JOURNAL OF VIROLOGY, Feb. 2009, p. 1911–1919
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 4
Attenuation of Rabies Virus Replication and Virulence by Picornavirus
Internal Ribosome Entry Site Elements?
Adriane Marschalek,1Stefan Finke,1† Martin Schwemmle,2Daniel Mayer,2Bernd Heimrich,3
Lothar Stitz,4and Karl-Klaus Conzelmann1*
Max von Pettenkofer-Institute and Gene Center, Ludwig-Maximilians-University, Feodor-Lynen-Str. 25, 81377 Munich,1Department of
Virology, Institute for Medical Microbiology and Hygiene, Hermann-Herder-Strasse 11,2Institute of Anatomy and Cell Biology, Albertstr.
23, University of Freiburg,379104 Freiburg, and Institute of Immunology, Friedrich-Loeffler-Institut Tu ¨bingen,
Paul-Ehrlich-Strasse 28, 72076 Tu ¨bingen,4Germany
Received 30 September 2008/Accepted 29 November 2008
Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and
relies on the canonical 5?-end-dependent translation of capped viral mRNAs. Here, we have used internal
ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the
neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape
from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and
human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a
generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2
IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic
nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons
of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-?) promoter, and
increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2
IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-? receptor
(IFNAR?/?). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demon-
strated only in newborn IFNAR?/?mice. Translational control of individual genes is a promising strategy to
attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study
the function of IRES elements in detail.
The order Mononegavirales, also known as nonsegmented
negative-strand (NNS) RNA viruses, includes the Rhabdoviri-
dae, Paramyxoviridae, Filoviridae, and Bornaviridae families
(46). Though diverse in host range, tropism, and pathogenesis,
these viruses share a highly conserved mode of gene expression
and gene order, which is 3?-N-P-M-G-L-5? (the nucleoprotein,
phosphoprotein, matrix protein, glycoprotein, and “large”
polymerase genes, respectively) in the prototype rhabdovi-
ruses, such as rabies virus (RV; genus Lyssavirus) or vesicular
stomatitis virus (genus Vesiculovirus). The hallmarks of their
gene expression are (i) obligatory sequential transcription of
monocistronic genes from a single 3?-terminal promoter, (ii)
attenuation of transcription at conserved gene borders, giving
rise to an mRNA transcript gradient, and (iii) 5?-cap-depen-
dent translation of the mRNAs. Accordingly, the gene order
and the steepness of the transcript gradient predetermine the
level of individual mRNAs and of viral protein synthesis (for a
recent review see reference 58).
Once inside a cell, RNA synthesis of mammalian NNS RNA
viruses appears not to be restricted by host species or cell type,
invariably leading to the canonical transcript gradient, i.e.,
their 3? promoters for RNA synthesis and the polymerase (P
plus L) are always “on.” The possibilities of manipulating the
expression of individual viral genes on the level of transcription
are therefore limited. Approaches have included shifting genes
to other positions in recombinant vesicular stomatitis virus and
RV, modifying gene border sequences to alter the abundance
of downstream gene transcripts, and engineering of terminal
promoters (reviewed in references 23 and 48). However, con-
ditional control of individual genes, or of virus replication, on
the transcription level has not been feasible so far.
In contrast to NNS RNA viruses, replication of many posi-
tive-strand RNA viruses like the Picornaviridae is often re-
stricted to a limited set of host cells and organs. This appears
to be governed largely by highly structured RNA sequences in
the untranslated terminal regions (UTRs) and their interaction
with specific host cell factors and viral proteins (3). Picornavi-
rus 5?UTRs contain sequences able to form internal ribosome
entry sites (IRES) which bypass the requirement for the ca-
nonical 5?-terminal cap structure of most eukaryotic mRNAs
and can mediate internal translation initiation (4, 13, 27,
The analysis of recombinant polioviruses (PV) provided
considerable evidence for the role of 5?UTR sequences in the
host range of picornaviruses. Chimeric PV containing the
IRES part of the 5?UTR sequences of human rhinovirus type
2 (HRV2) or hepatitis C virus grew to lower titers in cells of
neuronal origin and were attenuated for neurovirulence (24,
61). This was mostly attributed to cell-type-specific inhibition
of translation initiation of the IRES elements (11, 38). How-
* Corresponding author. Mailing address: Max von Pettenkofer In-
stitute & Gene Center, Feodor-Lynen-Str. 25, D-81377 Munich, Ger-
many. Phone: 49 89 2180 76851. Fax: 49 89 2180 76899. E-mail:
† Present address: Institute for Molecular Biology, Friedrich-Loef-
fler-Institute, Su ¨dufer 10, 17493 Greifswald-Insel Riems, Germany.
?Published ahead of print on 10 December 2008.
ever, data from other studies that included measurement of
protein synthesis from bicistronic reporter genes delivered by
recombinant DNA viruses suggested that HRV2 IRES-medi-
ated translation is not much restricted in neurons and there-
fore cannot represent the limiting step of picornavirus repli-
cation and organ tropism (31). Indeed, IRES elements of other
picornaviruses, like encephalomyocarditis virus, which is
widely used in DNA vectors, viruses, and in transgenic animals
(42), or the IRES of Theilers’s murine virus (51), are func-
tional in many cell types, indicating rather universal activity.
Here, we describe the use of IRES elements from PV and
HRV2 to direct gene expression of an NNS RNA virus, the
neurotropic RV, specifically, of the phosphoprotein P, which is
essential for both RNA synthesis and for dampening the host
interferon (IFN) response (7, 8, 55, 56). A neuron-specific
deficit of the HRV2 IRES-controlled RV was not observed.
While both IRES-controlled RVs replicated reasonably well in
IFN-negative in vitro systems, they exhibited an outstandingly
high degree of attenuation in vivo, illustrating the importance
of RV P protein to control innate host responses.
MATERIALS AND METHODS
Cells and viruses. HEK 293T, HEp-2, HepG2, Vero, NIH 3T3, NS20Y, NA,
and MDBK cell lines were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal calf serum, L-glutamine, and antibiotics. MHH-
NB11 and DK-MG cells were cultured in RPMI supplemented with 10% FCS,
L-glutamine, and antibiotics. BHK-21 and BSR T7/5 cells (9) were maintained in
Glasgow minimal essential medium supplemented with 10% normal calf serum
and 10% FCS, respectively, tryptose phosphate, amino acids, and antibiotics.
Recombinant RVs were rescued from transfected cDNA and grown in BSR T7/5
cells as described previously (22).
cDNA construction. In order to generate the dual luciferase virus pSAD
RL-IRES-FL, a bicistronic plasmid, containing the Renilla luciferase (RL) and
firefly luciferase (FL) open reading frames (ORFs) derived from pCMV-RL
(Invitrogen) and pSDI-CNPL (22), respectively, was cloned first (pCMV RL-
FL). The IRES elements of PV, HRV2, and foot-and-mouth disease virus
(FMDV) were PCR amplified from cDNA of PV (strain Mahoney) and HRV2
(kindly provided by E. Wimmer) or FMDV (pVITRO2) (Invitrogen) and cloned
into pCMV RL-FL to obtain pCMV RL-IRES-FL. The RL-IRES-FL cDNA
fragment was cut out from pCMV RL-IRES-FL and cloned into the full-length
RV pSAD DsRed (32). The junction sites of the IRES and the FL ORF were as
those in pSAD IRES-P (see below).
To generate pSAD PV-P and pSAD HRV2-P, the original N/P gene border
sequence was replaced by PCR-amplified IRES sequences in the full-length
pSAD L16 by standard cloning procedures. The resulting junction sites for pSAD
PV-P were 5?-GACTCATAAgaagttgaataaca.. (PV IRES)..attgttatcATGAGCAA
G-3? and 5?-GACTCATAAgaagttgaataaca..(HRV2 IRES)..attggcaccATGAGCA
AG-3? for pSAD HRV2-P (capital letters indicate RV sequence, underlined
portions are RV N and P stop/start codons, and italics indicates the IRES).
RNA analysis. RNA from cells was isolated with the RNeasy Mini kit
(Qiagen). Northern blot assays and hybridizations with [?-32P]dCTP-labeled
cDNAs recognizing the RV N and P gene sequences, respectively, were de-
scribed previously (15).
Immunoblotting. Western blot assays were performed as described previously
(8). N and P proteins were detected by using a polyclonal rabbit serum (S50)
raised against RV ribonucleoprotein (RNP) and a fluorescently labeled second-
ary antibody (Alexa 488–anti-rabbit immunoglobulin G [IgG]). For detection of
protein bands Western blots were imaged with the Typhoon 9400 variable mode
imager (Amersham Biosciences) at 500 V, and the intensities of bands were
quantified using the software ImageQuant 5.0.
Dual luciferase assays. For analyzing IRES activities, 2.5 ? 105cells were
seeded in 24-well plates and infected with the SAD RL-IRES-FL viruses at a
multiplicity of infection (MOI) of 3. Two days postinfection (p.i.) cell extracts
were subjected to the dual luciferase assay (Promega) in a luminometer
(Berthold) according to the supplier’s and manufacturer’s instructions. For in-
terferon assays involving reporter gene plasmids carrying the IFN-? promoter
(p125-Luc) or an IFN-stimulated response element (pISRE-Luc), dual luciferase
assays were performed on HEK 293T and BSR T7/5 cells, respectively, as de-
scribed previously (8).
Organotypic slice cultures. Hippocampi were dissected from neonate mouse
pups (day 0 to 1 [P0 to P1]) and cut into 400-?m horizontal sections with a tissue
chopper. The sections were placed into petri dishes filled with cold minimal
essential medium supplemented with 2 mM glutamine at pH 7.3. Obviously
intact slices were placed onto humidified porous membranes of cell culture
inserts (CM30; Millipore Corporation) and transferred sterile into six-well plates
containing 1.2 ml of medium (for details, see reference 5). Slices were cultivated
for 4 to 8 days at 37°C with 5% CO2in a humidified atmosphere and the medium
was changed every third day. Slice cultures were infected immediately after
preparation with 1.5 ?l of virus stock, corresponding to ca. 2.4 ? 104focus-
forming units (FFU).
Immunohistochemistry. Cultures selected for immunofluorescence analysis
and 4?,6-diamidino-2-phenylindole (DAPI) nuclear staining were fixed with 4%
paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 3 h. After several
rinses with PB for 1 h the Millipore membrane with the cultures on top was cut
off, mounted on an agar block, and resliced into 50-?m sections using a vi-
bratome. Free-floating sections were then incubated in PB containing 5% normal
goat serum, permeabilized with 0.1% Triton X-100 in PB for 30 min, and further
incubated with primary antibodies (anti-calbindin, a marker for dentate granule
cells, at a dilution of 1:10,000 [SWANT]; S50, recognizing RV RNP at 1:50) in
PB containing 1% normal goat serum overnight at 4°C. After five washes for 15
min each with PB, sections were incubated with secondary antibodies (Cy3-
conjugated goat anti-rabbit IgG, diluted 1:800, and Alexa 488-conjugated goat
anti-mouse IgG, diluted 1:200, respectively) for 2 h at room temperature in the
dark. Sections were extensively washed with PB, followed by DAPI staining for
2 min (dilution, 1:106), washed again in PB, and mounted onto gelatin-coated
slides and embedded with immunomount (Shandon). Sections were digitally
photographed (Zeiss ApoTome).
Mouse infection experiments. Wild-type (wt) and transgenic IFNAR?/?mice
were originally obtained from M. Aguet, Zurich, Switzerland, and kept in the
specific-pathogen-free facility at the Institute of Immunology, Friedrich-Loef-
fler-Institute, Tu ¨bingen, Germany. Age- and sex-matched adult wt or IFNAR?/?
mice (41) were infected intracerebrally (i.c.) into the left hemisphere with up to
105FFU in 20 ?l, and newborn mice received 10 ?l. The animals were observed
daily three times and scored for the appearance of neurological signs on an
arbitrary scale of 1 to 3 (level 1 for slight neurological signs, such as beginning
ataxia and slightly reduced motility; level 2 for increased neurological signs, such
as trembling and/or disorientation after tail spinning; level 3 for severe signs of
disease, such as ruffled fur, hunched position, and inability to move). Animals
scored twice at level 3 or at level 2 at noon and level 3 in the afternoon were
immediately sacrificed according to the German Animal Protection law and
serum and organs were preserved. The number of mice and experimental design
were approved by local authorities (Regional Board; permission no. FLI 223/05;
IRES-dependent reporter gene activity in cell lines. The
study of IRES translation initiation activity typically makes use
of bicistronic mRNAs in which an upstream reporter ORF is
translated in a 5?-cap-dependent manner and the downstream
ORF in an IRES-dependent manner. We therefore con-
structed recombinant RVs (SAD RL-IRES-FL) transcribing
an extra gene comprising the RL ORF, followed by an IRES
element derived from PV, HRV2, or FMDV, and the FL ORF.
Transcription of the extra gene was under the control of a copy
of the N/P gene border signals (Fig. 1). The respective recom-
binant viruses could be rescued from cDNA by standard trans-
fection experiments (22). The IRES-containing recombinant
viruses showed growth characteristics similar to those of the
parental RV SAD L16 and to a virus transcribing an extra
monocistronic gene downstream of G (SAD G eGFP) and
yielded maximal infectious titers of 1 ? 108to 2 ? 108FFU/ml
at 48 h p.i. (Fig. 2A).
To measure IRES-mediated translation of FL, dual lucifer-
ase assays were performed on a variety of cell lines infected
1912MARSCHALEK ET AL.J. VIROL.
with the recombinant RVs by comparing the activities of FL
and RL (Fig. 2B). The total RL activities varied from 6 ? 106
(mouse 3T3 cells) to 8 ? 108RLU (BSR T7/5 cells), reflecting
the capacity of RV to replicate in the diverse cell lines (data
not shown). As a standard for IRES-dependent FL activity, the
PV IRES was chosen (100%), since this IRES showed an
overall high activity and low variability. Of particular interest
was the comparison with the HRV2 IRES, because of its pro-
posed neuronal cell-specific restrictions. While the HRV2
IRES was less active than the PV IRES in all cells tested,
specific defects in neuronal cells were not confirmed. Rather,
the HRV2 IRES was noticeably active in cells of neuronal
origin, such as human HEK 293T or the mouse NA cells (30%
and 41% of PV, respectively) (Fig. 2B). In human MHH-
NB11and mouse NS20Y cells both IRES elements performed
poorly in absolute values but in comparison to PV, the HRV2
IRES still showed 35% and 33% activity, respectively (Fig. 2B).
In nonneuronal cells HRV2 IRES activity showed a broad
range of 4% to 56% of the PV IRES, with no species- or
organ-specific influence detectable. Thus, these data did not
support the reported specific restriction of HRV2 IRES trans-
lation initiation activity in neuronal cell types (11, 38); rather,
they indicated a general intrinsic low activity. In most cell lines
tested, the FMDV IRES showed the least activity with the
constructs used here.
IRES-controlled expression of RV phosphoprotein. To con-
trol expression of RV genes and to render replication of RV
dependent on the specific translation activity of an IRES, we
further used the IRES elements of PV and HRV2. The viral P
protein was considered the most indicative target, as RV P is
an essential cofactor of the viral polymerase (L) and acts as a
chaperone for encapsidation of viral RNA (for reviews see
references 14 and 58). Attenuation of P translation should
therefore result in attenuation of viral mRNA synthesis and of
virus replication. Moreover, RV P potently counteracts type I
IFN induction by targeting IRF3 and IRF7 phosphorylation
(7) and JAK/STAT signaling of type I and type II IFNs by
preventing access of phosphorylated STATs to the nucleus (8,
55, 56), and it thereby strongly interferes with the establish-
FIG. 1. Recombinant RV genomes. The genome of wt RV SAD
L16 comprises five cistrons defined by transcription stop/restart signals
(arrowheads) that give rise to five capped mRNAs as indicated. The
IRES reporter virus SAD RL-IRES-FL contains an extra gene from
which a bicistronic mRNA encoding RL and FL is transcribed. In SAD
HRV2-P and SAD PV-P, here represented by the genome of SAD
IRES-P, the transcription signal sequences of the N/P gene border are
replaced by the IRES elements of HRV2 and PV, such that P protein
expression depends on internal translation initiation by the IRES el-
FIG. 2. RV encoding bicistronic RL-IRES-FL reporter genes.
(A) Growth of IRES reporter RVs in BSR T7/5 cells. Cells were
infected with the indicated viruses at an MOI of 0.1, and infectious
supernatant virus titers were determined at the indicated time points.
As a control a virus containing an additional monocistronic gene
downstream of G (SAD G GFP) was used for comparison. (B) Cell
lines from nonprimate (upper panel) and primate species (lower
panel), including cells of neuronal origin (underlined) were infected
with the recombinant SAD RL-IRES-FL reporter viruses containing
the IRES of PV Mahoney (light gray), HRV type 2 (black), or FMDV
(dark gray). At 48 h p.i. RL and FL activities were measured using the
dual luciferase reporter system (Promega). The ratio of activity of FL
to RL with SAD RL-PV-FL was set as 100%. Data for every cell line
are from at least two independent experiments, each of which included
three parallel infections. Error bars indicate standard deviations. P ?
0.001 for all cells and viruses.
VOL. 83, 2009 ATTENUATION OF RABIES VIRUS BY IRES ELEMENTS1913
ment of an antiviral state. Reduction of P expression by limit-
ing its translation should therefore also increase the magnitude
of the host IFN response in IFN-competent systems and di-
minish the resistance of RV to IFN.
RV cDNAs were constructed in which the authentic N/P
gene border comprising the transcriptional stop/restart signal
sequences was replaced with the PV or HRV2 IRES (Fig. 1).
In order to obtain viable virus, P must be translated from the
bicistronic mRNA in an IRES-dependent manner. Recombi-
nant viable viruses SAD PV-P and SAD HRV2-P were rescued
by standard procedures, indicating that P was synthesized at
levels sufficient to support autonomous virus replication. Tran-
scription of bicistronic N/P mRNAs from the recombinant
RVs was confirmed in Northern blotting experiments. In both
cases, an RNA species of 3 kb comprising N and P sequences
was present in infected cells, whereas the typical monocistronic
N and P mRNAs of wt RV were not detectable (Fig. 3A). P
protein was readily detected by Western blotting in all virus-
infected cell cultures; however, it accumulated to discrete lev-
els (Fig. 3B). As determined by fluorescence imaging and com-
pared to wt SAD L16, which expresses P from a monocistronic,
capped mRNA (100%), P accumulated to 56% in SAD PV-P-
infected cells, followed by SAD HRV2-P with 34% of P at 48 h
p.i. (Fig. 3B). This confirmed the previously observed lower
reporter activity of the HRV2 IRES. Notably, the reduced
levels of P did not greatly affect overall virus gene expression,
as the reduction of other virus protein levels was less pro-
nounced (Fig. 3B, results with N). Growth curves in BSR T7/5
cells, which do not produce type I IFN in response to virus
infection (8, 14), revealed 10- and 20-fold reductions of peak
infectious virus titers for SAD PV-P and SAD HRV2-P, re-
spectively (Fig. 4A). These results corroborate the correlation
of specific IRES translational activity, levels of P protein, and
virus RNA synthesis in an IFN-deficient system. To examine
the growth of the viruses in an IFN-competent cell line, mouse
NA cells were infected in parallel. Indeed, a further reduction
of greater than 1 log10for both SAD PV-P and SAD HRV-P
was observed, indicating a further attenuation of virus growth
by the IFN system of the host cells.
Diminished IFN antagonism of IRES-P RV. Reduced P lev-
els should not only limit the capacity of RV to counteract
transcriptional induction of IFN but also increase the sensitiv-
ity of RV to exogenous IFN (7, 8). Reporter gene assays in
which FL is expressed under the control of the IFN-? pro-
moter from transfected p125-Luc in HEK 293T cells revealed
a markedly increased FL activity in cells infected with the
IRES-P viruses compared to wt RV-infected cells (Fig. 5A).
However, compared to previously described SAD ?PLP (7),
which expresses only trace amounts of P and which is therefore
a potent IFN inducer, FL activity was moderate. This indicates
a P dose-dependent deficiency in the capacity of the recombi-
nant RV to prevent activation of the IFN-? promoter. The
ability to counteract JAK/STAT signaling was examined in
IFN-incompetent BSR T7/5 cells to exclude effects of feedback
by endogenous IFN. In cells treated with recombinant IFN
A/D wt RV SAD L16 almost completely prevented FL expres-
sion from pISRE-Luc, whereas SAD HRV2-P and SAD PV-P
could restrain FL expression only to some extent (Fig. 5B).
Thus, IRES-mediated translational attenuation of P also limits
the virus’ ability to counteract the cellular IFN response.
FIG. 3. IRES-dependent expression of RV P. (A) BSR T7/5 cells
were infected with the indicated RVs, and total RNA was isolated 48 h
p.i. and analyzed by Northern blotting. Only bicistronic N/P mRNAs
are apparent after substitution of the RV N/P gene border with PV or
HRV2 IRES sequences. (B) Quantification of P protein levels by
fluorescence imaging. The Western blot shows expression of N and P
proteins in extracts from BSR T7/5 cells 48 h postinfection with the
indicated viruses. Numbers below the lanes indicate relative levels of
RV P after normalization with RV N and in comparison with P levels
of SAD L16 (100%).
FIG. 4. Growth of IRES-controlled RV in cell culture. IFN-incompetent BSR T7/5 cells (A) and IFN-competent NA cells (B) were infected
with the indicated viruses at an MOI of 0.1, and infectious supernatant virus titers were determined at the indicated time points. Titers for every
time point are from at least three independent experiments. Error bars indicate standard deviations.
1914 MARSCHALEK ET AL.J. VIROL.
Replication of IRES-controlled RVs in brain slice cultures.
To characterize growth and tropism of the IRES-controlled
RVs in relevant primary neuronal cell networks, organotypic
hippocampal slice cultures (34) were infected immediately af-
ter explantation with 2.4 ? 104FFU of recombinant RV. At 8
days p.i., slices of a SAD HRV2-P-infected or a SAD PV-P-
infected organ culture showed a normal cytoarchitecture (Fig.
6A, left panels). In contrast, a significant part of the SAD
PV-P-infected and the majority of wt SAD L16-infected cul-
tures could not be further analyzed due to intense tissue dam-
age (Fig. 6, graphs). At earlier time points (5 days p.i.) the
extent of tissue damage in SAD L16-infected cultures was
comparable, whereas most SAD PV-P- or SAD HRV2-P-in-
fected cultures showed no or only mild tissue damage. At 3 to
4 days p.i. only sparse viral antigen signals could be detected in
SAD HRV2-P-infected slices and slightly increased staining in
SAD PV-P-infected slices (Fig. 6B). In contrast, RV antigen
immunolabeling was prominent in SAD L16-infected cultured
hippocampi (Fig. 6B), suggesting that the observed tissue dam-
age at later time points of infection correlates with the growth
efficiency of these RVs.
In view of the reported nonpermissiveness of neurons for
HRV and for PV carrying the HRV2 IRES (10, 24, 37, 38), we
further analyzed the distribution of SAD HRV2-P and SAD
PV-P in the hippocampal slices. No obvious differences were
observed at 8 days p.i. (Fig. 6C). Independent of the virus used,
labeling of viral antigen often displayed a punctuate staining,
indicating virus-containing axonal elements (Fig. 6C). Some
calbindin-positive granule cell somata were also found to be
positive for virus antigen (Fig. 6C). Specific translational de-
fects of the HRV2 IRES in neurons were thus not apparent,
and therefore a limited tropism for neurons did not contribute
to the observed attenuation of SAD HRV2-P relative to SAD
PV-P. Rather, a generally lower translational activity of the
HRV2 IRES appears to be the basis of attenuation.
Severe attenuation of IRES-dependent RVs in vivo. To ex-
plore pathogenicity of viruses in which P translation and RV
replication are under IRES control, 3-week-old mice were
inoculated i.c. at doses ranging from 1 ? 102to 1 ? 105
FFU/mouse. The wt SAD L16 virus was lethal at all doses
within 11 days of incubation, with all animals showing similar
symptoms of pathogenicity, with ruffled fur and hunched back.
In striking contrast, both SAD HRV2-P and SAD PV-P were
completely nonpathogenic (Fig. 7A). Surprisingly, even
IFNAR?/?mice did not succumb to rabies after i.c. injection
of either SAD PV-P or SAD HRV2-P (Fig. 7C). Only in
newborn mice were these viruses able to cause disease. SAD
PV-P was lethal within 15 days, whereas all mice survived after
i.c. injection of SAD HRV2-P, indicating a more profound
attenuation of the latter (Fig. 7B). The attenuated phenotype
of SAD HRV2-P was still evident in newborn IFNAR?/?mice.
Although all three viruses were able to kill newborn
IFNAR?/?mice, differences in the time course of disease were
apparent. SAD L16 killed mice within 4 days, while mice in-
fected with SAD PV-P succumbed to rabies 8 days p.i. and
SAD HRV2-P-infected mice died only at 12 days p.i. (Fig. 7D).
Thus, as observed for virus replication in vitro, attenuation of
IRES-controlled RV in vivo was dependent on the degree of
IRES translation initiation.
Gene expression of NNS RNA viruses is regulated almost
exclusively on the transcriptional level (14, 58). We have here
mapped out a strategy to make the expression of an essential
viral protein dependent on translation, rather than transcrip-
tion, by replacing the canonical transcription signals of NNS
RNA viruses (specifically, the RV N/P gene border) with trans-
lation-active signals from positive-strand RNA viruses. It turns
out that RV is a particularly suitable heterologous viral system
to both explore and exploit translation initiation of IRES ele-
ments. As RV is an RNA virus that replicates in the cytoplasm,
the approach does not suffer from problems encountered with
DNA-based assays, such as the presence of cryptic promoters
in IRES DNA or from RNA splicing in the nucleus, which
obviously accounts for the ostensible IRES activity of many
cellular sequences (2, 28, 54). The broad host species and cell
range of RV in vitro and the lack of host cell shut down further
facilitate the investigation of IRES translation initiation in a
relatively “unbiased” cell.
The use of picornavirus IRES elements in this approach
appears promising in view of their relative high efficiencies of
internal translation initiation, and also possible cell-type-spe-
cific restrictions, which are attributed to differential use of
IRES trans-acting factors (25, 37, 38). However, IRES ele-
ments are an integral part of the terminal UTR structures that
are required for picornavirus replication (3, 6), and the role of
IRES trans-acting factors specifying the picornavirus host
range may therefore be beyond translation initiation (33, 47).
In contrast to picornaviruses and other positive-strand RNA
viruses (43, 57), the IRES elements in the RV replication
template and products are cotranscriptionally encapsidated in a
FIG. 5. Viral defects in counteracting IFN-? induction and JAK/
STAT signaling. (A) HEK 293T cells were transfected with the IFN-?
promoter-controlled plasmid p125-Luc and infected at an MOI of 3.
FL expression was determined using the dual luciferase reporter sys-
tem (Promega) at 24 h p.i. Both IRES-controlled RVs activate the
IFN-? promoter more efficiently than wt RV SAD L16. SAD ?PLP is
a gene shift control virus expressing trace amounts of P (7). (B) Inhi-
bition of IFN-stimulated gene expression by the indicated RV was
analyzed in IFN-negative BSR T7/5 cells. Cells infected at an MOI of
1 and transfected 6 h later with the ISRE promoter-controlled reporter
plasmid pISRE-Luc and pCMV-RL for normalization were treated
24 h p.i. with the indicated doses of IFN-? A/D. FL and RL activities
were determined 48 h p.i. Only wt RV SAD L16 was able to almost
completely abolish FL expression. Error bars indicate standard devi-
ations from at least three parallel experiments.
VOL. 83, 2009ATTENUATION OF RABIES VIRUS BY IRES ELEMENTS 1915
FIG. 6. Replication of IRES-controlled RVs in organotypic hippocampal slice cultures. Hippocampal slice cultures from newborn mice
(C57/BL6) were infected with 2 ? 104FFU of SAD HRV2-P, SAD PV-P, or SAD L16. After the indicated time periods, slices were fixed and
50-?m sections were prepared and immunostained for the RV N protein (green) or calbindin (red), a marker protein of granule cells of the dentate
gyrus. To visualize the cell nucleus and to evaluate tissue condition, the slices were counterstained with DAPI (blue). (A) Impact of RV infection
on hippocampus damage. Left panels: survey of a DAPI-stained hippocampal culture infected with SAD HRV2-P and SAD PV-P for 8 days,
showing intact cytoarchitecture similar to the uninfected control culture. Right graph: the extent of tissue damage induced by the different virus
strains 8 days p.i. based on DAPI staining. (-), severe; (?/?), partial; (?), no loss of hippocampal organization. n ? 16/group (the number of
1916 MARSCHALEK ET AL.J. VIROL.
tight RNP (1) and cannot form secondary structures. Thus, dis-
section of which IRES interactions and functions are important
the RV context.
The newly established dual luciferase reporter gene of RV,
SAD RL-IRES-FL, is particularly well-suited to rapidly com-
pare translation initiation activities of different IRES elements.
Both expression of luciferase in the reporter system and ex-
pression of P in the IRES-controlled RVs demonstrated a
universally high activity of the PV IRES element and a lower
activity (approximately one-third) of the HRV2 IRES. In nei-
ther RV system could a neuron-specific restriction of HRV2
IRES translation activity be observed. Particularly in HEK 293
cells, which are of neuronal origin (50), and in which chimeric
PV containing the HRV2 IRES are severely attenuated (10), a
quite high translational activity of the HRV2 IRES was ob-
served. While general organ- or species-specific preferences
(30, 31) were not observed for either IRES, it will be interest-
ing to determine the reason for the striking differences be-
tween PV and HRV2 IRES in individual cell lines, such as
As replication of RV is not affected by the presence per se of
the IRES elements in the viral genome, as evidenced by the
lack of attenuation of the reporter gene viruses (Fig. 2A),
reduced levels of P are responsible for the attenuation of SAD
PV-P and SAD HRV-P. Compared to 5?-cap-dependent trans-
lation from the standard RV monocistronic P mRNA, IRES-
dependent accumulation of P was reduced in infected cells, but
not by orders of magnitude. Yet, growth in the IFN-incompe-
tent BSR T7/5 cells of the PV IRES-controlled virus was de-
layed, and that of the HRV2 IRES-controlled virus even mo-
reso, indicating that virus RNA transcription and replication
are limited by the available P levels. In addition, low amounts
of P were limiting for the capacity of the viruses to counteract
transcriptional induction of the IFN-? promoter and to combat
Although the intriguing conception of neuron-specific inhi-
bition of RV replication by using the HRV2 IRES was appar-
ently not achievable, a strikingly high degree of attenuation
was observed not only of the HRV2 IRES-controlled RV but
also of SAD PV-P, as was obvious from a complete lack of
mortality after i.c. injection even in mice lacking a functional
IFNAR. A significant contribution of IFN signaling to the
antiviral host defense in response to wt RV was evidenced in
the experiments in which IFNAR?/?mice succumbed to wt
SAD L16 rabies much more rapidly than wt mice. However,
IFN feedback signaling in adult mice was obviously not neces-
sary to defeat the IRES-controlled RV, as evidenced by sur-
vival of the 3-week-old IFNAR?/?mice (Fig. 7B). In contrast
to wt RV, those RVs are not able to prevent activation of IRF3
and transcription of IFN-? (Fig. 5). The increased cell-auton-
omous IRF3-mediated host response appears to be potent
enough to defeat the viruses in the absence of IFN feedback.
Wild-type RV infection is detected in the cytosol predomi-
nantly by the pattern receptor RIG-I, whose regulatory domain
binds to viral triphosphate RNAs (16, 26), whereas infection
with the picornavirus encephalomyocarditis virus is sensed by
the related RNA helicase MDA5 (29), but the exact picorna-
virus signatures activating MDA5 are not yet defined. Al-
though it is likely that the observed increase in IFN-? induc-
tion by SAD PV-P and SAD HRV2-P is due to limiting
amounts of P and inefficient preclusion of RIG-I-like receptor
signaling, some contribution of the extra picornavirus IRES
sequences to recognition of the viruses by MDA5 cannot be
Only in the absence of a fully developed immune system, in
newborn mice, did the SAD PV-P and SAD HRV2-P cause
disease, and this revealed the predicted differences in the de-
gree of attenuation. In this setting, the IFN feedback by
IFNAR appeared to be critical, as IFNAR?/?mice were killed
by SAD HRV2-P, though for most animals a delay was ob-
served in comparison with SAD PV-P and SAD L16 (Fig. 7).
organotypic slice cultures investigated for each virus). (B) Virus spread of SAD HRV2-P, SAD PV-P, or SAD L16 in hippocampal slice cultures
3 to 4 days p.i. Note that cultures infected with SAD HRV2-P or SAD PV-P showed decreased viral spread. (C) Distribution of RV N antigen
in hippocampal cultures. Left panel: SAD HRV2-P-infected slices show typical organization. A calbindin-positive granule cell layer and the mossy
fiber projection are formed. At higher magnification few viral antigen signals are found on axonal profiles (arrowheads) and in calbindin-labeled
granule cell somata (arrow). Middle panel: in SAD PV-P-infected cultures the cellular organization is maintained. At higher magnification many
viral antigen signals are observed with a similar localization as in SAD HRV2-P-infected cultures. Right panel: cultures infected with SAD L16
are disorganized and only a few calbindin-positive granule cells are preserved. These cells also contain viral antigen. CA, cornu ammonis; dg,
dentate gyrus; gc, granule cells; mf, mossy fiber projection; pcl, pyramidal cell layer. Bars, 200 ?m (upper panels), 100 ?m (middle panel, SAD
HRV2-P and SAD PV-P), 50 ?m (middle panel, SAD L16), or 15 ?m (lower panels).
FIG. 7. Survival of mice after i.c. infection with recombinant RVs.
Three-week-old wt (A) or IFNAR?/?mice (C) were infected i.c. with
1 ? 105FFU/mouse, and newborn wt (B) or IFNAR?/?(D) mice were
infected with 100 FFU/mouse of the indicated viruses. In adult wt and
IFNAR?/?mice both SAD PV-P and SAD HRV2-P were completely
nonpathogenic (A and C), in contrast to wt RV SAD L16. In newborn
wt mice (B), SAD HRV2-P was still completely attenuated, whereas
SAD PV-P caused mortality, although with a delay compared to SAD
L16. Only newborn IFNAR?/?mice lacking IFNAR (D) succumbed to
VOL. 83, 2009 ATTENUATION OF RABIES VIRUS BY IRES ELEMENTS1917
The deficiency of young mice to combat infection is predom-
inantly attributed to the immature status of the adaptive im-
mune system, but a deficient innate immune response has also
been observed (17), which may explain the need for IFN feed-
back for protection against SAD HRV2-P. Age-dependent at-
tenuation in mice has also been observed for picornaviruses
(30, 51). It remains to be determined whether this relates to
the maturation of the immune system or to differences in IRES
The results provided here demonstrate a direct positive cor-
relation of IRES translation activity, accumulation of P pro-
tein, RV replication, and the capability of recombinant RV to
prevent IFN-mediated antiviral host responses. Thus, gene ex-
pression, replication, and pathogenicity of NNS RNA viruses
can be controlled by IRES elements from positive-strand RNA
viruses. As RV P is essential for viral gene expression, deletion
of the P gene leads to single-cycle viruses (21), which are thus
nonpathogenic for mice (12, 40). Replication-competent RV
attenuated in vivo have been generated previously by modifi-
cation of the G protein, which is responsible for the neuroin-
vasiveness of RV (20, 32, 39) and required for the spread
between neurons (19, 59, 60). In particular, an arginine residue
(R333) of the G has been shown to be responsible for RV
virulence for adult mice (18, 49, 53). Thus, attenuating 333
mutants are often incorporated into “second-generation” live
RV vectors considered as a basis for heterologous vaccines,
particularly against human immunodeficiency virus type 1 (35).
In addition, combinations of 333 variants with other mutations,
such as a deletion of an 11-aa stretch representing the LC8
binding site of the P protein, have been used to further atten-
uate the viruses (36). The latter modification may further at-
tenuate RV transcription in brain cells (52) while it does not
affect the ability of P to counteract IFN (unpublished results).
While these RVs are avirulent for adult mice, they still kill
suckling or newborn mice after i.c. inoculation (35, 36). To our
knowledge, the present HRV2 IRES-controlled RV is the first
example of a fully replication-competent recombinant RV that
has lost its pathogenic potential even after i.c. injection in
newborn mice, illustrating that the IFN antagonistic function
of P is a major virulence factor. The strategy of reducing
expression of virulence genes by the use of IRES elements is
particularly promising for development of safe live RV vac-
cines and vectors. Since not only RV but also many other
members of the Mononegavirales order, including emerging
viruses, have a propensity to grow in neurons and to damage
neuronal tissue, we also envisage the use of modified or other
IRES elements to further restrict replication of neurotropic
viruses specifically in neurons.
We thank Nadin Hagendorf and Uli Wulle for perfect technical
assistance and Geoffrey Chase for critical reading of the manuscript.
IRES cDNAs were kindly provided by E. Wimmer.
This work was supported by the DFG through SFB 455 (K.K.C.),
HE1520 (B.H.), and SCHW 632/10 (M.S.).
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