN resistance, which might allow it to
replicate at low levels. We therefore examined replication of
P1M, EMCV, and EP4 in cells treated with lower doses of IFN.
A dose of 10 units/ml IFN-? was chosen for growth curve
analysis, because EP4 replication was inhibited at higher doses
of IFN-? pretreatment (data not shown). Yields of P1M were
unaffected, while EMCV replication was completely blocked in
HeLa cells treated with 10 units/ml IFN-? (Fig. 9). Under the
same conditions, the yield of EP4 was approximately 40 PFU/
cell. To determine the efficiency of plaquing, HeLa cell mono-
layers were treated with IFN-? for 24 h, infected with P1M,
EMCV, or EP4, and covered with an agar overlay. EP4 failed
to form plaques on cells that were pretreated with 1,000 or
2,000 units/ml IFN-? (data not shown) but had a plaquing
efficiency of 1/450 on cells that had been pretreated with 100
units/ml IFN-?. The efficiency of plaquing of P1M at this
concentration was 1, while that of EMCV was zero.
We have shown that the enteroviruses EV70, HRV16, and
PV types 1, 2, and 3 can replicate in type I IFN-pretreated
cells. In contrast, replication of EMCV and VSV is completely
inhibited by IFN pretreatment. In our experiments, IFN is not
present postadsorption, allowing us to distinguish between an-
tagonism of IFN signaling and antagonism of ISG activity. At
least 3 h of IFN pretreatment is required for complete inhibi-
tion of EMCV replication. This observation indicates that the
ISGs that inhibit picornaviral replication are produced soon
after type I IFN treatment. Since IFN pretreatment leads to
the production of ISGs, and enteroviruses can replicate in cells
that have been pretreated with type I IFN, enteroviruses must
have some means of neutralizing the activity of ISGs that
target picornaviral replication. Infection of cells that do not
produce type I IFNs provides further evidence for abrogation
of ISG activity. Vero cells and K562 cells do not produce IFN
but are able to produce ISGs in response to the cytokine.
Replication of EMCV is completely inhibited in these cell lines
when they are pretreated with IFN-?, and PV replication is
inhibited to a level similar to that in cell types that produce
IFN. If the ability of PV to replicate in IFN-?-pretreated cells
were a consequence of blocking type I IFN production, we
would expect that viral replication would be unencumbered or
significantly less encumbered in IFN-?-pretreated K562 and
Enterovirus IFN resistance is dependent on MOI. At high or
intermediate MOIs (100, 10, or 1), PV replication is relatively
resistant to IFN, yielding ?100 PFU/cell. At a lower MOI
(0.1), resistance decreases and virus production is ?4 PFU/
cell. The number of virions that infect each cell may determine
the efficiency of replication in cells treated with IFN. At an
MOI of 100 or 10, more than 99.95% of cells receive more than
1 virus particle. In contrast, at an MOI of 0.1, only 9.5% of the
cells are infected and only 0.47% of cells receive more than
one particle. When more virions enter a cell, viral proteins
more rapidly accumulate to higher levels. Consequently, the
antiviral activities of ISGs are countered early in infection to
allow more robust viral replication.
FIG. 7. Replication of HRV16 and EV70 in cells treated with
IFN-?. HeLa cell monolayers were pretreated with 1,000 units/ml
IFN-? for 24 h or were left untreated before they were infected with
HRV16 (A) or EV70 (B) at an MOI of 10. Total virus was harvested
at the indicated hours postinfection, and viral titers were determined
by plaque assay on HeLa cells.
FIG. 6. EMCV replication in HeLa cells coinfected with PV. HeLa
cell monolayers were pretreated with 1,000 units/ml IFN-? for 24 h or
were left untreated before they were infected with EMCV (MOI, 10)
alone or coinfected with P1M (MOI, 1). Total virus was harvested at
the indicated times postinfection, and the EMCV titers were deter-
mined by plaque assay on 3T3 cells. P1M does not form plaques on this
4418 MORRISON AND RACANIELLOJ. VIROL.
PV replicates only in specific cells and tissues in primates,
even though the virus reaches many organs during the viremic
phase (reviewed in reference 52). In mice, the IFN response is
an important determinant of PV tissue tropism (50). PV infec-
tion of CD155 transgenic mice lacking the receptor for IFN-
?/? leads to viral replication in the liver, spleen, and pancreas,
in addition to the central nervous system. In contrast, PV
replication is limited to the central nervous system of wild-type
CD155 transgenic mice. Our finding that PV replicates in a
variety of human and monkey cell lines treated with IFN-?
suggests that the antiviral effect of the cytokine may vary de-
pending on the species or cell type. Understanding the mech-
anism by which enteroviruses limit the antiviral effects of IFN
will provide insight into the mechanism of viral pathogenesis.
Small-plaque PV mutants with changes at amino acid 88 in
2Aprocannot replicate in IFN-pretreated cells. Other small-
plaque PV mutants with changes elsewhere in the genome can
replicate under the same conditions, indicating that IFN sen-
sitivity is not conferred by any mutation that leads to a small-
plaque phenotype. These data suggest that 2Aprofunctions as
an IFN antagonist. When we attempted to select for IFN-
resistant viruses by plaque assay of the two 2Apromutants on
IFN-?-treated cells, the only viruses that were recovered had
changes at residue 88 of 2Aproto the tyrosine of the wild type
or to phenylalanine. Tyrosine and phenylalanine are structural
similar, and as expected, a Y88F 2Apromutant replicated to
wild-type levels in IFN-pretreated cells. These observations
strongly implicate 2Aproin IFN antagonism.
FIG. 8. Replication, eIF4GI cleavage, and translation in HeLa cells. (A and B) Diagrams of EMCV polyprotein showing the locations of PV
2Aproin EMCV chimeras EP4 and EP5. Duplicate monolayers of HeLa cells were pretreated with 1,000 units/ml IFN-? for 24 h or were left
untreated before they were infected at an MOI of 10 with EP4 or EP5. Total virus was harvested at different hours postinfection, and viral titers
were determined by plaque assay on HeLa cells. (C) Cytoplasmic extracts were prepared from the second set of monolayers described for panels
A and B and fractionated by electrophoresis on a 6% SDS-polyacrylamide gel, and Western analysis was carried out with rabbit anti-eIF4GI
antibody. (D) HeLa cell monolayers were infected at an MOI of 10 with EMCV, EP4, or EP5. At the indicated times postinfection, cells were
labeled with [35S]methionine for 15 min. Cytoplasmic extracts were fractionated by 12% SDS-polyacrylamide gel electrophoresis.
VOL. 83, 2009 ENTEROVIRUS INTERFERON RESISTANCE4419
Our results indicate that 2Aprois necessary for replication in
cells pretreated with IFN but that it is not apparently sufficient
for full expression of this phenotype. An EMCV recombinant
that synthesizes 2Aproof PV (EP4) has a higher level of IFN
resistance than wild-type EMCV, as measured by plaquing
efficiency on HeLa cells pretreated with 100 units/ml IFN-?
and by analysis of growth in cells pretreated with 10 units/ml
IFN-?. However, EP4 is unable to replicate in cells that have
been treated with high levels (1,000 units/ml) of IFN, in con-
trast to enteroviruses such as PV, EV70, and HRV16. This
observation suggests that other enteroviral proteins, in addi-
tion to 2Apro, are required for replication in cells treated with
high levels of IFN. Other viral proteins might be required
together with 2Aproto overcome the higher levels of ISGs
produced after treatment with 1,000 units/ml IFN-?. Alterna-
tively, efficient antagonism by 2Apromay require the protein-
ase to be part of a membrane-bound precursor polyprotein.
There are precedents for such a requirement. Initiation of PV
negative-strand synthesis in HeLa extracts does not occur
when a mixture of processed forms of 2A, 2B, 2C, 2AB, and
2BC are synthesized (29). Instead initiation occurs only when
the precursor, P23, or a mix of 2A and 2BCP3, or of P2 and P3,
is added to the extracts. The 3Cproof hepatitis A virus cleaves
the mitochondrial outer membrane protein MAVS only when
it is part of the precursor, 3ABC (65). The precursor is re-
quired for cleavage because the hydrophobic protein 3A tar-
gets the proteinase to the mitochondrial membrane.
Viral proteases have been implicated as antagonists of sev-
eral steps in the IFN response. The NS3/4A protease of hep-
atitis C virus cleaves MAVS to abrogate IFN production (19).
3Cproand 2Aproof PV induce apoptosis, which leads to
MDA-5 cleavage in a proteasome- and caspase-dependent
manner (3). 3Cproof hepatitis A virus, as part of the 3ABC
precursor, cleaves MAVS (65), and the p65-RelA component
of the NF-?B complex is cleaved by PV 3Cpro(49). Foot-and-
mouth disease virus leader proteinase also subverts type I IFN
production (8) by cleaving p65-RelA (9). These examples il-
lustrate antagonism of upstream events in the IFN response.
Until now, no viral proteases have been implicated in blocking
the events that occur after IFN binds to cells. We have shown
here that there is an inhibitory role for 2Aproin the most
downstream event in IFN signaling, ISG activity. Which ISG
proteins inhibit PV replication and how their activities are
blocked by 2Aproremain to be elucidated. It has been reported
that PKR, an ISG induced during infection with many viruses,
is degraded in cells infected with PV (5). However, we found
no change in the amount of PKR during PV infection of HeLa
cells when the protein was examined by Western blot analysis
(J. M. Morrison and V. R. Racaniello, unpublished data).
Because there are more than 300 ISGs, identification of spe-
cific proteins whose activities are modulated during enterovi-
rus infection will require the use of large-scale proteomic or
This work was supported in part by Public Health Service grants
AI50754, AI068017, and T32AI07161 from the National Institute of
Allergy and Infectious Diseases.
We thank Bert Semler for the PV 3Cpromutant Se1-3C-02 and Ann
Palmenberg for EMCV EC4.
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