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
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4422 MORRISON AND RACANIELLOJ. VIROL.