La autoantigen is necessary for optimal function of the poliovirus and hepatitis C virus internal ribosome entry site in vivo and in vitro.
ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5' untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.
- SourceAvailable from: Andras Donaszi-Ivanov[Show abstract] [Hide abstract]
ABSTRACT: The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defences through inhibition of IRF3. Here we use mass spectrometry to identify a new role for N(pro) through its interaction with 55 associated proteins, mainly ribosomal proteins and ribonucloproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumour protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3 and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoparticles and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a non-cytopathic BVDV strain Kyle. However, RNPs binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain, no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a pro-viral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins have roles in RISC function during RNA silencing. Using GAPDH silencing with siRNAs followed by Northern blot analysis of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex.Journal of Virology 06/2014; · 4.65 Impact Factor
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ABSTRACT: Nrf2 is a bZIP transcription factor regulating the expression of antioxidant and detoxification genes. We have found that Nrf2 knockout mice have an increased infarction size in response to regional ischemic reperfusion and have a reduced degree of cardiac protection by means of ischemic preconditioning. With cycles of brief ischemia and reperfusion (5'I/5'R) that induce cardiac protection in wild type mice, an elevated Nrf2 protein was observed without prior increases of Nrf2 mRNA. When an mRNA species is being translated into a protein, it is occupied by multiple ribosomes. The level of ribosome-associated Nrf2 mRNA increased following cycles of 5'I/5'R, supporting de novo Nrf2 protein translation. A dicistronic reporter assay indicated a role of the 5' Untranslated Region (5'UTR) of Nrf2 mRNA in oxidative stress induced Nrf2 protein translation in isolated cardiomyocytes. Western blot analyses after isolation of proteins binding to biotinylated Nrf2 5'UTR from the myocardium or cultured cardiomyocytes demonstrated that cycles of 5'I/5'R or oxidants caused an increased association of La protein with Nrf2 5'UTR. Ribonucleoprotein complex immunoprecipitation assays confirmed such association indeed occurring in vivo. Knocking down La using siRNA was able to prevent Nrf2 protein elevation by oxidants in cultured cardiomyocytes and by cycles of 5'I/5'R in the myocardium. Our data point out a novel mechanism of cardiac protection by de novo Nrf2 protein translation involving interaction of La protein with 5'UTR of Nrf2 mRNA in cardiomyocytes.Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 06/2014; · 5.09 Impact Factor
MOLECULAR AND CELLULAR BIOLOGY, Aug. 2004, p. 6861–6870
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 24, No. 15
La Autoantigen Is Necessary for Optimal Function of the Poliovirus
and Hepatitis C Virus Internal Ribosome Entry Site
In Vivo and In Vitro†
Mauro Costa-Mattioli, Yuri Svitkin, and Nahum Sonenberg*
Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Quebec, Canada H3G 1Y6
Received 9 March 2004/Returned for modification 2 April 2004/Accepted 30 April 2004
Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to
an internal ribosome entry site (IRES) in the mRNA 5? untranslated region. Translation initiation of these
RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La
autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in
vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we
demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show
that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative
form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent
translation and define the step of translation which is stimulated by La.
Translation initiation in eukaryotes is generally dependent
on a structure termed the cap (m7GpppN, where m is a methyl
group and N is any nucleotide), which is present at the 5?ends
of all nuclear transcribed mRNAs. The cap recruits the 40S
ribosomal subunit through its interaction with the cap binding
protein complex, eIF4F, which forms a bridge between the
mRNA and the 40S ribosomal subunit through eIF3 (19, 27).
Following its recruitment to the mRNA 5?end, the 40S ribo-
somal subunit in association with several initiation factors, is
thought to migrate along the 5? untranslated region (5?UTR)
until an initiation codon is encountered (27). However, in a
sizeable fraction of eukaryotic cellular mRNAs (9, 25, 70), the
40S ribosomal subunit is recruited to the mRNA via an alter-
native mechanism using an internal ribosome binding site
(IRES). IRESs were first discovered in picornavirus mRNAs
(poliovirus and encephalomyocarditis virus), which are natu-
rally uncapped (38, 54), and subsequently identified in many
cellular mRNAs (reviewed in references 25 and 70; see the
Internal Ribosome Entry Site database  for an updated list).
Strikingly, cellular IRESs are mainly found in mRNAs that are
translated under stress conditions (31). These conditions in-
clude hypoxia, apoptosis, serum starvation (26, 29, 32, 43, 45),
and mitosis (10, 58). Thus, control of IRES-dependent trans-
lation plays a critical role in regulation of cell growth and
To understand the mechanism of control of IRES function,
it is imperative to study the mode by which the 40S ribosome
subunit is recruited to the IRES. To this end, we and others
undertook the identification and characterization of cellular
proteins that interact with the poliovirus IRES. The studies of
Meerovitch et al. led to the identification of the first IRES
trans-acting factor (ITAF), the La autoantigen (47, 48). Sub-
sequently, La was shown to bind to the hepatitis C virus (HCV)
IRES near the initiation codon and to stimulate HCV RNA
translation in a rabbit reticulocyte lysate at low concentrations
(1, 2). The HCV IRES-La interaction has been recapitulated
in a Saccharomyces cerevisiae three-hybrid system (57). In ad-
dition, La binds to cellular IRESs, such as X-linked inhibitor of
apoptosis (XIAP) (30) and BiP (42).
Initiation of poliovirus translation from the correct initia-
tion site in a rabbit reticulocyte lysate is feeble, and aberrant
products are readily detected (8, 14), possibly due to limiting
amounts of one or more translation factors. Interestingly, the
amount of La protein present in reticulocyte lysate is very low
compared to extracts from nucleated cells (48), and the addi-
tion of recombinant La enhances poliovirus RNA translation
and eliminates the production of aberrant proteins (48, 65). It
was thus concluded that La protein is a bona fide ITAF. How-
ever, the physiological significance of these studies was ques-
tioned (35, 36), because of the large amounts of recombinant
La that had been used (10-fold more recombinant La than the
amount of La protein present in HeLa cell extract). Thus, the
possibility that La is not a physiological ITAF but rather mim-
ics a genuine poliovirus ITAF, owing to its RNA binding prop-
erties was raised (35, 36, 72). To address this issue, we studied
the requirement of La autoantigen for poliovirus translation in
vivo. Using small interfering RNA (siRNA) and a dominant-
negative mutant of La (LaDN), we demonstrate that La is
required for optimal translation of poliovirus. In addition, we
demonstrate that LaDN inhibits the initiation of formation of
48S ribosome complexes on HCV and poliovirus mRNAs.
MATERIALS AND METHODS
Cell culture and viruses. HeLa CLL2 cells (American Type Culture Collec-
tion) were cultured under standard conditions in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and anti-
biotics. Cells were passaged by a 1:7 dilution before reaching confluency to
maintain exponential growth. Poliovirus type 1 (Mahoney) was produced after
transfection of HeLa cells with the T7 RNA polymerase transcripts of pPV1 as
* Corresponding author. Mailing address: Department of Biochem-
istry and McGill Cancer Center, McGill University, McIntyre Medical
Building, Montreal, Quebec, Canada H3G 1Y6. Phone: (514) 398-
7274. Fax: (514) 398-1287. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://mcb
described previously (55). Adenovirus dl309 (39) was kindly provided by P.
Branton (McGill University) and HCV-poliovirus chimera was obtained from F.
Lahser and E. Wimmer (44, 77). Poliovirus was propagated in HeLa cells, and
the virus titer was determined by a plaque assay as described previously (61).
siRNA transfection and bicistronic reporter cotransfection. Target sequences
for siRNA were designed by using the Ambion web-based criteria and purchased
from Dharmacon. The positions and sequences of the siRNAs used in the
present study are given in Table 1. One day prior to transfection, the cells were
trypsinized, resuspended in medium without antibiotics, and transferred to 24-
well plates at a density of 5 ? 105cells per well in a volume of 500 ?l. Trans-
fection experiments were performed using Lipofectamine 2000. For each trans-
fection, 3 ?l of siRNA duplex (20 ?M annealed duplex from Dharmacon) was
mixed with 50 ?l of OPTIMEM (Invitrogen). In a separate tube, 3 ?l of Lipo-
fectamine 2000 per reaction mixture was added to 50 ?l of OPTIMEM and
incubated for 5 min at room temperature. Both solutions were mixed and incu-
bated for an additional 20 min at room temperature to allow the formation of
complexes. The solutions were then added to the cells in 24-well plates. Cells
were incubated at 37°C in the presence of the transfection solution for 24 h,
washed with DMEM, and used for virus infection. For Western blotting and
metabolic radiolabeling experiments, cells were lysed directly in 24-well plates
with 1? Laemmli sample buffer. The lysates were incubated at 95°C for 5 min,
and an equal amount of protein was resolved on sodium dodecyl sulfate (SDS)–
15% polyacrylamide gels. For in vivo translation experiments, HeLa cells at 90%
confluency were cotransfected in 24-well plates using Lipofectamine 2000 as de-
scribed above. Briefly, 500 ng of bicistronic reporter pcDNA3-RLuc-PolioIRES-
FLuc (56) and 3 ?l of indicated siRNA duplex (20 ?M annealed duplex from
Dharmacon) were used. Cell extracts were prepared in passive lysis buffer (Pro-
mega) 20 h after transfection and assayed for Renilla reniformis luciferase (RLuc)
and firefly luciferase (FLuc) activity in a Lumat LB9507 bioluminometer (EG&G
Bertold) using a dual-luciferase reporter assay system (Promega) according to
the manufacturer’s instructions.
Virus infections and metabolic radiolabeling. HeLa cells were first transfected
with siRNA and then infected with the Mahoney strain of poliovirus type 1 or
adenovirus type dl309 at a multiplicity of infection (MOI) of 5 PFU per cell.
Following virus adsorption at room temperature for 30 min, cells were incubated
in methionine-free DMEM at 37°C. At different times postinfection indicated in
the figure legends, the medium was replaced with medium containing 10 ?Ci of
[35S]methionine per ml. After further incubation for 30 min at 37°C, the cells
were washed with phosphate-buffered saline and lysed in 1? Laemmli sample
buffer. Radiolabeled lysates were subjected to SDS–15% polyacrylamide gel
electrophoresis (SDS–15% PAGE) as previously described (67).
LaDN mutant expression. DNA transfection was performed using Lipo-
fectamine Plus reagent (Gibco) according to the protocol provided by the man-
ufacturer. Briefly, cells were seeded at a density of 5 ? 104cells/ml in 24-well
plates and transfected 24 h later in serum-free Opti-MEM medium (Gibco) with
500 ng of pcDNA3-myc-La226-348 (kindly provided by Martin Holcik) or 500 ng
of control plasmid (pcDNA3-myc), 4 ?l of Lipofectamine Plus reagent, and 2 ?l
of Lipofectamine 2000 per well. The transfection mixture was replaced 3 h later
with fresh DMEM supplemented with 10% fetal bovine serum. Twenty-four
hours after transfection, cells were washed with DMEM and then infected with
poliovirus. Proteins were metabolically labeled as described above.
[3H]uridine labeling of viral RNA. HeLa cells previously transfected with
pcDNA3-myc-La226-348 and control plasmids were infected with poliovirus at a
MOI of 20. At various times postinfection, cells were treated with 5 ?g of
actinomycin D per ml for 1 h prior to the addition of [3H]uridine for 1 h. Ten
microliters of cytoplasmic extract was spotted onto Whatman 3MM filter paper,
dried for 20 min at room temperature, incubated for 5 min with cold 10%
trichloroacetic acid (TCA), and washed three times with cold 5% TCA. Radio-
activity was counted using a Beckman scintillation counter.
In vitro translation. Poliovirus RNA was translated in vitro using micrococcal
nuclease-treated HeLa S3 cell extracts that had not been subjected to dialysis (4,
50). The composition of the in vitro translation reaction mixture was similar to
that described previously for the Krebs-2 cell-free system (67). Translation re-
action mixtures (30 ?l) contained 15 ?l of extract and the following components
(final concentrations are given) as follows: 1 mM ATP, 0.2 mM GTP, 0.2 mM
CTP, 0.2 mM UTP, 10 mM creatine phosphate, 0.1 mg of creatine phosphoki-
nase per ml, 20 ?M concentrations of all L-amino acids except for L-methionine,
0.5 mCi of [35S]methionine per ml, 25 mM HEPES-KOH (pH 7.3), 15 mM KCl,
125 mM potassium acetate, 3 mM MgCl2,250 ?M spermidine, and 15 ?g of
poliovirus (type 1, Mahoney) RNA per ml. Following incubation (34°C, 4 h),
reactions were stopped by the addition of 2 volumes of 1.5? Laemmli sample
buffer. Translation products were detected following SDS–15% PAGE and au-
toradiography. To assay for poliovirus replication, reaction mixtures were sup-
plemented with 20 ?M unlabeled methionine and incubated at 34°C for 16 h.
Reaction mixtures were treated with a mixture of RNase A and RNase T1(50),
diluted with phosphate-buffered saline, and assayed for plaque formation on
HeLa cell monolayers (61).
Plaque assay. The plaque assay was performed as previously described (61).
Two hundred fifty microliters of cell extract in which poliovirus was produced
was used to infect HeLa monolayer cells (2 ? 106cells in 60-mm-diameter
plates). After 3 days of incubation at 37°C, plaques were detected by staining the
cells with 1% crystal violet.
Ribosome binding assays. HCV IRES-containing RNA was transcribed in
vitro with T7 RNA polymerase using plasmid pHCV(40-372) NS? (60) that had
been linearized at the junction of the HCV and nonstructural (NS?) sequence by
BamHI. Poliovirus RNA was obtained as described previously (64), and rabbit
globin mRNA was purchased commercially (Invitrogen). RNAs were 3? end
labeled to specific activities of about 107cpm/?g for HCV IRES and 5 ? 105for
globin and poliovirus mRNAs using [?-32P]cordycepin-5?-triphosphate (Perkin-
Elmer) and yeast poly(A) polymerase as recommended by the manufacturer
(Amersham Biosciences). Ribosome binding experiments were performed using
a cycloheximide-supplemented HeLa S10 cell extract (41) in a total volume of 40
?l for 15 min at 34°C. Reaction mixtures contained 20 ?l of micrococcal nucle-
ase-treated HeLa S10 cell extract, 0.6 mM cycloheximide, 4 ?l of a 10? master
mix (10 mM ATP, 2 mM GTP, 100 mM creatine phosphate, 1 mg of creatine
phosphokinase per ml, 19 unlabeled L-amino acids [lacking L-methionine; 0.2
mM each], and 125 mM HEPES-KOH [pH 7.3]) (68), 2 ?l of L-methionine (0.4
mM), 4 ?l of a salt solution (0.8 M KCl, 5 mM MgCl2, 2.5 mM spermidine), and
HCV IRES (?106cpm) or poliovirus (?105cpm) RNA. Following incubation,
samples were chilled on ice, diluted fourfold with ice-cold high-salt buffer, and
analyzed on 5-ml 15 to 30% linear sucrose gradients as described previously (66).
Assembly of 48S ribosome initiation complexes on HCV IRES was measured
in a similar way, except that the GTP analogue ?,?-imido-GTP (GMP-PNP) (1
mM) was substituted for GTP in the reaction mixture and a low-salt buffer (23)
was used to prepare 10 to 30% sucrose gradients. Gradients were centrifuged for
180 min at 54,000 rpm using a Beckman SW55 rotor. Ribosome binding was cal-
culated by two methods. The total area under the 80S or 48S ribosome initiation
complex curve was measured using the histogram function of Adobe Photoshop.
Alternatively, the graphs were printed out, and the areas under the curves were cut
out and weighed. No significant differences between the two methods were noted.
Specific inhibition of poliovirus translation by siRNA against
La autoantigen. To examine the requirement of La for polio-
virus translation in vivo, we used the RNA interference (RNAi)
method (16, 17, 76). La-85-siRNA (named according to its
position in the human La cDNA) was selected for use (from
three different siRNA sequences), as it elicited the strongest
level of La knockdown (see below). To examine the effect of La
knockdown on poliovirus translation, cells were transfected
with siRNA and then infected with type 1 poliovirus (Mahoney
strain) at a MOI of 5. Cells were pulse-labeled with [35S]me-
thionine at various times postinfection, and extracts were an-
alyzed by SDS–15% PAGE. Infection with poliovirus resulted
in a reduction of host cell protein synthesis (Fig. 1A) as ex-
TABLE 1. Positions and sequences of the siRNAs used for
gene expression knockdown in this study
bNuc-siRNA, siRNA against nucleolin.
6862COSTA-MATTIOLI ET AL.MOL. CELL. BIOL.
FIG. 1. La knockdown diminishes translation and replication of poliovirus. Transfection of siRNA in HeLa cells (CLL2) was performed in
24-well plates. Twenty-four hours after transfection, cells were infected with poliovirus (A) or adenovirus (B) or mock infected, and protein
synthesis was examined by pulse-labeling at various times as described in Materials and Methods. Cells were transfected with a nonspecific siRNA
(control siRNA [Ctlr-siRNA]), La85-siRNA, or siRNA against nucleolin (Nuc-siRNA). (C) Western blot analysis of La protein. (D) Poliovirus
yield is affected by La knockdown. A plaque assay was performed on a confluent monolayer of HeLa cells using serial dilutions of samples obtained
from the lysates shown in panel A (5 h postinfection.).
VOL. 24, 2004 La PROTEIN REQUIRED FOR HCV AND POLIOVIRUS TRANSLATION6863
pected (5, 75). Poliovirus proteins were conspicuous 5 h postin-
fection in cells transfected with a nonspecific siRNA (lane 8),
or siRNA against nucleolin (lane 7). In sharp contrast, polio-
virus proteins were barely detected at 5 h postinfection in cells
that had been treated before with siRNA against La autoanti-
gen (compare lane 6 to lanes 7 and 8). To further assess the
specificity of the effect of the La knockdown on poliovirus trans-
lation, HeLa cells were infected with adenovirus, whose mRNAs
are translated via a cap-dependent mechanism (reviewed in ref-
erence 12), which is not known to require La. La-85-siRNA
failed to inhibit translation of adenovirus proteins (Fig. 1B, com-
pare lanes 9 and 10), indicating that the La requirement is spe-
cific for poliovirus translation. The reduction of La protein by
siRNA treatment was specific inasmuch as treatment of HeLa
cells with La-85-siRNA (Fig. 1C, lanes 2, 4, and 6), but not with
a nonspecific siRNA against the inverted sequence of 4E-T
(15) (lanes 1, 3, 5, and 8) or an siRNA directed against nucleo-
lin (lane 7), resulted in a reduction of endogenous La protein
(?50%). Moreover, transfection with siRNA had no effect
on the intracellular distribution of La protein (data not shown).
To examine the effect of La knockdown on poliovirus yield,
a plaque assay was performed 5 h postinfection. La-85-siRNA
treatment resulted in a fivefold reduction in virus yield (Fig.
1D). Taken together, these data demonstrate that the La pro-
tein is required for efficient poliovirus infection in vivo.
La85-siRNA inhibits poliovirus IRES-driven protein syn-
thesis. To determine whether the effect of La siRNA on viral
protein synthesis in vivo is a consequence of inhibition of
translation, the effect of La85-siRNA on in vivo expression of
a bicistronic reporter mRNA, RLuc-PolioIRES-FLuc (56), was
examined. The bicistronic mRNA contains the poliovirus IRES
inserted between the RLuc and FLuc open reading frames
(ORFs) (Fig. 2). Thus, translation of RLuc is cap dependent,
whereas translation of the second cistron (FLuc) is IRES de-
pendent. Cotransfection of HeLa cells with pcDNA3-RLuc-
PolioIRES-FLuc and La-siRNA significantly reduced the FLuc/
RLuc ratio (more than threefold) (Fig. 2). In striking contrast,
the nonspecific siRNA (Ctlr-siRNA) failed to inhibit FLuc
synthesis, indicating a specific reduction of IRES-dependent
translation by La85-siRNA. Importantly, RLuc synthesis,
FIG. 2. IRES-driven translation of poliovirus RNA is inhibited by La85-siRNA. A schematic diagram of the bicistronic construct pcDNA3-
RLuc-PolioIRES-FLuc is shown at the top of the figure (CMV, cytomegalovirus). The bicistronic construct pcDNA3-RLuc-PolioIRES-FLuc was
cotransfected into HeLa cells with either La85-siRNA or control siRNA (Ctlr-siRNA). Twenty hours after transfection, cells were analyzed for
FLuc and RLuc activities. The ratios of RLuc/FLuc activity are presented by the bars in the histogram. Absolute levels of RLuc and FLuc activities
(in relative light units) are presented below the histogram.
6864 COSTA-MATTIOLI ET AL.MOL. CELL. BIOL.
which is cap dependent, did not vary significantly among cells
transfected with La85-siRNA, cells transfected with a nonspe-
cific siRNA (Ctlr-siRNA), or mock-transfected cells (Fig. 2),
indicating that the decrease in IRES-dependent translation
does not reflect a general decrease in translation. Taken to-
gether, these results bolster the conclusion that La protein en-
hances IRES activity in vivo.
A LaDN, La(226-348), inhibits poliovirus IRES activity in
vitro. To provide further evidence for the importance of La in
poliovirus translation in vivo, a LaDN was used. This mutant,
La(226-348), consists of the C-terminal fragment of La protein
(amino acids 226 to 348) (11), which was previously shown to
inhibit La-stimulated translation of several viral and cellular
mRNAs (11, 30, 69). First, we wished to examine whether the
mutant form inhibits translation of poliovirus in a HeLa cell
extract, as it was tested previously only in a reticulocyte lysate
(11). The HeLa cell extract contains much larger amounts of
La than the reticulocyte lysate does (48). In agreement with
previous data (50), translation of poliovirus RNA in a HeLa
cell extract yielded all the known authentic virus proteins (Fig.
3A, lane 1). When increasing amounts of glutathione S-trans-
ferase-tagged La(226-348) [GST-La(226-348)] (see Fig. 3B for
expression) were added to the HeLa cell extract, a dramatic
dose-dependent inhibition of poliovirus translation was evident
(Fig. 3A and C, compare lane 1 to lane 5, ?90% inhibition at
the highest concentration). La(226-348) neither affected the
spectrum of virus-specific proteins nor caused the appearance
of any aberrant translation products (Fig. 3A). Craig et al.
reported previously that in reticulocyte lysate, GST-La(226-
348) preferentially inhibited the appearance of P1, compared
to the synthesis of the products resulting from initiation at the
“spurious” initiation sites (11). In contrast to GST-La(226-
348), GST alone failed to inhibit poliovirus translation even at
a very high concentration (10 ?M; compare lanes 1 to 6).
Consistent with its inhibition of translation, La(226-348) dra-
matically reduced virus yield. Indeed, La(226-348) at a con-
FIG. 3. Effects of LaDN on translation of poliovirus RNA and infectious virus synthesis in HeLa S10 cell extracts. (A) Effects of LaDN on
poliovirus RNA translation in vitro. Translation reactions were performed in a total volume of 30 ?l and contained HeLa S10 cell extract, poliovirus
RNA (0.45 ?g), [35S]methionine, and other components as described in Materials and Methods and previously (67). La(226-348) was added at the
following final concentrations: 1.25 ?M (lane 2), 2.5 ?M (lane 3), 5 ?M (lane 4), and 10 ?M (lane 5). In lanes 1 and 6, reaction mixtures were
supplemented with control buffer and GST (10 ?M), respectively. Reaction mixtures were incubated at 34°C for 4 h, and the reactions were stopped
by the addition of Laemmli sample buffer. Proteins were separated by SDS–15% PAGE, blotted onto a nitrocellulose membrane, and analyzed by
autoradiography. The positions of the major virus proteins are indicated to the right of the gel. (B) The membrane from panel A was probed with
antibodies against GST. The positions of GST-La(226-348) and GST are indicated at the sides of the gel. (C) TCA-insoluble radioactivity assay
of 1-?l aliquots of the reaction mixtures from panel A. (D) Plaque assay for poliovirus infectivity. Reaction mixtures lacking [35S]methionine were
programmed with poliovirus RNA for 16 h as described above for panel A. Virus titers were determined as described in Materials and Methods.
VOL. 24, 2004 La PROTEIN REQUIRED FOR HCV AND POLIOVIRUS TRANSLATION6865
centration of 5 ?M essentially abolished plaque formation
(Fig. 3D). It is noteworthy that at this concentration of La(226-
348), some residual virus translation (?15%) was still detected
(Fig. 3A, lane 5). Thus, the magnitude of inhibition at the level
of protein synthesis is dramatically amplified at the down-
stream step of replication. The specific effect of La(226-348)
was confirmed in an in vitro translation extract from HeLa cells
using the bicistronic construct RLuc-poliovirus-Fluc mRNA in
which only poliovirus IRES-driven translation was reduced
(data not shown). Taken together, our data show that inacti-
vation of La renders HeLa cell extract defective in supporting
poliovirus translation and in turn virus replication.
Poliovirus IRES-directed translation is impaired in cells
transfected with La(226-348). Next, the effect of La(226-348)
on poliovirus IRES-mediated translation in cultured cells was
examined. Cells were transfected with an expression plasmid
encoding LaDN (pcDNA3-mycLa226-348) (30) and an empty
vector as a control. Cells were subsequently infected with po-
liovirus (MOI of 20) and incubated for 30 min with [35S]me-
thionine at different times after infection (Fig. 4A). In cells
expressing LaDN, viral protein synthesis was inhibited, espe-
cially at early times after infection (compare lanes 1 and 2),
compared to control, indicating that La(226-348) functions in a
dominant-negative manner in vivo.
We also wished to determine the effect of the LaDN on the
processes downstream of translation, such as RNA synthesis
and virus assembly. Cells were labeled with [3H]uridine for 1 h
in the presence of actinomycin D. As expected, the synthesis of
cellular RNA was inhibited in noninfected cells treated with ac-
tinomycin D (Fig. 4B). Viral RNA synthesis in the control cells
was detected at 3 h postinfection compared with 4 h for cells
expressing the LaDN mutant. Levels of viral RNA synthesis in
cells expressing La(226-348) reached 33% of wild-type levels
after 5 h (Fig. 4B). Thus, RNA synthesis is retarded by La(226-
348), consistent with an inhibition of translation by the LaDN
mutant. Moreover, La(226-348) caused a nearly 10-fold-lower
yield of virus compared to the control (Fig. 4C). Taken to-
gether, the decrease in [3H]uridine and [35S]methionine incorpo-
ration indicate that the expression of La(226-348) affects transla-
tion and subsequently RNA replication and virus assembly.
FIG. 4. LaDN reduces poliovirus replication in vivo. (A) The kinetics of synthesis of poliovirus proteins was determined by pulse-labeling as
described in Materials and Methods. Twenty-four hours after transfection with pcDNA3-myc-La226-348 (mutant) (LaDN) and pcDNA3-myc
(control), cells were infected with poliovirus or mock infected and labeled with [35S]methionine. The positions of the major virus proteins are
indicated to the right of the gel. (B) Kinetics of poliovirus RNA synthesis. At various times postinfection, control and LaDN-transfected cells were
treated with 5 ?g of actinomycin D per ml for 1 h and then labeled with [3H]uridine for 1 h. Noninfected cells (n.i) treated with actinomycin D
are also shown. TCA-precipitated radioactivity was determined as described in Materials and Methods. (C) Plaque assays of lysates derived from
LaDN- and control poliovirus-infected cells were performed as described in Materials and Methods.
6866 COSTA-MATTIOLI ET AL.MOL. CELL. BIOL.
HCV IRES-driven translation is impaired in La85-siRNA-
treated cells. Since HCV does not grow in cultured cells (3), we
used a poliovirus-HCV(IRES-core) chimeric model (44, 77) to
assess the requirement of La for HCV IRES activity in vivo.
The poliovirus-HCV chimera (P/H 701-2A) contains the 5?
cloverleaf structure of poliovirus, followed by the HCV
IRES (nucleotides 9 to 332), the first 369 nucleotides of the
HCV core region, the entire poliovirus ORF, 3?UTR, and the
poliovirus mRNA HCV-IRES mRNA
FIG. 5. LaDN inhibits the initiation of formation of 80S ribosome complexes. RNA-ribosome binding assay was performed in a cycloheximide
(0.6 mM)-treated HeLa cell extract (40 ?l) with poliovirus (?105cpm) (A), HCV IRES-poliovirus chimera (?106cpm) (B), and rabbit globin
(?105cpm) (C) 3?-end-labeled mRNAs. The extract was incubated with GST-La226-348 (4 ?g) or GST (4 ?g) at 34°C for 15 min and diluted with
ice-cold high-salt buffer (66). Ribosome complexes were analyzed as described in Materials and Methods.
VOL. 24, 2004 La PROTEIN REQUIRED FOR HCV AND POLIOVIRUS TRANSLATION 6867
poly(A) tail (77). In HeLa cells that were depleted of La by
siRNA, the P/H 701-2A chimera grew to a titer that was ap-
proximately sevenfold lower than that for control cells (see Fig.
S1C and D in the supplemental material). In addition, LaDN
drastically inhibited translation of the poliovirus-HCV chimera
mRNA in HeLa cell extracts (data not shown).
La protein promotes the initiation of formation of 48S and
80S ribosome complexes on poliovirus and HCV IRESs. To
further characterize La function in translation initiation of
poliovirus mRNA, 80S ribosome binding assays were per-
formed with poliovirus mRNA, and the effect of the La(226-
348) dominant-negative mutant was examined. Initiation of
formation of poliovirus 80S complexes, albeit inefficient, in
agreement with earlier data (22), was inhibited by the addition
of the La(226-348) dominant-negative mutant (Fig. 5A) (the
position of the 80S ribosome initiation complex was deter-
mined by sedimenting a pure preparation of 80S ribosomes in
a parallel tube). Thus, La plays a critical role in the initiation
of formation of 80S ribosome complexes. Next, to determine
the importance of La in the initiation of formation of 80S
ribosome complexes on HCV IRES, the effect of the LaDN
mutant on ribosome binding was examined. The addition of
the GST-La(226-348) mutant resulted in a significant decrease
(?60%, three replicate samples) in viral RNA binding to ri-
bosomes (Fig. 5B). In sharp contrast, binding of globin mRNA
to ribosomes, which is not known to be dependent on La, was
not affected by the LaDN mutant (Fig. 5C) (the second sedi-
menting complex [fractions 18 to 25] in Fig. 5C most likely
represents disomes , but this complex has not been char-
acterized further). To rule out the possibility that the ribosome
binding inhibition seen by GST-LaDN is due to the GST tag,
we cleaved the tag from the protein, and after purification, the
untagged LaDN was tested for inhibiting the initiation of for-
mation of 80S ribosome complexes. Untagged LaDN also in-
hibits HCV 80S ribosome binding in a dose-dependent manner
(see Fig. S2 in the supplemental material). Thus, La protein is
a specific ITAF for poliovirus and HCV IRESs but is not
required for globin mRNA translation.
To determine whether inhibition of 80S initiation complex
formation was a consequence of reduced 48S ribosome initia-
tion complex formation, we performed ribosome binding stud-
ies on HCV mRNA in the presence of GMP-PNP. The GTP
nonhydrolyzable analogue GMP-PNP competes with GTP for
incorporation into the ternary complex (eIF2-GTP-tRNAi),
thus inhibiting the release of eIF2 from the 40S subunit, and
consequently preventing 60S ribosomal subunit joining (28).
The addition of GMP-PNP caused the accumulation of
48S ribosome preinitiation complexes (compare 48S ribo-
some peaks in the presence [control and GST] and absence
[no treatment] of GMP-PNP in Fig. 6; the sucrose gradient was
10 to 30% and the centrifugation time was 180 min to improve
the resolution of the 48S mRNA ribosome complex). Consis-
tent with the results seen with 80S ribosome initiation complex
assembly on HCV IRES mRNA, GST-La(226-348) decreased
(by ?45%, two replicate samples) the binding of the 40S ribo-
somes to the HCV IRES (Fig. 6). Thus, the LaDN mutant in-
hibits the initiation of formation of complexes by preventing
the recruitment of the 40S ribosomal subunit. In contrast, GST
had no effect on 48S ribosome complex formation (Fig. 6).
Similar results were obtained with the 48S ribosome initiation
complex for poliovirus mRNA (data not shown). These results
indicate that the La protein is directly involved in the initiation
of formation of 48S ribosome complexes on poliovirus and
In this study, a combination of genetic and biochemical
methods consisting of RNAi and a LaDN mutant were used to
demonstrate that La is required for IRES-dependent transla-
tion of poliovirus in vivo (Fig. 1, 2, and 4) and in vitro (Fig. 3).
Adenovirus translation, which is not dependent on IRES func-
tion, was not affected by RNAi-mediated depletion of La (Fig.
1B). These results validate and extend earlier conclusions that
the La protein binds to an internal region in the 5?UTR (47,
65) that is functionally important for poliovirus translation (46,
51, 53, 55). The La protein also interacts with both the 5?UTR
and 3?UTR of HCV RNA (63) and was reported to stimulate
HCV IRES-mediated translation (2, 13). In agreement with
these findings, cells depleted of La by siRNA exhibited a de-
crease in virus yield of greater than sevenfold compared to
control cells when infected with a poliovirus chimeric cDNA in
which the poliovirus IRES is replaced by the HCV IRES (44, 77).
The physiological significance of earlier results demonstrat-
ing the importance of La in vitro was questioned because of the
FIG. 6. LaDN inhibits the initiation of formation of 48S ribosome
complexes. HCV IRES-labeled mRNA (?106cpm) was incubated for
10 min with a HeLa S10 cell extract and 4 ?g of GST-La(226-348).
GMP-PNP was included in the reaction mixtures where indicated. Sam-
ples were analyzed by sucrose gradient centrifugation (see Materials
and Methods). Radioactivity was determined by scintillation counting.
6868COSTA-MATTIOLI ET AL.MOL. CELL. BIOL.
large amounts of La used in the earlier studies (35–37, 72).
There are several possible explanations for this unusual re-
quirement. First, the recombinant protein may have been ren-
dered partially inactive during the purification process. Sec-
ond, the effector domain (C-terminal region) of La is known to
undergo phosphorylation (18), and this modification (which
does not occur in the recombinant protein) could be important
for optimal activity of the protein. Indeed, different RNAs are
preferentially associated with the phosphorylated form of La
compared to the nonphosphorylated form, and a fraction of
the phosphorylated form of La is cytoplasmic (33). Finally,
poliovirus causes the cleavage of La (62), which is then redis-
tributed to the cytoplasm (34, 48), raising the possibility that
cleaved La is more potent than wild-type La in translation.
What is the molecular mechanism by which La stimulates
40S ribosome recruitment? Our data demonstrate that a LaDN
mutant protein inhibits 80S (Fig. 5) and 48S initiation complex
formation (Fig. 6) on HCV and poliovirus mRNAs. The use of
a highly fractionated system in combination with a toe-printing
assay would allow further characterization of molecular mech-
anisms underlying this inhibition. Originally, it was suggested
that La functions as an RNA chaperone to fold the IRES into
a structure that can recruit initiation factors, such as eIF4G (6,
49, 65). Indeed, studies in yeast show that the yeast homologue
of La protein (LHP1) functions as a molecular chaperone as it
promotes the refolding of a mutant Met-tRNA (74). Alterna-
tively, it is also possible that La stimulates translation indirectly
by displacing an inhibitory protein from the IRES. In addition,
La might aid in the recruitment of ribosomes in a more direct
fashion, since human La protein has been reported to sediment
with the 40S ribosomal subunit and to bind the 18S rRNA (52).
In summary, we determined that La functions to recruit the
40S ribosome subunit to the viral mRNA. In addition, we
provided direct evidence based on in vivo studies that La pro-
tein is a bona fide ITAF for poliovirus and HCV. These results
have important implications for studies on inhibition of virus
replication in cells. Inhibition of poliovirus and HCV replica-
tion has been reported for siRNAs directly targeting poliovirus
(21) or HCV RNA (40, 59, 71, 73). However, viruses are likely
to evade siRNA by adaptive mutations of the target sequences
(20). To circumvent this problem, it would be important to
design siRNAs against cellular factors, such as La protein,
which are required for optimal virus replication. We expect
that the knowledge gained from this study will aid in the un-
derstanding of how cellular proteins may regulate IRES activ-
ity and might also be important in the design of new IRES-
targeted antiviral strategies.
We thank Jerry Pelletier and Andrea Brueschke for critical reading
of the manuscript, Karen Meerovitch for helpful discussions, Martin
Holcik for providing the pcDNA-3-myc-La226-348 mutant, F. Lahser
and E. Wimmer for the HCV-poliovirus chimera, and Colin Lister and
Sandra Perrault for excellent assistance.
This work was supported by a grant from the Canadian Institute of
Health Research (CIHR) to N.S., who is recipient of a CIHR Distin-
guished Scientific Award and a Howard Hughes Medical Institute
International Scholarship. M.C.-M is a postdoctoral fellow supported
by a CIHR fellowship.
ADDENDUM IN PROOF
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