Dengue virus utilizes a novel strategy for translation initiation when cap-dependent translation is inhibited.
ABSTRACT Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus (DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5' m7GpppN-capped positive-sense RNA genome with a nonpolyadenylated 3' untranslated region (UTR) that has been presumed to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-dependent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular translation. We further show that under cellular conditions in which translation factors are limiting, DEN can alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal ribosome entry site but requires the interaction of the DEN 5' and 3' UTRs for activity, suggesting a novel strategy for translation of animal viruses.
- [show abstract] [hide abstract]
ABSTRACT: We have shown previously that polypyrimidine tract binding protein 1 (PTB) binds and activates the Apaf-1 internal ribosome entry segment (IRES) when the protein upstream of N-ras (unr) is prebound. Here we show that the Apaf-1 IRES is highly active in neuronal-derived cell lines due to the presence of the neuronal-enhanced version of PTB, nPTB. The unr and PTB/nPTB binding sites have been located on the Apaf-1 IRES RNA, and a structural model for the IRES bound to these proteins has been derived. The ribosome landing site has been located to a single-stranded region, and this is generated by the binding of the nPTB and unr to the RNA. These data suggest that unr and nPTB act as RNA chaperones by changing the structure of the IRES into one that permits translation initiation.Molecular Cell 04/2003; 11(3):757-71. · 15.28 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In vitro replication of dengue virus requires the presence of cis-acting elements within the 5' end and the 3' UTR of the viral genome. Some, like the putative cyclization sites (PCS), may promote interaction at both ends of the viral genome. To investigate whether viral or cellular proteins could be involved in this interaction, UV-induced cross-linking assays using extracts from the monocytic cell line U937 were performed. Our data demonstrate that the 5' end and the 3' UTR with the PCS interact with five cellular proteins with the same molecular weight. When both regions were differentially labeled, with biotin and 32P, respectively, the interaction of at least seven proteins with both ends could be demonstrated. Immunoprecipitation assays also demonstrate that La protein binds to the 5' end and with the 3' UTR. Moreover, these proteins also interact with the nonstructural proteins NS5 and NS3. The role of the NS5-La and NS3-La interaction in U937 cells remains to be established.Virus Research 07/2004; 102(2):141-50. · 2.75 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Unlike other positive-stranded RNA viruses that use either a 5'-cap structure or an internal ribosome entry site to direct translation of their messenger RNA, calicivirus translation is dependent on the presence of a protein covalently linked to the 5' end of the viral genome (VPg). We have shown a direct interaction of the calicivirus VPg with the cap-binding protein eIF 4 E. This interaction is required for calicivirus mRNA translation, as sequestration of eIF 4 E by 4 E-BP 1 inhibits translation. Functional analysis has shown that VPg does not interfere with the interaction between eIF 4 E and the cap structure or 4 E-BP 1, suggesting that VPg binds to eIF 4 E at a different site from both cap and 4 E-BP 1. This work lends support to the idea that calicivirus VPg acts as a novel 'cap substitute' during initiation of translation on virus mRNA.EMBO Reports 11/2005; 6(10):968-72. · 7.19 Impact Factor
JOURNAL OF VIROLOGY, Mar. 2006, p. 2976–2986
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 80, No. 6
Dengue Virus Utilizes a Novel Strategy for Translation Initiation
When Cap-Dependent Translation Is Inhibited
Dianna Edgil, Charlotta Polacek, and Eva Harris*
Division of Infectious Diseases, School of Public Health, University of California, Berkeley, Berkeley, California 94720-7360
Received 7 June 2005/Accepted 21 December 2005
Viruses have developed numerous mechanisms to usurp the host cell translation apparatus. Dengue virus
(DEN) and other flaviviruses, such as West Nile and yellow fever viruses, contain a 5? m7GpppN-capped
positive-sense RNA genome with a nonpolyadenylated 3? untranslated region (UTR) that has been presumed
to undergo translation in a cap-dependent manner. However, the means by which the DEN genome is
translated effectively in the presence of capped, polyadenylated cellular mRNAs is unknown. This report
demonstrates that DEN replication and translation are not affected under conditions that inhibit cap-depen-
dent translation by targeting the cap-binding protein eukaryotic initiation factor 4E, a key regulator of cellular
translation. We further show that under cellular conditions in which translation factors are limiting, DEN can
alternate between canonical cap-dependent translation initiation and a noncanonical mechanism that appears
not to require a functional m7G cap. This DEN noncanonical translation is not mediated by an internal
ribosome entry site but requires the interaction of the DEN 5? and 3? UTRs for activity, suggesting a novel
strategy for translation of animal viruses.
Protein synthesis consists of an intricate series of events
requiring components that are too numerous to be encoded by
viral genomes. Therefore, viruses depend on the host cell
translation machinery for the production of viral proteins and,
as a result, have developed novel mechanisms to compete with
cellular mRNAs for limiting translation factors. Specifically,
many viral RNAs are able to bypass dependency upon an
m7GpppN cap structure for translation initiation. During cap-
dependent translation initiation, the eukaryotic initiation fac-
tor 4E (eIF4E), a component of the cap-binding complex
eIF4F, recognizes an m7GpppN cap structure at the 5? end of
viral and cellular mRNAs. The eIF4F cap-binding complex
consists of eIF4E, an adaptor protein (eIF4G), and a helicase
complex (eIF4A plus cofactor eIF4B). Only when bound to
the cap structure can the eIF4F complex recruit the ribosome
complex to the mRNA (25). For animal viruses and a number
of eukaryotic mRNAs, cap-independent translation initiation
is achieved through the use of an internal ribosome entry site
(IRES) (21), examples of which are found in the 5? untrans-
lated region (UTR) of the genomes of picorna- and hepacivi-
ruses (50). Cap-independent translation initiation in plant vi-
ruses that lack an IRES structure does exist and involves the
use of structures located in the viral 5? or 3? UTR (51). For
instance, RNA structures in the 3? UTR of the Luteovirus
barley yellow dwarf virus interact with eIF4F and presumably
deliver them to the viral 5? UTR via long-range base pairing
(31, 51), and eIF4F binds to the 3? UTR of the Tombusvirus
satellite tobacco mosaic virus to facilitate cap-independent
translation (23). In addition, RNA-RNA interactions between
the 5? and 3? UTRs of certain Necrovirus and Dianthovirus
RNAs also mediate cap-independent translation (47, 49, 57).
Despite the multiple mechanisms of cap-independent transla-
tion initiation found in plant virus families, the IRES is the
only form of cap-independent translation that has been de-
scribed for animal viruses.
In response to cellular stresses, such as nutrient deprivation,
heat shock, and viral infection, and to normal cellular pro-
cesses, such as mitosis and differentiation, eukaryotic cells can
precisely and reversibly modify the activity of the translation
machinery. A key point of control is the availability of eIF4E
(26). One important method of regulating eIF4F binding to
capped mRNAs is via sequestration of eIF4E from the cap-
binding complex by the hypophosphorylated form of the
eIF4E-binding proteins (4E-BPs) (25). This interaction, in
turn, modulates the level of cap-dependent translation.
During short periods of inhibition of cellular cap-dependent
translation, such as apoptosis or entry into mitosis, several
cellular transcripts (e.g., ornithine decarboxylase, cellular in-
hibitors of apoptosis, and p34Cdc2-related protein kinases)
have been shown to undergo a switch from cap-dependent to
cap-independent translation (10, 54, 60). Similarly, some viral
mRNAs have been shown to switch to more efficient forms of
translation initiation during certain stages of the viral life cycle
or when cellular translation factors are limiting (often due to
inactivation by viral proteases). For example, translation from
the picornavirus IRES has been shown to be upregulated in
certain cell types (6, 18). One cap-dependent mechanism by
which viral translation is initiated more efficiently than eukary-
otic mRNA is through shunting of the ribosome. Viruses that
undergo ribosome shunting (e.g., adenovirus, Sendai virus, and
cauliflower mosaic virus) directly translocate the ribosome
from the upstream initiation complex to the AUG initiator
codon without requiring the eIF4A helicase to unwind RNA
secondary structure (33, 43, 63). For adenovirus, the viral 5?
UTR directs both the ribosome scanning and shunting mech-
anisms when eIF4F is abundant but exclusively uses the ribo-
some shunting mechanism during late adenovirus infection,
* Corresponding author. Mailing address: Division of Infectious Dis-
eases, School of Public Health, 140 Warren Hall, University of California,
Berkeley, Berkeley, CA 94720-7360. Phone: (510) 642-4845. Fax: (510)
642-6350. E-mail: email@example.com.
when eIF4F is inactivated (63). Characterization of the spe-
cialized circumstances involved in the developmentally regu-
lated translation of multifunctional viral mRNAs will likely
reveal important factors that define pathogenesis.
a major public health threat worldwide (7). It is an enveloped
with a type I cap structure at the 5? end, a 96-nucleotide (nt) 5?
UTR, and a 451-nt nonpolyadenylated 3? UTR (8). DEN and
other flaviviruses, such as West Nile, yellow fever, Kunjin, and
Japanese encephalitis viruses, are presumed to undergo cap-de-
pendent translation due to the presence of both a 5? cap structure
and virally encoded methyltransferase and 5? RNA-triphos-
cellular translation factors, DEN has been shown to infect differ-
entiated cells, such as those of the myeloid lineage, which are
known to contain limiting amounts of translation factors (29, 41).
In view of the fact that DEN does not shut off host cell protein
synthesis, the mechanism by which the viral genome competes
successfully for cellular translation factors to establish a produc-
tive infection is unclear. Here we report that under conditions
that inhibit cellular cap-dependent translation, the DEN genome
can be translated without a functional cap structure by a novel
non-IRES-mediated mechanism that requires both the DEN 5?
and 3? UTRs.
MATERIALS AND METHODS
Plaque assay and cell infection. Virus titers of DEN2 strain 16681 (provided
by the Centers for Disease Control and Prevention, Fort Collins, CO) in PFU/ml
were determined by plaque assay using baby hamster kidney (BHK) 21 clone 15
cells as described previously (16). For infections in the presence of inhibitors of
cap-dependent translation, BHK or Vero monkey kidney cells were exposed to
viral strains at a multiplicity of infection (MOI) of 1 for 24 h in medium
containing either 40 ?M LY294002 (Sigma Chemical Co., St. Louis, MO) or 1
?M wortmannin (Sigma).
Western blot assay. BHK cells (2 ? 105) were exposed to DEN at an MOI of
1 (for visualization of eIF4E, eIF4G, and NS1) or 10 (for 4E-BP1) in the
presence of 40 ?M LY294002 or 1 ?M wortmannin for 12 h. Cells were then
harvested, lysed in a Triton X-100 solution, and separated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were trans-
ferred to a nitrocellulose membrane and visualized by Western blotting with
polyclonal anti-4E-BP1 (Cell Signaling Technologies, Beverly, MA), anti-eIF4E
(Cell Signaling Technologies), anti-eIF4G (Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA), anti-actin (Santa Cruz Biotechnology, Inc.), or anti-DEN NS1
monoclonal antibodies (P. R. Beatty and E. Harris, unpublished results). Equal
amounts of protein (25 ?g) were loaded per lane except for 4E-BP1 blots, where
equal numbers of cells were loaded in each lane. Quantitation was performed
using a Bio-Rad Chemi-Doc system (Bio-Rad, Hercules, CA).
Infectious clone and reporter constructs. Infectious DEN RNA was tran-
scribed from the pD2/IC infectious clone (gift of Richard Kinney, Centers for
Disease Control and Prevention, Fort Collins, CO) from the T7 promoter using
the RiboMAX large-scale RNA production system (Promega, Madison, WI) to
incorporate either an m7GpppA (New England Biolabs, Beverly, MA) or an
ApppA (Sigma) as the 5?-terminal nucleotide. RNA reporter constructs used in
this study were generated using the pGL3 vector backbone (Promega) and either
the 5? UTR of the human ?-globin gene or the DEN2 5? UTR sequence fused
to the luciferase (Luc) gene, followed by either the DEN2 3? UTR sequence or
a 268-nt vector sequence plus a 60-nt polyadenylated [poly(A)] tail (34). As
above, the RNA was transcribed from the T7 promoter to contain either an
m7GpppA, m7GpppG, ApppG (New England Biolabs), or an ApppA as the
5?-terminal nucleotide using the RiboMAX large-scale RNA production system.
The dicistronic constructs (35) used in the IRES experiments were in vitro
transcribed from the T7 promoter using the RiboMAX large-scale RNA pro-
Metabolic labeling of DEN-infected cells. For experiments in which translation
of DEN NS5 was measured in comparison to cellular proteins, cells were either
treated with inhibitors during infection or transfected with a small interfering
RNA (siRNA)-generating plasmid for 48 h prior to infection. Cells were then
exposed to DEN at an MOI of 100 and incubated in RPMI supplemented with
5% fetal bovine serum (FBS) for 12 or 24 h. Prior to harvest, cells were incubated
for 1 h in 500 ?l of cysteine- and methionine-deficient RPMI medium with 5%
dialyzed FBS. Newly synthesized proteins were labeled with [35S]cysteine-methi-
onine (100 ?Ci) for 30 min. The cells were detached with Hank’s balanced salt
solution plus 5 mM EDTA, washed twice in cold PBS, and counted, and then
equal numbers of cells were resuspended in 25 ?l of SDS sample buffer. Proteins
were separated on an 8% SDS-polyacrylamide gel, which was then exposed to a
Molecular Dynamics PhosphorImager detection system (Amersham Biosciences,
Piscataway, NJ). Proteins were quantitated using ImageQuant image analysis
software (Amersham Biosciences).
In vitro translation assay. In vitro translation extracts were generated from
BHK cells by a previously published method (19). To compare translation of
RNA reporter constructs in the presence of m7GpppA or ApppA cap analog
competitor, in vitro translation extracts were prepared and then incubated with
increasing concentrations (0 to 1.25 mM) of m7GpppA cap analog (NEB) or
ApppA (Sigma) for 15 min at room temperature. These extracts were then
programmed with molar equivalents of each RNA reporter construct transcribed
to contain an m7GpppN cap structure at its 5? end, as indicated above. The
lysates were incubated at 30°C for 60 min, and Luc activity was measured using
the luciferase assay reagent (Promega) and a TD20/20 luminometer (Turner
Designs, Sunnyvale, CA).
RNA transfection. Infectious viral RNA generated from the pD2/IC DEN
infectious clone and RNA reporter constructs was transfected into cells using
Lipofectamine 2000 (Gibco BRL, Carlsbad, CA). Immediately prior to transfec-
tion, cell monolayers were washed with Optimem medium (Gibco BRL). In a
polystyrene tube, 1 ml of Optimem was mixed with 5 ?l of transfection reagent
and 50 ?l of concentrated viral RNA per well of a 12-well plate. For pD2/IC
RNA, the solution was added to each well, and cells were incubated at 37°C for
4 to 6 h. Cells were then washed two times with 2 ml of RPMI plus 10% FBS, 40
?M LY294002 or 1 ?M wortmannin was added, and cells were further incubated
at 37°C for 24 h. For RNA reporter constructs, monolayers were washed 1 h
posttransfection, and 40 ?M LY294002 or 1 ?M wortmannin was added. Cells
were incubated at 37°C for 8 to 12 h, and Luc activity was assayed by lumino-
metry. For all experiments, efficiency of translation was determined via real-time
reverse transcription-PCR (RT-PCR) of RNA extracted from cells when trans-
fection reagent was removed 1 h after its addition to the cells.
siRNA expression. An eIF4E-specific siRNA construct was generated from
the pRF42 plasmid backbone (46). An oligonucleotide encoding sequence from
the eIF4E gene (nt ?9 to ? 12; GenBank accession no. BC010759) (13) in the
sense and the antisense directions separated by a 10-nt hairpin was inserted into
the BbsI site of the pRF42 vector. BHK cells were transfected with 4 ?g of either
an RNA inhibitory (RNAi) construct targeting eIF4E, a control reverse-sense
RNAi construct, or empty vector in the presence of Lipofectamine 2000 liposo-
mal reagent and transfection medium (Invitrogen, Carlsbad, CA). After 2 h,
monolayers were washed, cells were incubated at 37°C, and siRNA inhibition at
48 h was determined by quantitation of eIF4E suppression by Western blotting
using anti-eIF4E antibodies. An equal number of cells was loaded in each lane.
Dicistronic reporter constructs. The dicistronic constructs used in the IRES
experiments were derived from the dicistronic encephalomyocarditis virus
(EMCV)-IRES construct and the ?EMCV-IRES construct (gift of Peter Sar-
now, Stanford University) (35). For dicistronic constructs containing DEN se-
quences, the DEN 5? UTR was inserted between the mutant EMCV IRES and
the firefly Luc gene using a PCR-derived fragment (EcoRI-NarI). The 451-nt
DEN 3? UTR was inserted between the NarI and the XbaI sites at the 3? end of
the firefly Luc gene in the construct containing the DEN 5? UTR.
DEN translation is resistant to suppression of cap-depen-
dent translation. To delineate the ability of DEN to compete
with eukaryotic mRNAs for translation factors involved in
cap-dependent translation, several strategies were pursued.
First, the compounds LY294002 and wortmannin were used to
mimic cellular conditions under which eIF4E activity is sup-
pressed. These drugs inhibit the phosphoinositol-3 kinase path-
BP1), which results in sequestration of the cap-binding protein
VOL. 80, 2006 NOVEL STRATEGY FOR DENGUE VIRUS TRANSLATION INITIATION2977
eIF4E from eIF4F and inhibition of cap-dependent translation
(61). When BHK cells were incubated with DEN in the presence
of LY294002 or wortmannin, equivalent virus titers were ob-
served at 24 h postinfection in treated and mock-treated cells
(Fig. 1A). The phosphorylation state of 4E-BP1 in these cells was
examined, and the predominant species in the cells treated with
LY294002 or wortmannin for 24 h was found to be the hypophos-
phorylated (alpha) form (27), as determined by Western blotting
(Fig. 1B). The inhibition of cellular protein synthesis was verified
by biosynthetically labeling cells that were either untreated or
treated with LY294002 or wortmannin (Fig. 1C). To further con-
firm these results, the experiments were repeated in Vero cells
treated with LY294002 or wortmannin, and similar results were
obtained (Fig. 1A). Additionally, DEN propagation in the pres-
FIG. 1. DEN replication and translation are resistant to inhibitors of cap-dependent translation. (A) DEN replicates in cells exposed to
inhibitors for 24 h. BHK or Vero cells (2 ? 105) were exposed to DEN2 strain 16681 and simultaneously treated with 40 ?M LY294002 or 1 ?M
wortmannin per well or mock treated. Cells were incubated for 24 h at 37°C, and then cell supernatants were collected, and infectious virus titer
was determined using BHK21 cells (PFU/ml). The data are expressed as an average of three experiments. Error bars indicate standard errors of
the means. (B) Treatment of BHK cells with LY294002 or wortmannin results in hypophosphorylation of 4E-BP1. Cells treated as described above
were harvested 24 h postinfection. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane, and 4E-BP1 was
visualized by immunoblotting with polyclonal anti-4E-BP1 antibody. Indicated are the three phosphorylation states of 4E-BP1, ?, ?, and ?; ?
designates the lowest state of phosphorylation. (C) Treatment of BHK cells with LY294002 or wortmannin inhibits cellular protein synthesis. Cells
were treated with wortmannin or LY294002 or mock treated as for panel A for 12 h and then metabolically labeled. Cells were starved in
cysteine-methionine-free medium 1 h prior to labeling and then pulsed with 150 ?Ci of [35S]cysteine-methionine for 30 min, harvested, and
analyzed by SDS-PAGE. (D) DEN infection does not inhibit cellular translation. To metabolically label total cellular protein in uninfected cells
or cells infected with DEN2 for 24 h, BHK cells were starved in cysteine-methionine-free medium and pulsed with [35S]cysteine-methionine, harvested,
and analyzed by SDS-PAGE as for panel C. DEN NS5 and NS3 are indicated by arrows. Data are representative of three experiments. (E) DEN
RNA is translated in inhibitor-treated cells. Cells were treated with LY294002 or wortmannin or mock treated as described for panel A and were
infected with DEN or mock infected. At 12 h postinfection, total cellular protein was metabolically labeled as described for panel C. Lysates from
equal numbers of cells were analyzed by SDS-PAGE. DEN NS5 (arrow) and representative cellular proteins were quantitated as percent protein
relative to the untreated control. Brackets indicate cellular proteins that were quantitated. The gel is representative of four experiments, and the
average of the four experiments was computed and is presented ? the standard deviation. (F) Synthesis of DEN NS1 is not reduced in
inhibitor-treated cells. Cells treated as for panel E were harvested 12 h postinfection. Proteins were separated by SDS-PAGE and transferred to
a nitrocellulose membrane, and NS1 was visualized by immunoblotting with monoclonal anti-NS1 antibody. Actin was visualized on the same blot,
and NS1 levels were normalized to actin. G. eIF4G and eIF4E levels are not significantly affected by treatment with wortmannin. BHK cells were
treated with 1 ?M wortmannin and harvested 4 to 24 h posttreatment. eIF4G and eIF4E were visualized by Western blotting with polyclonal
anti-eIF4G antibody or anti-eIF4E antibody, respectively. Actin was included as a loading control. h.p.t., hours posttreatment.
2978 EDGIL ET AL.J. VIROL.
target of rapamycin pathway to dephosphorylate 4E-BP1, again
resulted in equal amounts of viral progeny as in mock-treated
cells (data not shown).
Cellular infection with the majority of animal viruses results
in the inhibition of host protein synthesis through a variety of
mechanisms, including cleavage of certain translation factors,
modulation of the ratio of cellular message to viral RNA levels,
or prevention of the export of cellular messages from the
nucleus (55, 58). However, when metabolically labeled total
cellular proteins from uninfected BHK cells or those infected
with DEN for 24 h were analyzed, DEN infection appeared to
exert no effect on cellular protein synthesis (Fig. 1D), consis-
tent with earlier reports of other flaviviruses (4). The identity
of DEN protein NS5 was previously confirmed by Western
blotting (17) (data not shown). To determine the ability of DEN
to translate in the presence of wortmannin and LY294002, total
cellular proteins from DEN-infected cells were metabolically la-
beled and examined. Treatment with wortmannin (Fig. 1E, lanes
2 and 3) or LY294002 (Fig. 1E, lanes 4 and 5) suppressed cellular
protein synthesis equivalently, regardless of infection status. In
these experiments, in contrast to the approximately 50% reduc-
tion of cellular protein synthesis resulting from LY294002 or
wortmannin treatment, synthesis of DEN proteins, represented
by NS5, was less affected (0 to 20% reduction) (Fig. 1D). Fur-
thermore, quantitation of DEN NS1 in treated versus untreated
cells by Western blotting revealed uniform levels of protein ex-
pression (Fig. 1F). Levels of eIF4F components remained stable
in cells over the course of treatment (Fig. 1G). Likewise, transla-
tion of DEN NS5 was relatively unaffected in cells depleted of
of 4E-BP1 (44), whereas translation of cellular proteins was re-
duced (data not shown). Together, these results indicate that,
despite the presence of a 5? cap structure on the DEN genome,
DEN replication and translation are resistant to inhibition of
As an alternative and more specific approach to inhibit cap-
dependent translation, siRNA-mediated gene silencing was em-
ployed to suppress expression of the prototypical form of mam-
malian eIF4E, eIF4E-1 (38). A plasmid encoding a 21-nt hairpin
targeting a sequence at the 5? end of eIF4E-1 (13, 46), a control
plasmid containing the eIF4E-1 sequence in reverse, and a con-
trol empty vector were transfected into BHK cells. Expression of
the eIF4E-1-specific siRNA reduced levels of eIF4E-1 by approx-
imately 90% in comparison with the control plasmid, as deter-
mined by Western analysis 48 h after transfection (Fig. 2A). Cells
were infected with DEN at 48 h posttransfection, and the virus
titers were measured at 24 h postinfection. Similar to results
obtained with LY294002 and wortmannin (Fig. 1A), suppression
of eIF4E-1 via siRNA had no effect on the ability of DEN to
replicate (Fig. 2B). Also consistent with the LY294002 and wort-
mannin results (Fig. 1E), total cellular protein synthesis was de-
creased by 60% in the eIF4E-1-depleted cells in comparison with
cells containing the control siRNA plasmids, whereas DEN NS5
FIG. 2. DEN replication and translation resist siRNA-mediated depletion of eIF4E. A. Treatment with an eIF4E-targeted siRNA for 48 h
reduces eIF4E expression in cells. Cells were transfected with an RNAi construct targeting eIF4E, a control RNAi construct, or empty vector. After
2 h, monolayers were washed and incubated for 48 h at 37°C. Cell lysates were harvested at 48 h and separated by SDS-PAGE, and proteins were
transferred to a nitrocellulose membrane. eIF4E was visualized by Western blotting with polyclonal anti-eIF4E antibody and quantitated. eIF4E
is presented as a percentage of protein relative to the empty vector negative control ? the standard deviation from three experiments. B.
siRNA-mediated suppression of eIF4E does not affect DEN replication after 24 h. Cells described above were exposed to DEN2 strain 16681 at
an MOI of 10. After 2 h, monolayers were washed, and the cells were incubated for an additional 24 h at 37°C. Cell supernatants were collected,
and titers were determined using BHK cells (PFU/ml). The data are expressed as an average of three experiments. Error bars indicate standard
deviations. C. Suppression of eIF4E does not affect DEN translation. Cells were transfected with an RNAi construct targeting eIF4E, a control
RNAi construct, or empty vector for 48 h preinfection. siRNA-treated cells were exposed to DEN at an MOI of 100 for 12 h. One hour prior to
labeling, cells were starved in cysteine-methionine-free medium. Cells were then pulsed with 150 ?Ci of [35S]cysteine-methionine for 30 min, cell
lysates were harvested, and proteins were separated by SDS-PAGE. Cellular and viral NS5 (arrow) proteins were analyzed, and data are presented
as described for Fig. 1E. Brackets indicate cellular proteins that were quantitated.
VOL. 80, 2006 NOVEL STRATEGY FOR DENGUE VIRUS TRANSLATION INITIATION2979
protein expression was only decreased by ?10% (Fig. 2C). These
results confirm that DEN translation can occur when levels of
eIF4E are reduced.
DEN translates independently of the cap structure under
conditions of reduced eIF4E. The resistance of DEN to inhi-
bition of eIF4E activity suggests that DEN may be capable of
cap-independent translation. To determine more directly the
effects of eIF4E sequestration on DEN translation, a series of
reporter constructs was generated containing either the DEN
5? UTR or the human ?-globin 5? UTR fused to the firefly
luciferase (Luc) gene, followed by either the DEN 3? UTR or
a 268-nt vector sequence that ends with a 60-mer poly(A) tail
(Fig. 3A). With these constructs, competition assays were per-
formed using an m7GpppA cap analog, which binds to the
cap-binding pocket of eIF4E, removing it from the pool of
functional translation factors. Thus, this treatment should sup-
press cap-dependent translation in a dose-dependent manner.
In addition, the same competition was performed with ApppA
cap analog to control for specificity and Mg2?-chelating prop-
erties of the cap compounds. In vitro translation extracts pre-
pared from BHK cells were incubated with increasing concen-
trations of either m7GpppA or ApppA cap analog for 15 min
and then programmed with molar equivalents of each m7GpppN-
capped RNA reporter construct. As expected, in the control
ApppA experiments, translation of all four constructs was
equally affected in a nonspecific manner by increasing doses of
cap analog (Fig. 3B). In contrast, at increasing concentrations
of m7GpppA cap analog, translation of the construct contain-
ing both the DEN 5? and 3? UTRs exhibited less sensitivity to
the sequestration of eIF4E than the other constructs (Fig. 3C).
Compared with the 5DLuc3D construct, translation from the
constructs containing the ?-globin 5? UTR was suppressed
80% at approximately eightfold-lower concentrations of cap
analog, irrespective of the sequence of the 3? UTR. The con-
tribution of the DEN 5? UTR to this effect is evidenced by the
intermediate phenotype of the construct containing the DEN
5? UTR and a poly(A) tail. These results demonstrate that
DEN translation, mediated by the presence of both the 5? and
3? UTRs, has a reduced dependence on eIF4E.
To directly assess the influence of a functional 5?
m7GpppN cap structure on DEN translation, the reporter
constructs described above were transcribed in vitro to contain
FIG. 3. Translation of DEN reporter RNAs is resistant to competition for eIF4E in vitro. (A) Schematic diagram of RNA reporter constructs.
RNA reporter constructs contain either the DEN2 5? UTR or the human ?-globin (?g) 5? UTR fused to the firefly Luc gene, followed by either
the DEN2 3? UTR or a vector sequence plus a 60-mer poly(A) tail. RNA transcripts were generated from a T7 promoter. (B) ApppA cap analog
does not differentially affect translation of DEN reporter constructs. In vitro translation extracts were incubated with increasing concentrations of
ApppA cap analog for 15 min at room temperature and then programmed with molar equivalents of RNA reporter constructs, as indicated. Luc
activity was measured after 1 h and is presented as a function of cap concentration. Data shown are representative of four experiments. (C) DEN
translation resists competition for eIF4E. In vitro translation extracts were incubated with increasing concentrations of m7GpppA cap analog for
15 min at room temperature and then programmed with molar equivalents of RNA reporter constructs, as indicated. Luc activity was measured
after 1 h and is presented as a function of cap concentration. Data shown are representative of six experiments.
2980EDGIL ET AL.J. VIROL.
either a functional m7GpppN cap structure or a nonfunctional 5?
ApppN cap, which stabilizes the RNA but is unable to bind
eIF4E. The m7GpppN- and ApppN-capped RNA transcripts
wortmannin. The different RNA constructs have been shown by
Northern blot analysis to have similar stability in transfected cells
(34); likewise, the stability of the RNAs was unaffected by
treatment with the inhibitors, as determined by real-time RT-
PCR analysis (?5% of untreated control). When Luc activity
was measured 12 h posttransfection of m7GpppN-capped
RNAs, only the construct containing both the DEN 5? and 3?
UTRs was fully resistant to the effects of the drugs, whereas
translation of all other constructs was reduced by at least 50%
(Fig. 4A). Translation from a control reporter construct con-
taining the 5? UTR of the human actin gene was similarly
reduced in the presence of translational inhibitors (data not
shown). When ApppN-capped RNAs were examined, transla-
tion of the four constructs in untreated cells was equally inef-
ficient but was slightly improved in cells treated with inhibitors
(Fig. 4B), presumably due to the increased availability of
eIF4G (3, 59). However, in the presence of inhibitors of cap-
dependent translation, Luc activity from only the ApppN-
capped construct containing both the DEN 5? and 3? UTRs
was substantially increased (Fig. 4B) to levels similar to that of
the m7GpppN-capped DEN construct (Fig. 4C). These results
suggest that, contrary to prior belief (45), the capped DEN
genome can be translated independently of the cap structure
under conditions that inhibit translation of cap-dependent cel-
lular messages. Moreover, both the DEN 5? and 3? UTRs are
necessary to mediate this effect. As the DEN genome likely
contains an m7G cap throughout its infectious life cycle, we will
refer to the alternative mechanism of DEN translation that can
function in the absence of an m7G cap as noncanonical DEN
FIG. 4. Translation of DEN reporter constructs is resistant to inhibition of cap-dependent translation. (A) The m7G-capped DEN reporter construct
is translated in inhibitor-treated cells. Cells were transfected with equal amounts of the m7GpppN-capped RNA reporter constructs described for Fig.
3A. After 1 h, monolayers were washed, 40 ?M LY294002 or 1 ?M wortmannin was added, and the cells were incubated at 37°C for 12 h. Luc activity,
assayed after 12 h, was normalized to the amount of RNA 1 h posttransfection and then to constructs translated in untreated cells. The data are expressed
as an average of four experiments. Error bars indicate standard deviations. (B) The nonfunctionally (ApppA-) capped DEN reporter construct is
translated in inhibitor-treated cells. Cells were treated as for panel A, except that the ApppN-capped RNA reporter constructs were examined. The data
are expressed as an average of three experiments. Error bars indicate standard deviations. (C) Translation from ApppA-capped DEN RNA reporter
constructs in inhibitor-treated cells is similar to that of m7GpppA-capped DEN constructs. Cells were treated as for panels A and B. The data are shown
in relative luciferase units and are representative of three experiments. Error bars indicate standard deviations.
VOL. 80, 2006NOVEL STRATEGY FOR DENGUE VIRUS TRANSLATION INITIATION 2981
The DEN genome can switch from canonical cap-dependent
translation to a noncanonical translation initiation mecha-
nism. The observation that noncanonical DEN translation is
upregulated from ApppA-capped DEN reporter constructs
under conditions of reduced eIF4E (Fig. 4B and C) led us to
examine the ability of the DEN genome in its entirety to
replicate in the absence of a functional cap structure. In this
experiment, RNA was transcribed from the DEN2 infectious
clone to contain either a functional 5? m7GpppA cap structure
or a nonfunctional 5? ApppA cap and was transfected into
untreated or inhibitor-treated cells. Equal amounts of the
m7GpppA-capped and the ApppA-capped DEN2 RNAs were
observed by real-time RT-PCR at both 1 h and 12 h posttrans-
fection (data not shown). When supernatants of untreated cells
were analyzed by plaque assay 24 h posttransfection, no infec-
tious progeny were observed following transfection of the
ApppA-capped viral RNA, compared with titers of 103PFU/
ml obtained following transfection of m7GpppA-capped viral
RNA (Fig. 5, columns 1 and 4), indicating that DEN translates
exclusively via its 5? m7GpppA cap structure in cells competent
for cap-dependent translation. Consistent with earlier obser-
vations (Fig. 1A), viral titers from cells transfected with the
m7GpppA-capped viral RNAs were unaffected by the presence
of inhibitors of cap-dependent translation (Fig. 5, columns 2
and 3). Importantly, as predicted by the previous results ob-
tained with reporter RNAs, viral titers from inhibitor-treated
cells transfected with the nonfunctionally capped viral ge-
nomes were increased from 0 PFU to 20 to 30% of that ob-
tained following transfection of the m7GpppA-capped genome
(Fig. 5, columns 5 and 6). This indicates that a productive DEN
infection can be initiated independently of the cap structure
under conditions of reduced eIF4E.
DEN does not contain an IRES element. The discovery of
the ability of DEN to translate and replicate independently of
a cap structure led us to investigate the existence of IRES
activity in the DEN UTRs. A series of dicistronic reporter
constructs (35) were generated that encode a cap-dependent 5?
UTR fused to the Renilla luciferase gene, followed by a mu-
tated EMCV IRES to prevent read-through (36). The second
cistron consists of the DEN 5? UTR, a functional EMCV IRES
(positive control), or the mutated EMCV IRES (negative con-
trol) fused to the firefly luciferase gene, and finally the DEN 3?
UTR or a 60-mer poly(A) tail (Fig. 6A). If DEN RNA contains
IRES activity, firefly Luc should be expressed from the dicis-
tronic construct containing the internal (uncapped) DEN 5?
UTR. When in vitro-transcribed RNAs derived from the di-
cistronic constructs were transfected into cells, cap-dependent
Renilla Luc activity was equivalent for all constructs (Fig. 6B).
However, IRES activity was apparent only with the construct
containing the positive control (Fig. 6C). Neither the presence
of the DEN 3? UTR nor the addition of inhibitors of cap-
dependent translation initiation altered these results (data not
shown), indicating that noncanonical DEN translation is not
mediated by internal ribosome entry.
We describe a situation wherein the flavivirus DEN appears
to have evolved a novel mechanism to effectively recruit trans-
lation initiation factors under conditions in which these factors
are limiting within the cell. We show that treatment of cells
with inhibitors of cellular cap-dependent translation (e.g.,
LY294002, wortmannin, or eIF4E-specific siRNAs) leads to
inhibition of cellular translation but does not affect the yield of
infectious DEN viral progeny, the level of DEN protein syn-
thesis, or the translation of RNA reporter constructs contain-
ing both the DEN 5? and the 3? UTRs. Experiments in which
increasing concentrations of m7G cap analog were used to
deplete eIF4E in translation extracts indicated that, in com-
parison to cellular 5? leaders, translation of reporter constructs
containing both the DEN 5? and 3? UTRs is resistant to com-
petition for eIF4E. These results suggest that the presence
and/or interaction of both the DEN 5? UTR and the DEN
3?UTR is necessary to mediate resistance to the effects of
eIF4E suppression. Furthermore, although the nonfunction-
ally capped viral genome is unable to replicate independently
of an m7G cap structure under normal cellular conditions,
when eIF4E is inhibited, replication of the ApppA-capped
DEN infectious clone is upregulated. However, despite its re-
sistance to inhibitors of cap-dependent translation, we find that
the DEN UTRs do not contain IRES activity. In summary, our
results support a model in which the interaction of the DEN 5?
and 3? UTRs allows the virus to translate in either a canonical
cap-dependent or a noncanonical alternative manner in re-
sponse to cellular conditions.
Viruses that undergo cap-dependent translation have evolved a
variety of mechanisms through which to compete for compo-
nents of eIF4F. For example, adenovirus and cauliflower mo-
saic virus initiate translation via a cap-dependent process
called ribosome shunting, whereby the ribosome is directly
translocated from the upstream initiation complex to the AUG
initiator codon without requiring the eIF4A helicase to unwind
RNA secondary structure (33, 64). Similarly, the capped vesic-
ular stomatitis virus (VSV) mRNAs, which are translated un-
der conditions of virally induced suppression of eIF4F, have
adapted to this condition by encoding very short 5? leaders, and
three out of five VSV mRNA 5? UTRs are less than 14 nt long
(9). Alternatively, certain viruses compete for the limiting
amounts of translation initiation factors within the cell through
the use of viral proteins bound to the 5? or 3? ends of the
genome that are able to recruit initiation factors more effec-
tively than other capped mRNAs (12). For example, like the
DEN genome, RNA elements of the alfalfa mosaic virus
(AMV) and rotavirus genomes possess a 5?-terminal cap struc-
FIG. 5. DEN alternates between canonical cap-dependent and
noncanonical translation. Equal amounts of functionally (m7GpppA)
or nonfunctionally (ApppA) capped in vitro transcripts generated from
the DEN2 infectious clone were transfected into cells for 4 h. Cells
were washed and incubated with LY294002 or wortmannin for 24 h.
Supernatants were then collected, and titers were determined by
plaque assay. The data are expressed as an average of three experi-
ments. Error bars indicate standard deviations.
2982 EDGIL ET AL.J. VIROL.
ture but lack 3?-terminal poly(A) tails. In the case of AMV, the
viral coat protein (CP) binds the 3? end of AMV RNAs and
mimics the function of the poly(A)-binding protein in transla-
tion of cellular mRNAs by interacting with eIF4G to bring
about efficient translation of the viral RNAs (40). Similarly, the
3?-end sequences of the rotavirus genome bind the rotavirus
NSP3 protein, which both confers stability to the viral mRNAs
(15) and interacts with eIF4G with extremely high affinity, thus
recruiting it away from cellular messages (53). Furthermore,
the influenza virus NS1 protein binds both the influenza virus
5? UTR and eIF4G, directly recruiting the 43S ribosomal com-
plex to the viral 5? terminus (1). Finally, the 5? end of the
calicivirus genome is covalently linked to the viral protein VPg,
which was found to interact directly with the translation initi-
ation factors eIF3 and eIF4E, acting as a cap substitute and
promoting translation from Vpg-linked viral RNA while inhib-
iting the translation of capped mRNAs (12, 28).
In contrast to the majority of capped mRNAs, we show that
the DEN genome, under conditions of reduced eIF4E, can be
induced to translate independently of a cap structure. That the
DEN UTRs failed to initiate translation via IRES activity
implies that initiation of DEN translation may be end depen-
dent. Experiments were performed to test a reporter construct
containing a stable hairpin immediately downstream from the
5? end of the DEN 5? UTR, which should inhibit end-depen-
dent translation initiation by blocking ribosome entry (14, 31,
39). A dramatic decrease in translation of the DEN hairpin-
containing construct was observed in untreated as well as
eIF4E-depleted in vitro translation extracts that support non-
canonical DEN translation (S. Paranjape, K. Holden, M. Lee,
and E. Harris, unpublished results). These results are consis-
tent with a requirement for a free 5? end for both canonical and
noncanonical DEN translation. Such a requirement is not un-
precedented; cap-independent translation of members of the
plant Luteovirus family requires the interaction of the viral 5?
and 3? UTRs, which facilitates recruitment of the ribosome to
the 5? end of the genome (31, 51). The absence of an IRES in
the DEN 5? UTR combined with the requirement for the DEN
3? UTR for cap-independent translation suggest a similar type
of communication between the viral UTRs and argue against
Although DEN is able to be translated under conditions of
low levels of eIF4E, we have obtained data suggesting that
DEN requires intact eIF4G for full activity. In extracts treated
with the coxsackie B virus 2A protease, which cleaves eIF4G,
DEN translation is reduced by 75%, as is the ?-globin control
FIG. 6. The DEN 5? and 3? UTRs do not confer IRES activity. A. Dicistronic reporter constructs contain a cap-dependent 5? UTR fused to
the Renilla Luc gene followed by a mutated (?EMCV) IRES and then the DEN 5? UTR or a wild-type EMCV IRES upstream of the firefly Luc
gene. The constructs terminate with either the 451-nt DEN2 3? UTR or a 60-mer poly(A) tail. B. Translation of the cap-dependent Renilla Luc
is equivalent for all constructs. C. DEN 5? and 3? UTRs do not support IRES activity. Cells were transfected with equal amounts of RNA from
the dicistronic construct described above. After 1 h, monolayers were washed and 40 ?M LY294002 or 1 ?M wortmannin was added per well.
Renilla and firefly Luc activities were measured at 12 h. Data are an average of four experiments. Error bars indicate standard deviations.
VOL. 80, 2006 NOVEL STRATEGY FOR DENGUE VIRUS TRANSLATION INITIATION2983
construct (data not shown). This is consistent with reduction of
Kunjin virus translation by ?75% upon coinfection with po-
liovirus (56). While the N terminus of eIF4G that is removed
by 2A protease includes an eIF4E binding site, the require-
ment for this fragment for efficient DEN translation may not
suggest that eIF4E is necessary but rather may indicate the
importance of other binding sites, such as the previously iden-
tified PABP binding site or potentially additional binding sites
for proteins that are involved in translational enhancement.
Alternatively, the N-terminal segment of eIF4G may be nec-
essary for maintaining the proper conformation of eIF4G (30).
Additionally, to determine the intrinsic affinity of DEN mRNA
for eIF4F relative to an “average” cellular mRNA, filter bind-
ing assays and surface plasmon resonance experiments are
We propose a model in which the DEN genome is able to
alternate from standard cap-dependent translation to a form
of noncanonical translation initiation under conditions of re-
duced eIF4E (Fig. 7). In this model, in the presence of eIF4E,
the DEN 3? UTR functionally replaces a poly(A) tail to en-
hance translation efficiency via a canonical cap-dependent scan-
ning mechanism (34). However, a decrease in the concentra-
tion of eIF4E (or possibly other cellular translation initiation
factors) prompts the reorganization of the viral RNP com-
plexes bridging the DEN 5? and 3? UTRs. In this conformation,
RNA structures or sequences in the 3? UTR deliver or stabilize
translation initiation factors at the 5? end of the RNA, allowing
factors such as eIF4G and eIF4A to be recruited while bypass-
ing the requirement for eIF4E. Several proteins have been
reported to associate with the DEN 3? UTR, including eEF1A,
La, PTB, YB-1, and hnRNP A1 and Q (5, 22, 32) (S. M.
Paranjape and E. Harris, unpublished data). These are candi-
dates for proteins that bridge the DEN 5? and 3? UTRs to
enhance canonical and/or noncanonical DEN translation.
A likely trigger for noncanonical DEN translation is a re-
duction in eIF4E levels, due to the critical role of eIF4E in
regulation of protein synthesis (26) and as suggested by our
results. The biological relevance of the reduced dependence of
DEN translation on eIF4E may correlate with the ability of
DEN to translate under conditions in which cellular translation
is limited, for example, in differentiated cells such as myeloid
and dendritic cells, known to be in vivo targets of DEN, that
contain lower levels of available eIF4E (29, 41). Consistent
with this hypothesis, we have previously shown that an increase
in DEN replication coincides with differentiation in human
myeloid cells (42), and we are currently evaluating whether
translation of DEN proteins and functionally and nonfunction-
ally capped DEN RNA constructs also increases under these
conditions. In addition, mammalian cellular stress response
and immune functions, such as the interferon antiviral re-
sponse (24), may compel viral translation by one mechanism
over the other. Finally, noncanonical DEN translation may be
elicited only in certain host target cells by the presence of cell-
or tissue-specific factors. For example, cap-independent trans-
lation of the picornavirus genome has been observed to be
specifically regulated in neuronal cells (6, 18). Similarly, cap-
independent translation initiation from certain cellular genes
has been shown to be dependent upon the presence or activity
of specific factors for expression (11, 37, 48, 52).
The existence of two mechanisms by which the DEN ge-
nome may be translated is of potential significance for many
viruses, such as flaviviruses, that maintain complex life cycles in
disparate hosts (i.e., mosquito and human), as well as in mul-
tiple cell types within the same host. We find that DEN can
switch between cap-dependent translation initiation and a non-
IRES-mediated form of noncanonical translation initiation
that requires the presence of the 5? and 3? UTRs. Further
characterization of this noncanonical mechanism of DEN
translation should reveal essential components of the viral
life cycle. Ultimately, the report of a capped viral RNA that
can alternate between mechanisms of translation in re-
sponse to cellular environment has implications for cellular
tropism, viral transmission, vector and host competence,
and antiviral strategies.
FIG. 7. Model of 5?-3? interactions in canonical (cap-dependent) and noncanonical DEN translation initiation. When eIF4E is abundant, a
cap-dependent scanning mechanism of translation initiation occurs (A). When eIF4E is limiting, the DEN 3? UTR interacts with host proteins to
deliver and/or stabilize key translation initiation factors at the 5? UTR (B).
2984EDGIL ET AL. J. VIROL.
We thank Karen Clyde, Jennifer Doudna, Anna-Marija Helt, Kather-
ine Holden, Allen Miller, Suman Paranjape, Jose Pen ˜a, and Sondra and
Milt Schlesinger for helpful discussions and editorial comments. We are
grateful to Michael Diamond for the siRNA plasmid and advice, to Peter
Sarnow for the dicistronic constructs, to Karen Clyde for assistance with
quantification of radioactively labeled gels, and to Peter Sarnow, John
Hershey, and Loy Volkman for general scientific counsel.
This work was supported by the Pew Charitable Trusts, NIH
(AI052324), the Committee on Research at the University of Califor-
nia, Berkeley (E.H.), and the Soroptimist International Founder Re-
gion Fellowship (D.E.).
We declare that we have no competing financial interests.
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