JOURNAL OF VIROLOGY, Mar. 2006, p. 2589–2595
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 6
Age-Dependent Poliovirus Replication in the Mouse Central Nervous
System Is Determined by Internal Ribosome
Entry Site-Mediated Translation
Steven Kauder,† Sherry Kan,‡ and Vincent R. Racaniello*
Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York 10032
Received 2 June 2005/Accepted 21 December 2005
Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role
of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus
(P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant
virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not
limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in
neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to
selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2
IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used
to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates
translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated trans-
lation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES
determines the species specificity of HRV2 infection.
Human rhinoviruses (HRVs) are nonenveloped, positive-
stranded RNA viruses of the family Picornaviridae (8). Approx-
imately 100 HRV serotypes have been identified and divided
into two groups based on receptor usage. The receptor for
major group HRVs is human intracellular adhesion molecule 1
(hICAM-1), and the receptor for minor group HRVs is human
low-density lipoprotein receptor (8). HRVs replicate in the
epithelium of the human respiratory tract (1, 2, 11, 41) and are
responsible for the majority of common cold infections of
humans (36). Only humans develop clinical symptoms after
HRV infection; experimental asymptomatic infections have
been documented in chimpanzees (10) and gibbons (35). A
rodent model for infection with wild-type HRV has not been
A small-animal model of HRV infection would be useful for
elucidating the mechanisms of HRV-induced pathogenesis and
to develop therapeutic interventions. The limited host range of
most HRV serotypes (8) has hindered establishment of such a
model. Mouse cells are not permissive for the replication of
minor group serotypes, despite the ability of these viruses to
enter these cells after binding the murine homolog of human
low-density lipoprotein receptor. Two exceptions are HRV1A
(37) and a variant of HRV2 (HRV2/L) selected by passage in
mouse cells (43). The changes required for growth of HRV2/L
in mouse cells have not been identified (43). HRV2/L produces
altered P2 proteins in infected cells, suggesting that the block
to HRV2 replication in murine cells could be due to a defect
in RNA replication (29). This conclusion is supported by the
finding that HRV2/L is less sensitive than HRV2 to chemical
inhibitors of RNA replication (43). It is believed that mouse
cell lines are neither permissive nor susceptible to infection
with most major group HRVs. While the block to replication
of HRV14 and HRV16 in mouse L cells is relieved upon
synthesis of hICAM-1, HRV39 fails to replicate (17). Passage
of HRV39 in mouse cells producing hICAM-1 led to the iden-
tification of a virus, HRV39/L, that can replicate in these cells
(16). Amino acid changes in viral proteins 2B and 3A mediate
HRV39 growth in mouse cells.
HRV host range may be determined in part by translation of
the viral mRNA. The 5? ends of picornaviral mRNAs are not
linked to a 7-methylguanosine cap structure and cannot be
translated by 5? end-dependent initiation as are most cellular
mRNAs (6, 24, 25). Instead, initiation on picornaviral RNA is
mediated by the viral internal ribosome entry site (IRES), a
cis-acting RNA sequence that binds ribosomes in the absence
of an mRNA cap or free 5? end (3, 4, 7, 32, 33). It has been
suggested that the limited tropism of poliovirus, a member of
the same virus family as HRV, is determined by organ-specific
differences in IRES-mediated translation (5, 14, 42). This hy-
pothesis has been disproved by the finding that the IRES of
poliovirus and other picornaviruses mediates translation in
organs that are not permissive for virus replication (9, 20, 39).
Results obtained with a transgenic mouse model for polio-
myelitis have implicated the HRV2 IRES as a determinant of
HRV2 host range (14, 15). Poliovirus infection of mice trans-
genic for the human poliovirus receptor gene, CD155 (TgPVR
mice) leads to virus replication in neurons of the brain and
spinal cord and flaccid paralysis (23, 38). Unlike wild-type
poliovirus, a recombinant poliovirus strain with the IRES of
HRV2 does not replicate or cause disease in CD155 transgenic
* Corresponding author. Mailing address: Department of Microbi-
ology, Columbia University College of Physicians and Surgeons, 701
W. 168th St., New York, NY 10032. Phone: (212) 305-5707. Fax: (212)
305-5106. E-mail: email@example.com.
† Present address: Gladstone Institute of Virology and Immunology,
University of California, San Francisco, CA 94141-9100.
‡ Present address: The Rockefeller University, New York, NY
mice. These results were interpreted to indicate that HRV2
IRES-mediated translation is specifically blocked in the central
nervous system (CNS) and that, by extension, organ-specific
differences in IRES-mediated translation can determine viral
organ tropism. An alternative explanation is that activity of the
HRV2 IRES is blocked in all murine organs. These hypotheses
cannot be reconciled solely by the infection of mice with re-
combinant poliovirus strains, because poliovirus replication in
organs other than the CNS is blocked by the alpha/beta inter-
feron response (19).
In this study, we examined the role of the HRV2 IRES in
viral host range by analyzing HRV2 IRES-mediated transla-
tion in the context of both a bicistronic mRNA and P1/HRV2,
a recombinant poliovirus strain that is dependent on the
HRV2 IRES. Direct measurement of HRV2 IRES-mediated
translation in murine organs was achieved by quantitation of
the reporter proteins encoded by a bicistronic mRNA. HRV2
IRES-mediated translation was observed in the brain and spi-
nal cord in neonatal mice, and P1/HRV2 was found to repli-
cate in these organs in neonatal CD155 transgenic mice. In
contrast, HRV2 IRES-mediated translation was not observed
in the brain and spinal cord in adult mice, consistent with the
absence of P1/HRV2 replication in these organs. The lack of
HRV2 IRES-mediated translation in adult mice may explain
the block to HRV2 replication in this host. Neonates are gen-
erally more susceptible to infection with neurotropic viruses
(13, 26–28), and our findings suggest that IRES-mediated
translation may be a novel determinant of this trait.
MATERIALS AND METHODS
Cells, plasmids, and viruses. S3 HeLa, A549, and L20B (31) cells were grown
in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, Calif.), 10% bovine
calf serum (HyClone, Logan, Utah), and 1% penicillin-streptomycin (Invitro-
gen). Neuro-2A and SH-SY5Y cells were grown in the same medium except that
10% fetal bovine serum (Invitrogen, Carlsbad, Calif.) was used. For plaque
assays HeLa cells were grown in Dulbecco’s modified Eagle medium (Specialty
Media, Philipsburg, N.J.), 0.2% NaHCO3, 5% bovine calf serum, 1% penicillin-
streptomycin, and 0.9% Bacto-agar (Difco, Franklin Lakes, N.J.).
Plasmid pP1/HRV2, an infectious poliovirus DNA clone in which nucleotides
108 to 745 are replaced with HRV2 sequence, was created as follows. Nucleo-
tides 108 to 610 of the HRV2 genome were amplified by PCR from an infectious
HRV2 DNA clone (12) using primers SK95 (5?-CGC GAA TTC TAG AAG
TTT TTC ACA AAG-3?) and SK96 (5?-GCG GAG CTC GGT GCC AAT ATA
TAT ATT-3?), cleaved with EcoRI/SacI, and used to replace the 640-bp EcoRI/
SacI fragment from pP1/CVB3 (20).
A plasmid encoding a bicistronic mRNA with the HRV2 IRES flanked by
firefly and Renilla luciferase coding regions (Fig. 1A) was created as follows. The
HRV2 IRES was amplified by PCR from pP1/HRV2 using primers SK1 (5?-CGC
GTCGACTTAAAACAGCTCTGGGGT-3?) and SK96 (5?-GCGGAGCTCGGT
GCCAATATATATATT-3?), cleaved with SalI, blunt-ended with T4 DNA poly-
merase, cleaved with SacI, and ligated to an EcoRV/SacI-cleaved pDC516 (Mi-
crobix, Toronto, Ontario, Canada)-based bicistronic expression plasmid that has
been described previously (20). The creation of a plasmid encoding a bicistronic
mRNA with the poliovirus IRES flanked by the firefly and Renilla luciferase
coding regions has also been described previously (20).
Recombinant human adenoviruses encoding bicistronic reporter mRNA
were produced using the Admax system (Microbix, Toronto, Ontario, Canada).
Viruses encoding bicistronic mRNAs were created by recombination in 293
cells between the calcium phosphate-transfected adenovirus genome plasmid
pBHGfrt?E1,3FLP and the pDC516-based plasmids described above that en-
code bicistronic mRNA. Recovered virus was subjected to two rounds of plaque
purification as described by the manufacturer (Microbix, Toronto, Ontario, Can-
FIG. 1. IRES-mediated translation in cultured cells. (A) Schematic of bicistronic reporter DNA encoded by reporter plasmids and recombinant
adenoviruses. The arrow indicates the transcription initiation site of the murine cytomegalovirus immediate early promoter. Firefly luciferase and
Renilla luciferase have independent translation initiation and termination codons. SV40 An, simian virus 40 polyadenylation signal. The bicistronic
reporter DNA is included in bicistronic plasmids used for DNA transformation of cultured cells and in recombinant adenoviruses used for assay
of IRES-mediated initiation in mouse organs. (B) Translation mediated by the IRES of poliovirus (filled bars) or HRV2 (open bars) in the
indicated cell lines transformed with a plasmid encoding the bicistronic mRNA. To control for transformation efficiency, Renilla luciferase
expression was normalized to firefly luciferase expression. IRES activity (y axis) is the relative increase (n-fold) in Renilla luciferase translation
compared with results obtained using a plasmid that does not have an IRES. Data points are the means of three transformations, and error bars
indicate standard deviations. (C) Genome structure of poliovirus type 1 strain Mahoney (P1/M) and recombinant strain P1/HRV2. Poliovirus
polyprotein (open box), predicted AUG initiation codons, and IRES are indicated. HRV2 sequence is shaded. (D) Single-cycle replication of
poliovirus type 1 strain Mahoney (filled symbols) and P1/HRV2 (open symbols) in HeLa (square), Vero (circle), SH-SY5Y (triangle), and L20B
(diamond) cell lines. Data points are the means of two infections.
2590KAUDER ET AL.J. VIROL.
ada). Virus stocks were established by infection of 293 cells, and virus was
purified from cells by centrifugation onto a cushion of cesium chloride as de-
scribed by the manufacturer (Microbix, Toronto, Ontario, Canada).
To produce poliovirus strains P1/M and P1/HRV2, viral DNA clones pT7M
and pP1HRV2 were linearized by restriction enzyme cleavage and used as
templates for runoff transcription by T7 RNA polymerase (Promega, Madison,
Wis.). RNA was transfected into HeLa cells using DEAE-Dextran, and after 3
days intracellular virus was released by three freeze-thaw cycles and passed once
in HeLa cells. Virus was subjected to two rounds of plaque purification, and virus
stocks were produced in HeLa cells.
Poliovirus replication in cultured cells. Monolayers of adherent cells were
infected at a multiplicity of 10 PFU per cell. At different times after infection,
cells were scraped into tubes and subjected to three freeze-thaw cycles to release
intracellular virus. The virus titer in each sample was determined by plaque assay
of HeLa cell monolayers.
Assay for IRES-dependent translation in continuous cell lines. Cells at 70%
confluence in 35-mm dishes were transformed with 3 ?g of plasmids encoding
bicistronic mRNAs using Lipofectamine Plus (Invitrogen, Carlsbad, Calif.).
Growth medium was replaced after 3 h. After 24 h, cells were washed with 1 ml
of phosphate-buffered saline (PBS) and lysed in 1 ml of passive lysis buffer
(Promega, Madison, Wis.). A dual luciferase assay (Promega, Madison, Wis.)
and a Lumat LB9507 luminometer (EG&G Bertold, Oak Ridge, Tenn.) were
used to determine firefly luciferase and Renilla luciferase activity levels in lysates.
To control for variations in transformation or transcription, the ratio of firefly
luciferase activity to Renilla luciferase activity was determined and defined as
IRES activity. To control for IRES-independent Renilla luciferase translation,
the activity of each IRES was normalized to the ratio determined in lysates from
cells transfected with a plasmid lacking an IRES. The concentration of luciferase
protein was calculated with reference to a standard curve generated by using
known concentrations of recombinant firefly luciferase (Fisher Scientific, Spring-
field, N.J.) and Renilla luciferase (Chemicon, Temecula, Calif.).
Assay for IRES-dependent translation in murine organs. C57BL/6J mice (The
Jackson Laboratory, Bar Harbor, Maine) were injected (at 4 weeks or 1 to 2 days
old) as follows: intraperitoneally with 109PFU of recombinant human adeno-
virus for assays of heart, lung, liver, kidney, and ileum; intramuscularly with 5 ?
108PFU for assays of muscle; and intracerebrally with 5 ? 108PFU for assays of
brain and spinal cord. The volume of the inoculation was 50 ?l for 4-week-old
mice or 15 ?l for 1- to 2-day-old mice. Sixteen to 24 h after infection, mice were
sacrificed, and organs were removed and homogenized with a PowerGen 125
homogenizer (Fisher Scientific, Springfield, N.J.) in 0.5 ml of passive lysis buffer.
Crude protein extracts were prepared, and the dual luciferase assay and the
Lumat LB9507 luminometer were used to determine firefly luciferase and Renilla
luciferase activities in the extracts. To control for variation in adenovirus infec-
tion or transcription, the ratio of firefly luciferase activity to Renilla luciferase
activity was determined and defined as IRES activity. To control for IRES-
independent Renilla luciferase translation, the activity of each IRES was nor-
malized to the ratio determined in organs from mice infected with an adenovirus
lacking an IRES. All experimental mouse protocols adhered to Institutional
Animal Care and Use Committee guidelines and were approved by the Institu-
tional Animal Care and Use Committee of Columbia University Medical Center,
New York, N.Y.
Infection of TgPVR mice with poliovirus. TgPVR mice transgenic for the
human poliovirus receptor (38) were genotyped to ensure that they carried the
human poliovirus receptor gene. Mice lacking the CD155 gene were used as
negative controls. Tail fragments were incubated overnight at 55°C in 0.2 ml of
50 mM KCl, 10 mM Tris, pH 8.3, 2.5 mM MgCl2, 0.1 mg/ml gelatin, 0.45%NP-40,
0.45% Tween 20, and 60 ?g of proteinase K (The Jackson Laboratory, Bar
Harbor, Maine). Two microliters of the digestion product were used as a tem-
plate for PCR under standard conditions using primers 20A1C (5?-CTCACC
ACTGTACTCTAGTCTG-3?) and 20A1W (5?-AGAAGGACTCACTAGACT
CAGG-3?). A 350-bp PCR product indicated the presence of the human polio-
virus receptor gene.
To assay viral replication, the following inoculations were performed. TgPVR
mice (either 4 weeks old or 1 to 2 days old) were inoculated intraperitoneally
with 105PFU or 103PFU of poliovirus type 1 Mahoney strain in a volume of 50
?l (4-week-old mice) or 15 ?l (1- to 2-day-old mice). TgPVR mice (either 4
weeks old or 1 to 2 days old) were inoculated intraperitoneally with 2 ? 107PFU
or 5 ? 106PFU of poliovirus strain P1/HRV2 in a volume of 50 ?l (4-week-old
mice) or 15 ?l (1- to 2-day-old mice). At different times after infection, mice were
sacrificed, and organs were removed and homogenized in PBS with 0.2% bovine
calf serum with a PowerGen 125 homogenizer. Intracellular virus was released by
three freeze-thaw cycles, and cellular debris was removed by centrifugation at
16,100 ? g for 15 min at 4°C. The titer of infectious virus in the supernatant of
each sample was determined by plaque assay of HeLa cell monolayers.
Generation of virulent P1/HRV2. TgPVR mice were genotyped as described
above. Neonatal TgPVR mice were inoculated intracerebrally with 5 ? 106PFU
of P1/HRV2. Mice were sacrificed after 3 days, and brains were homogenized in
1 ml of PBS with 0.2% bovine calf serum. Intracellular virus was released by
three freeze-thaw cycles, and cellular debris was removed by centrifugation at
16,100 ? g for 15 min at 4°C. The recovered virus was amplified by infection of
HeLa cells in 35-mm dishes with 100 ?l of supernatant. This process was re-
peated three times. The titer of infectious virus in the supernatant of each brain
lysate was determined by plaque assay of HeLa cell monolayers.
To assay P1/HRV2 virulence, TgPVR mice (1 to 2 days old) were inoculated
intracerebrally with 5 ? 106PFU of virus in a volume of 15 ?l. Eleven TgPVR
mice were inoculated with P1/HRV2, 16 were inoculated in the second round of
infection, and 14 were inoculated in the third round of infection. Eight TgPVR
mice (4 weeks old) were inoculated with 2 ? 107PFU of P1/HRV2 recovered
from the second round of infection in a volume of 50 ?l. Mice were observed
daily for paralysis or death, and paralyzed mice were sacrificed immediately.
To determine the nucleotide sequence of viral genomes recovered from paralyzed
mice, total RNA was extracted from 0.2 ml of virus stock grown in HeLa cells
using Trizol (Invitrogen, Carlsbad, Calif.). First-strand cDNA was produced
using 1/20 of the RNA preparation with the Improm II reverse transcription
system (Promega, Madison, Wis.) and oligonucleotide primers SCK7, SCK14,
SCK20, or oligo(dT) anchor primer (5?/3? RACE Kit; Roche Diagnostics
GmbH, Mannheim, Germany) (Table 1). These four cDNAs were amplified
in a PCR using 5 ?l of each reverse transcription reaction and oligonucleotide
pairs SK103/SCK7, SCK6/SCK14, SCK13/SCK20, or SCK18/oligo(dT) anchor
primer (Table 1), respectively. Nucleotide sequencing reactions used oligonucleo-
tides SKC1, SCK2, SCK3, SCK4, SCK5, SCK8, SCK9, SCK10, SCK11, SCK12,
SCK15, SCK16, SCK17, SCK19, SK104, and SK105 (Table 1). The nucleotide
sequences of PCR products were determined by the Herbert Irving Comprehensive
HRV2 IRES-mediated translation in cultured cells. To de-
termine whether HRV2 IRES-directed translation might reg-
ulate viral replication in cultured cells, internal initiation me-
TABLE 1. Oligonucleotides used for sequence analysis of the
Name Sequence (5?–3?)a
aV represents A, G, and C.
VOL. 80, 2006 RHINOVIRUS IRES AND HOST RANGE2591
diated by the HRV2 IRES was quantitated in human and
murine cell lines. Human HeLa and A549 cells and murine
L20B (L cells that produce CD155) (31) and neuroblastoma
2A (N2A) cells were transformed with plasmids that produce
bicistronic mRNAs encoding firefly and Renilla luciferases sep-
arated by the HRV2 IRES (Fig. 1A). The same cell lines were
also transformed with plasmids encoding bicistronic mRNAs
possessing the poliovirus IRES. As previously demonstrated
(4), the HRV2 IRES mediates translation of the second open
reading frame of the bicistronic mRNA. In the human cell
lines tested, the HRV2 IRES confers a greater than 10-fold
increase in relative Renilla luciferase expression over a control
plasmid that lacks an IRES (Fig. 1B). In the murine cell lines,
activity of the HRV2 IRES is significant but less pronounced.
In L20B and N2A cells, presence of the IRES results in a five-
and twofold increase, respectively, in relative Renilla lucif-
erase expression compared with a control plasmid that lacks
an IRES (Fig. 1B). In all cell lines tested, the activity of the
poliovirus IRES is greater than that of the HRV2 IRES
The recombinant poliovirus strain P1/HRV2 was created to
study HRV2 IRES-mediated translation in the context of viral
replication. In the genome of this virus, the poliovirus IRES is
replaced with the cognate sequence from the HRV2 genome
(Fig. 1C). As previously demonstrated (14), the HRV2 IRES
can functionally replace the poliovirus IRES. Transfection of
HeLa cells with P1/HRV2 RNA results in generation of infec-
tious virus that produces plaques on HeLa cell monolayers
(data not shown). Single-step growth analysis of P1/HRV2 in
cell lines of primate and murine origin reveals a defect in
replication compared to poliovirus type 1, consisting of an
initial delay in virus production and decreased final yield (Fig.
1D). In murine L20B cells, the delay in virus production per-
sists at least 4 h longer than in primate cells (Fig. 1D).
Age-dependent replication of P1/HRV2 in murine CNS. P1/
HRV2 replication is defective in the murine L20B cell line
(Fig. 1D). To determine if dependence on the HRV2 IRES
causes a similar replication defect in vivo, infections were car-
ried out in TgPVR mice, which are transgenic for the human
poliovirus receptor gene CD155 (38). After intraperitoneal
inoculation, no significant rise in the titer of P1/HRV2 was
observed in the brain and spinal cord in adult TgPVR mice
(Fig. 2B). In contrast, poliovirus type 1 replicates to high titers
in the brain and spinal cord in adult TgPVR mice (Fig. 2A).
After inoculation of neonatal TgPVR mice, titers of P1/HRV2
increased 1,300-fold and 110-fold, respectively, in the brain
and spinal cord (Fig. 2D). Titers of poliovirus type 1 increased
10,000-fold and 1,000-fold, respectively, in the brain and spinal
cord in neonatal TgPVR mice (Fig. 2C). P1/HRV2 is cleared
from the brain and spinal cord in nontransgenic neonates (Fig.
2D). The human poliovirus receptor is required for increases
in P1/HRV2 titer in the neonatal brain and spinal cord, which
indicates that P1/HRV2 replicates in these organs.
Age-dependent HRV2 IRES-mediated translation in the mu-
rine CNS. To determine whether the failure of P1/HRV2 to
replicate in the brain and spinal cord in adult TgPVR mice is
caused by a block of viral protein synthesis, HRV2 IRES-
mediated translation was measured in adult and neonatal mice
by infecting mice with recombinant human adenovirus vectors
that produce bicistronic mRNAs encoding firefly and Renilla
luciferases separated by the HRV2 or poliovirus IRES (20).
Poliovirus IRES-mediated translation was observed in the
brain and spinal cord in both adult and neonatal mice (Fig. 3).
In contrast, translation mediated by the HRV2 IRES is highly
age dependent. In adult mice, HRV2 IRES-mediated trans-
lation in the brain or spinal cord was not detected (Fig. 3). In
newborn mice, HRV2 IRES-mediated translation was ob-
served in both the brain and spinal cord (Fig. 3). HRV2 IRES-
mediated translation in these neonatal organs is approximately
FIG. 2. Poliovirus replication in murine CNS. Virus titers in spinal
cord (circle) and brain (triangle) in TgPVR (filled symbols) or non-
transgenic (open symbols) mice were determined at the indicated days
postinfection. (A) Virus titers in adult TgPVR mice infected with
poliovirus type 1 strain Mahoney. (B) Virus titers in adult TgPVR mice
infected with P1/HRV2. (C) Virus titers in neonatal TgPVR mice
infected with poliovirus type 1 strain Mahoney. (D) Virus titers in
neonatal TgPVR and nontransgenic mice infected with P1/HRV2.
Data points are the geometric means of titers in organs of at least three
FIG. 3. IRES-mediated translation in brain and spinal cord in adult
and neonatal mice. Animals were infected with recombinant adeno-
viruses that produce bicistronic mRNA with the IRES of poliovirus
(PV) or HRV2 (Fig. 1A). To control for infection efficiency, Renilla
luciferase expression was normalized to firefly luciferase expression.
IRES activity (y axis) is the relative increase (n-fold) in Renilla lucif-
erase translation compared with results obtained using an adenovirus
vector that does not have an IRES. Data points are the means of
results for five mice, and error bars indicate standard deviations.
2592KAUDER ET AL.J. VIROL.
10- and 7-fold greater, respectively, than in adults. Poliovirus
IRES activity in neonatal brain and spinal cord is also higher
than in the same adult organs (six- and fivefold greater, re-
Isolation of a P1/HRV2 variant that causes disease in neo-
natal mice. In the murine model of poliomyelitis, poliovirus
infection is followed by virus replication in the CNS and the
development of flaccid paralysis (23, 38). Infection of neonatal
mice with P1/HRV2, a recombinant virus dependent on the
IRES of HRV2, is followed by increased virus titer in the CNS
(Fig. 2D), but paralysis is not observed. To determine if viru-
lence of P1/HRV2 is blocked absolutely, viral variants that
replicate more efficiently in the CNS were selected by the serial
infection of newborn mice with P1/HRV2. Passage of P1/
HRV2 in neonatal mice led to the isolation of viruses with
increased virulence. The initial stock of P1/HRV2 did not
cause paralysis in TgPVR neonates (Fig. 4A). Virus recovered
from the CNS of one of these mice caused flaccid paralysis in
19% of inoculated TgPVR neonates (Fig. 4A). After a second
passage in neonatal mice, the virus caused flaccid paralysis in
64% of inoculated TgPVR neonates (Fig. 4A). The virulence
of this virus is attenuated: the dose that causes paralysis in
64% of neonates is approximately 25,000 times the 50% lethal
dose of a virulent poliovirus strain in neonates. Furthermore,
this virus does not cause disease in adult TgPVR mice (data
not shown). The virulent virus recovered from the third round
of infection replicated to similar titers in the neonatal CNS as
P1/HRV2 (Fig. 2D and 4B). Nucleotide sequence analysis of
the genome of the third-passage virus did not reveal changes
from the P1/HRV2 sequence (data not shown).
Age-dependent HRV2 IRES-mediated translation in extra-
neural murine organs. To determine whether the age-depen-
dent block to HRV2 IRES-mediated translation is specific for
the CNS, translation was assayed in extraneural organs by
infecting mice with adenovirus vectors that encode bicistronic
mRNAs. The poliovirus IRES mediates high levels of transla-
tion in the liver and kidney in both adult and neonatal mice
(Fig. 5). HRV2 IRES-mediated translation was observed in
neonatal but not adult liver or kidney (Fig. 5). HRV2 IRES-
mediated translation in neonatal liver and kidney is approxi-
mately 14- and 16-fold greater, respectively, than in adults,
while poliovirus IRES-dependent translation in neonatal liver
and kidney is sixfold greater than in adults (Fig. 5). These
results demonstrate that HRV2 IRES-mediated translation in
the mouse is age dependent and is detected in the neonatal but
not adult brain, spinal cord, liver, and kidney.
The experiments reported here were designed to determine
whether the block to HRV2 replication in mice and in mouse
cell cultures is at the level of IRES-mediated initiation. Using
a bicistronic reporter plasmid, we observed HRV2 IRES-de-
pendent translation in cultured mouse L cells. Furthermore,
the recombinant poliovirus P1/HRV2, which is dependent on
the HRV2 IRES, replicates in L cells. The block to HRV2
replication in L cells must therefore be at a stage after viral
translation, such as RNA replication or virion assembly. The
study of HRV2 strains selected for growth in L cells should
identify the nature of the block to replication.
To determine whether the block to HRV2 replication in
mice is a consequence of restricted IRES-dependent transla-
tion, we made use of a transgenic mouse model for poliovirus
pathogenesis (38). Poliovirus type 1 replicates to high titers
and causes paralytic disease in TgPVR mice. When the polio-
virus IRES is replaced with the cognate sequence from HRV2,
the recombinant virus does not replicate or cause disease in
adult TgPVR mice. However, P1/HRV2 does replicate in the
brain and spinal cord in neonatal TgPVR mice, although pa-
ralysis does not occur. The block to replication of P1/HRV2 in
the brain and spinal cord in adult mice is associated with a
defect in HRV2 IRES-mediated translation that is relieved in
the brain and spinal cord in neonatal mice.
Our findings are compatible with the conclusions of a pre-
vious study, which demonstrated that a poliovirus recombinant
dependent on the HRV2 IRES does not replicate or cause
disease in adult CD155 transgenic mice (14). The authors of
that study asserted that attenuation of viral virulence was due
to a block to HRV2 IRES-mediated translation that is specific
FIG. 4. Virulence and replication of P1/HRV2 recovered from se-
rial infections of TgPVR mice. (A) P1/HRV2 virulence. Percentages of
mice that are not paralyzed after infection with P1/HRV2 (square),
second-passage infection (triangle), and third-passage infection (cir-
cle). (B) Titers of third-passage P1/HRV2 infection in murine brain
and spinal cord. Virus titers in spinal cord (circle) and brain (triangle)
in TgPVR (filled symbols) or nontransgenic (open symbols) mice were
determined at the indicated days postinfection. Data points are the
geometric means of titers in organs of at least three mice.
FIG. 5. IRES-mediated translation in kidney and liver in adult and
neonatal mice infected with recombinant adenoviruses. To control for
infection efficiency, Renilla luciferase expression was normalized to
firefly luciferase expression. IRES activity (y axis) is the relative in-
crease (n-fold) in Renilla luciferase translation compared with results
obtained using an adenovirus vector that does not have an IRES. Data
points are the means of results for five mice, and error bars indicate
VOL. 80, 2006 RHINOVIRUS IRES AND HOST RANGE 2593
for the CNS and concluded that organ-specific differences in
IRES activity can determine viral tropism. By directly measur-
ing translation, we have shown that HRV2 IRES-mediated
translation is defective not only in the CNS but also in kidney
and liver. This result is not surprising, for direct studies of viral
IRES-mediated translation in mice have not identified an
IRES that determines viral organ tropism or is active only in
certain organs (9, 20, 39).
Neonatal mice are more susceptible than adult mice to viral
infection, a property that has been attributed to differences in
their immune and apoptotic responses (13, 26–28). Our results
indicate that IRES activity may also be a determinant of in-
creased susceptibility of neonates to viral infection. The results
of previous studies have shown that IRES-mediated trans-
lation is greater in neonatal mice (9, 39), but no biological
consequences were attributed to this pattern. Our results in-
dicate that HRV2 IRES-mediated translation is higher in or-
gans of neonatal mice than in adults, and this activity is asso-
ciated with the increased susceptibility of neonates to virus
infection. IRES-mediated translation in neonates may be fa-
cilitated by greater expression of IRES trans-acting factors
(ITAFs), cellular proteins necessary for IRES function (3). For
example, the translation initiation by the IRES of foot-and-
mouth disease virus requires ITAF45, a protein that is pro-
duced in proliferating, nondifferentiated cells (34). The HRV2
IRES may similarly depend on an ITAF that is synthesized
during neonatal development. The translation deficiency was
also observed in liver and kidney in adult mice, suggesting that
mouse tissues are generally unable to support HRV2 IRES-
dependent initiation. P1/HRV2 did not replicate in nonneural
organs in neonatal TgPVR mice (data not shown), most likely
as a consequence of the alpha/beta interferon response that
limits poliovirus replication to the brain and spinal cord in
In many animal models of picornavirus pathogenesis, dis-
ease is closely associated with virus replication (18, 21, 30, 40).
However, replication of unpassaged P1/HRV2 in the neonatal
brain does not lead to disease, indicating that other determi-
nants may modulate picornavirus virulence. The absence of
disease is not likely a consequence of a general defect in virus
replication, because in cultured cells the growth of P1/HRV2,
although decreased, is similar to that of the neurovirulent
strain P1/HCV (20). Serial passage of P1/HRV2 in neonatal
mice led to the isolation of a virus that causes paralytic disease;
however, attempts to identify the mutations responsible for
acquisition of virulence were unsuccessful. Such mutations
may be selected against in HeLa cells and might be lost during
the amplification of recovered virus in HeLa cells. Alterna-
tively, the population of virulent virus recovered after two
serial infections in mice may be relatively small. Mutations
responsible for virulence may be identified after further en-
richment for virulent strains by additional rounds of serial
infection. The identification of such mutations should provide
information on the mechanisms by which P1/HRV2 induces
paralytic disease in mice.
This work was supported by Public Health Service grant AI50754
from the National Institute of Allergy and Infectious Diseases.
We thank Dieter Blass for his gift of a cloned HRV2 cDNA.
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