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Journal of General Virology (2004), 85, 1565–1569
Short
Communication
DOI 10.1099/vir.0.79992-0
The Rhopalosiphum padi virus 59 internal ribosome
entry site is functional in Spodoptera frugiperda 21
cells and in their cell-free lysates: implications for
the baculovirus expression system
Elizabeth Royall,13 Kathryn E. Woolaway,134 Jens Schacherl,2
Stefan Kubick,2 Graham J. Belsham3 and Lisa O. Roberts1
1
School of Biomedical and Molecular Sciences, University of Surrey, Guildford,
Surrey GU2 7XH, UK
Correspondence
Lisa O. Roberts
2
[email protected]
RiNA GmbH, Takustraße 3, 14195 Berlin, Germany
3
BBSRC Institute for Animal Health, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK
Received 27 January 2004
Accepted 19 February 2004
Cap-independent internal initiation of translation occurs on a number of viral and cellular
mRNAs and is directed by internal ribosome entry site (IRES) elements. Rhopalosiphum padi
virus (RhPV) is a member of the Dicistroviridae. These viruses have single-stranded, positive-sense
RNA genomes that contain two open reading frames, both preceded by IRES elements. Previously,
the activity of the RhPV 59 UTR IRES has been demonstrated in mammalian, Drosophila and
wheat germ in vitro translation systems. It is now shown that this IRES also functions within
Spodoptera frugiperda (Sf21) cells which are widely used in the baculovirus expression system,
and in a novel Sf21 cell-based lysate system. Inclusion of the RhPV IRES in a dicistronic
reporter mRNA transcript increased translation of the second cistron 23-fold within Sf 21 cells.
In contrast, the encephalomyocarditis virus IRES was inactive in both systems. The RhPV IRES
therefore has the potential to be utilized in insect cell expression systems.
Initiation of protein synthesis on most cellular mRNAs
involves the recognition of the 59 cap structure (m7GpppN)
by translation initiation factors and subsequent recruitment of the 43S pre-initiation complex which includes
the 40S small ribosomal subunit (reviewed by Hershey &
Merrick, 2000). However, translation initiation on certain
viral mRNAs (e.g. from picornaviruses) occurs by a capindependent mechanism. An internal ribosome entry site
(IRES) element present within the 59 untranslated region
(UTR) directs initiation of protein synthesis at an internal
position hundreds of nucleotides downstream from the 59
terminus. The viral IRES elements are highly structured
and position the ribosome at, or just upstream of, the
initiation codon. The presence of IRES elements has also
been reported within certain cellular mRNAs, such as those
involved in apoptosis, cell cycle regulation or stress response (Carter et al., 2000; Hellen & Sarnow, 2001).
The best-characterized IRES elements are those from the
mammalian picornaviruses (reviewed by Belsham & Jackson,
3E. R. and K. E. W. contributed equally to this work.
4Present address: BBSRC Institute for Animal Health, Ash Road,
Pirbright, Woking, Surrey GU24 0NF, UK.
0007-9992 G 2004 SGM
2000). The picornavirus IRES elements are grouped into
two major classes based on their predicted secondary
structure and their activity in vitro. One class contains IRES
elements from the entero- and rhinoviruses (e.g. poliovirus) while the second contains the cardio- and aphthovirus IRES elements (e.g. encephalomyocarditis virus,
EMCV). The cardio-/aphthovirus IRES elements function
efficiently in the rabbit reticulocyte lysate (RRL) translation
system. However, the poliovirus and rhinovirus IRES elements are inefficient in this system unless the reaction is
supplemented with HeLa cell extracts (Brown & Ehrenfeld,
1979; Dorner et al., 1984). Similarly, the activity of the
hepatitis A virus IRES (which forms a third class of IRES)
is stimulated by the addition of liver cell, but not HeLa
cell, extracts (Glass & Summers, 1993). It is clear that
cellular trans-acting factors play an important role in the
mechanism of IRES action and may contribute to the
cellular tropism of picornaviruses. Indeed, it has been
demonstrated that different IRES elements function with
different efficiencies in different cell types (Borman et al.,
1997; Roberts et al., 1998).
We have previously demonstrated that the 59 UTR of
Rhopalosiphum padi virus (RhPV) mRNA contains an
IRES element (Woolaway et al., 2001). This virus belongs
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1565
E. Royall and others
to the family Dicistroviridae, which also includes cricket
paralysis virus (CrPV), Drosophila C virus (DCV) and
Plautia stali intestine virus (PSIV). The single-stranded,
positive-sense RNA genome of these viruses contains two
open reading frames (ORFs) that encode two polyproteins
(Moon et al., 1998). ORF1 encodes the non-structural
proteins and ORF2 the structural proteins. All of these
proteins possess sequence similarity with mammalian
picornavirus proteins. It has been shown that the 59 UTRs
and the intergenic regions (IGRs) of these virus genomes
contain IRES elements (Sasaki & Nakashima, 1999; Domier
et al., 2000; Wilson et al., 2000a; Woolaway et al., 2001).
The IGR IRES elements are unusual in that they direct
translation initiation from non-AUG codons and they do
not require any of the canonical initiation factors for
assembly of initiation complexes on the mRNA (Wilson
et al., 2000b; Nishiyama et al., 2003). In contrast, the 59 IRES
of RhPV directs initiation from AUG codons. It has been
shown to function in a Drosophila cell-based in vitro
translation system and also functions efficiently in RRL
and wheat germ lysates (Woolaway et al., 2001). The ability
of the RhPV 59 IRES to function in insect translation
systems suggests potential utility of this IRES in insect
cell expression systems. To explore this further, we have
tested the function of this IRES in Sf 21 cells and in a novel
in vitro Sf 21 cell-based lysate system (Kubick et al., 2003).
These cells are commonly used with baculovirus expression systems. We report here that the RhPV 59 IRES displays efficient activity in these cells and in the in vitro
system.
To examine the activity of the RhPV 59 IRES within Sf 21
cells, reporter plasmids of the form T7:CAT/IRES/LUC
containing the RhPV IRES (previously referred to as
RhPVD1; nt 1 to 579) or the mammalian picornavirus
EMCV IRES were used, as described by Woolaway et al.
(2001). Cap-dependent translation was monitored by measuring chloramphenicol acetyltransferase (CAT) expression
and activity of the IRES element was assessed from the
expression of luciferase (LUC). Mutated versions of the
RhPV IRES plasmid containing 59 and 39 end deletions
[named RhPVD2 (with nt 1 to 463), D3 (nt 1 to 374) and
D4 (nt 100 to 588)] have also been described previously
(Woolaway et al., 2001). Sf 21 cells were grown at 28 uC
in TC100 medium (Gibco-BRL) supplemented with fetal
bovine serum (FBS; 10 %), penicillin (5000 units penicillin
G sodium ml21), streptomycin (5000 mg streptomycin
sulphate ml21 in 0?85 % saline) and 2 mM L-glutamine.
The dicistronic reporter plasmids were assayed by transfection into Sf 21 cells (60 mm dishes) using lipofectin
(20 ml; 1 mg ml21 stock; Gibco-BRL). T7 RNA polymerase
was expressed in the cells by prior infection with a
recombinant baculovirus (AcT7N; gift from Dr J. Vlak,
University of Wageningen, The Netherlands; van Poelwijk
et al., 1995). After 48 h incubation at 28 uC, the cells
were harvested in 400 ml cell lysis buffer (Promega) and
the lysates clarified by centrifugation at 14 000 r.p.m. for
5 min at 4 uC. CAT assays were performed using the CAT
1566
Fig. 1. Analysis of the RhPV 59 IRES activity in Sf21 cells.
Plasmids of the form T7:CAT/IRES/LUC were transfected into
AcT7N-infected Sf21 cells as described in the text. After 48 h,
cell extracts were prepared and analysed for CAT (cap-dependent)
and LUC (IRES-dependent) expression as described in the
text. Values are shown relative to the RhPV IRES (nt 1 to
579), which was set at 100. IRES-containing plasmids are indicated by the name of the IRES insert. Results are the means
(±standard error) from four separate experiments.
ELISA kit (Boehringer Mannheim) as described in the
manufacturer’s instructions. Colour development was measured on an ELISA plate reader (Labsystems Multiscan
Bichromatic) at 405 nm. As expected, each of the constructs induced a similar level of CAT expression (Fig. 1).
LUC expression was measured using a LUC assay kit
(Promega) and a Bio-orbit luminometer. The RhPV 59
IRES was able to direct LUC expression at a level 23-fold
higher than the background LUC expression seen with
pGEM-CAT/LUC (no IRES) and 400-fold higher than seen
from the construct containing the EMCV IRES (Fig. 1).
Presumably, the presence of the structured EMCV RNA
sequences inhibited any translational read-through and
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Journal of General Virology 85
RhPV IRES activity in Sf21 cells and lysate
http://vir.sgmjournals.org
pSP72LUC
No IRES
EMCV
RhPV
No RNA
LUC
CAT
(B)
600
LUC expression
400
200
Rh
PV
EM
C
V
no
IR
ES
pS
P7
2L
UC
N
o
RN
A
0
(C)
80
60
40
20
Rh
PV
EM
C
V
no
IR
ES
pS
P7
2L
UC
RN
A
0
N
o
An Sf 21 cell-based in vitro translation system has recently
been described using monocistronic constructs (Kubick
et al., 2003). Since the RhPV IRES displayed activity in Sf 21
cells, we wanted to explore the properties of the RhPV 59
IRES within this system since a cell-free system can be
more readily manipulated to define parameters important
for RhPV IRES activity. We tested the ability of the RhPV
IRES to direct translation of the downstream luciferase
ORF in both untreated and nucleased Sf 21 lysates. Capped
transcripts from the linearized dicistronic plasmids were
made in vitro using T7 RNA polymerase (T7 Cap-Scribe;
Roche). In vitro translation reactions contained 5 ml 106
buffer (30?6 mM HEPES/KOH pH 7?95; 1?5 mM magnesium acetate; 100 mM potassium acetate; 2?5 mM DTT;
0?25 mM spermidine); 1 ml 0?1 M creatine phosphate;
1?0 ml 0?1 M ATP; 0?5 ml 0?1 M GTP; 1?5 ml 1 mM total
amino acid mix; 0?5 ml 10 mg creatine kinase ml21; and
1?25 ml 50 mM magnesium acetate. The mRNA was added
at a final concentration of 100 ng ml21 in a total volume of
25 ml, containing Sf 21 lysate (50 % by volume), and the
reactions were incubated at 27 uC for 90 min. Translation
reactions in the nucleased lysate incorporating [35S]methionine were analysed for CAT and LUC expression by SDSPAGE and autoradiography. In addition, LUC activity
was measured as above. Each of the dicistronic RNA transcripts containing the CAT gene expressed CAT at a similar
level (Fig. 2A). However, only the dicistronic transcripts
containing the RhPV IRES were able to direct efficient
expression of luciferase, the EMCV IRES was essentially
inactive in this system (Fig. 2A, B). Luciferase was also
expressed from a monocistronic RNA control (pSP72LUC).
Translational efficiencies were compared with those in
untreated (un-nucleased) Sf 21 lysate (Fig. 2C) to assess if
the IRES could function in the presence of competing
RNAs. All transcripts expressed CAT at a similar level as
expected (data not shown). The RhPV IRES was able to
direct expression of luciferase in both systems (Fig. 2A–C).
In neither system was the EMCV IRES able to direct
translation of luciferase better than the control plasmid
lacking any IRES. The capped monocistronic luciferase
control mRNA functioned well in the nucleased lysate
(Fig. 2A, B) but, in the untreated lysate, very little luciferase
expression was observed (Fig. 2C). Presumably, the RNA
transcripts were unable to compete efficiently for the
translational machinery in the presence of endogenous
mRNAs (Fig. 2C). Indeed, the expression of luciferase was
higher in the nucleased lysate than in the untreated lysate
in each case, e.g. the RhPV IRES-directed luciferase expression was more than three-fold higher in the nucleased
lysate. Consistent with these observations, translation of
(A)
LUC expression
hence LUC expression. As we have seen previously in
in vitro translation systems (Woolaway et al., 2001), the
59 and 39 end deletion mutants directed less-efficient
expression of LUC in cells. However, the mutants still
retained about 40 to 50 % activity of the wild-type RhPV
IRES within cells (Fig. 1). The antisense version of the RhPV
IRES was unable to direct LUC expression as expected.
Fig. 2. The RhPV 59 IRES functions in Sf21 lysates. Capped
mRNA transcripts of the form CAT/IRES/LUC were translated
in nucleased (A and B) or untreated Sf21 lysates (C) as
described in the text. Samples from the nucleased lysate reactions were analysed by SDS-PAGE and autoradiography (A)
using a phosphorimager (Personal Molecular Imager FX; BioRad) and by LUC assay (B). Samples from the untreated lysate
reactions were analysed by LUC assay alone (C). The IREScontaining dicistronic transcripts are referred to by the name of
the IRES insert. The results shown are from one complete
experiment but are representative of three separate experiments.
CAT from the capped transcripts was also elevated in
nucleased lysate as compared with that in untreated lysate
(results not shown).
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E. Royall and others
These results show that the RhPV 59 IRES functions efficiently in Sf 21 cells and in Sf 21 lysates; the RhPV 59 IRES
also functions well in a Drosophila lysate (Woolaway et al.,
2001). In contrast, the EMCV IRES is inactive in each of
these systems (Finkelstein et al., 1999; Woolaway et al.,
2001). It may be that insect cells lack an important transacting factor that is required for EMCV IRES function, or
alternatively they may contain an inhibitor of EMCV IRES
activity. Recently, Domier & McCoppin (2003) demonstrated the ability of the RhPV 59 and IGR IRES elements
to function in Sf 9 cells. In their system the 59 IRES seemed
to function as efficiently as the IGR IRES to direct internal
initiation. However, it should be noted that these authors
reported just a three- to fourfold increase in expression
directed by the 59 IRES in Sf 9 cells compared to a negative
control containing a defective RhPV IGR IRES. In Sf 21
cells, we observed a 23-fold increase in expression when
the 59 IRES was inserted into a dicistronic mRNA (compared to a dicistronic mRNA lacking an IRES). We have also
observed a 180-fold increase in LUC expression from the
RhPV IRES in mosquito cells using the same plasmids (Kohl
et al., 2004). In a recent report, Masoumi et al. (2003)
demonstrated that the 59 and IGR IRES elements of CrPV
display different levels of activity in a range of different
insect cells. Surprisingly, neither of the IRES elements from
the CrPV genome could function in Sf 9 cells. This suggests
that subtle differences may exist between the IRES elements
from these related dicistroviruses.
Insertion of the RhPV 59 IRES into an uncapped monocistronic mRNA increased the translational efficiency in the
Sf 21 lysate by fourfold (Kubick et al., 2003). In this study
we have also demonstrated that the RhPV IRES directed
LUC expression between three- and 10-fold over that seen
from the CAT/no IRES/LUC control in vitro. However,
within Sf 21 cells, the increase in expression from the
RhPV IRES was 23-fold. This is likely due to a lower
background of internal initiation from the ‘no IRES’
dicistronic mRNA within cells compared with that seen
in vitro. This is analogous to the mammalian systems in
which a mutant EMCV IRES displayed around 0?25 % of
the activity of a wt EMCV IRES within cells, whereas in
the in vitro system the activity was around 5 % (van der
Velden et al., 1995).
These data suggest that use of this IRES element within an
insect cell-based expression vector can enhance translation,
without the need for making capped transcripts in vitro.
Similarly, insertion of the IRES between two ORFs may
allow for expression within the baculovirus expression
system of a protein of interest along with a reporter or
selectable marker, or expression of two subunits of a protein
within the same cells at the same time.
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K. E. W. gratefully acknowledges a BBSRC studentship award.
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