Download Internal ribosome entry in the coding region of murine hepatitis virus

Journal of General Virology (1994), 75, 3041-3046. Printed in Great Britain
3041
Internal ribosome entry in the coding region of murine hepatitis virus
mRNA 5
Volker Thiel* and Stuart G. Siddell
Institute o f Virology, University o f Wiirzburg, Versbacher Strasse 7, 97078 Wiirzburg, Germany
The unique region of murine hepatitis virus (MHV)
mRNA 5 has two open reading frames, ORF 5a and
ORF 5b, that encode small proteins of unknown
function. In the experiments described here, we have
used the in vitro translation of synthetic mRNAs to
examine the expression of these ORFs. Our results show
that a synthetic mRNA containing both ORFs is
functionally bicistronic. More importantly, the expres-
Introduction
Mouse hepatitis virus (MHV), a member of the Coronaviridae, has a positive-strand RNA genome of about
31300 nucleotides (Pachuk et al., 1989; Lee et al., 1991 ;
Bonilla et al., 1994). In the infected cell, the expression of
viral proteins is mediated by the genomic-length RNA,
also known as mRNA 1, together with seven subgenomic
mRNAs. These mRNAs form a Y-co-terminal nested set
and they contain a common leader sequence of about 70
nucleotides at their 5' end (Lai et al., 1983). Only the 5'
unique region of each mRNA, i.e. the region that is
absent from the next smallest mRNA, is translationally
active (Leibowitz et al., 1982; Siddell, 1983).
Most MHV mRNAs contain only a single open
reading frame (ORF) in their 5' unique region. These
mRNAs appear to be functionally monocistronic. However, mRNA 1 and the subgenomic mRNA 5 are
exceptions (Skinner et al., 1984; Budzilowicz & Weiss,
1987; Pachuk et al., 1989). The unique region of MHV
mRNA 1 contains two large ORFs that constitute the
coronavirus RNA polymerase locus. Expression of viral
proteins from this locus involves both ribosomal frameshifting and autoproteolytic processing (Baker et al.,
1989; Bredenbeek et al., 1990; Denison et al., 1991,
1992). MHV mRNA 5 also contains two ORFs in its
unique region, designated ORF 5a and ORF 5b. In vitro
translation studies suggest that this mRNA is functionally bicistronic and the ORF 5b gene product has
been detected in MHV (strain A59)-infected cells
Leibowitz et al., 1988). On the basis of sequence
0001-2691 © 1994SGM
sion of ORF 5b, but not ORF 5a, is maintained in a
tricistronic mRNA containing an additional Y-proximal
ORF. Thus, in the context of the MHV mRNA 5 unique
region, the initiation of protein synthesis on ORF 5b can
occur independently of ribosomes that enter from the 5'
end of the mRNA. We conclude that the translation of
ORF 5b is mediated by the internal entry of ribosomes.
similarities, it is likely that the MHV ORF 5b gene
product is equivalent to the small membrane (sM)
proteins of infectious bronchitis virus (IBV) and transmissible gastroenteritis virus particles (Liu & Inglis,
1991; Godet et al., 1992).
Two mechanisms can be proposed for the translation
of the MHV ORF 5b. The leaky scanning model, as
proposed by Kozak (1989), is one possibility. In this
case, the initiation of ORF 5b translation would be
mediated by ribosomes that fail to recognize the ORF 5a
initiation codon and scan to the next available AUG
triplet, which is the ORF 5b initiation codon. An
alternative model is a cap-independent mechanism
involving ribosome entry at an internal position on the
MHV mRNA 5. An analogous mechanism has been
described for a variety of picornavirus RNAs and
hepatitis C virus RNA (Jackson et aI., 1990; Brown et
al., 1992). If this model is correct, the initiation of MHV
ORF 5b translation should occur independently of
ribosomes that bind to the 5' cap structure of the
mRNA.
In the experiments reported here, we have analysed the
in vitro translation of synthetic mRNAs that contain the
unique region of MHV mRNA 5 (minus the leader RNA
and some 5' non-translated sequences) preceded by an
ORF derived from the fl-galactosidase gene of Escherichia coli. The results show that the fl-galactosidase ORF
is an effective barrier to the movement of ribosomes from
the 5' end of the mRNA but, nevertheless, ORF 5b is
efficiently translated. Our conclusion is that translation
ofORF 5b is mediated by the internal entry of ribosomes.
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3042
V. T h i e l a n d S. G. S i d d e l l
p5ab/BstElI
p5ab/BamHI
5a (107)
5a (107)
5b (88)
T70RF5a
ORF5b
BamHI
BstEIl
T70RF5b
p5b/BamHl
5b
(88)
BamHI
pZ5ab/BamHI Z (376)
T7
ORFZ
ORF5a ORFSb
5a (107) - - ~
5b (88)
BamHI
T7
pZ/BamHI
ORFZ
Z (376)
BamHI
ORF5b ~
p5bN/HindIII 5bTM(337)
T7
p5abN/HindIII 5a ( 1 0 7 )
+ - - - ' ~ J ~
~
5bTM( 3 3 7 )
I
II
pZ5abN/HindIII Z (376)
5a (107)
5bTM(337)
p5bl°/BamHI
5b I° (99)
HCVN
T7
ORF5b t°
p5abl°lBamHI 5a (107)
5bj° (99)
pZ5abl°/BamHI Z (376)
5a (107)
5b I° (99)
C,C]'YAGCAAGAATGCTT(ATG)
7ACATTA~.~CrA~'TCC
t
BamHI
Fig. 1. Structure of the transcription plasmids. The plasmids used in
this study are illustrated. The position of the T7 promoter is shown (D)
and ORFs are indicated as boxes; ORF Z, (IS]); ORF 5a, (D); ORF
5b, (m); HCV-N, ([]). The positions of relevant restriction enzyme
recognition sites are shown, and the size, in amino acids, of potential
translation products encoded in mRNAs derived from the plasmids are
given in parentheses. The broken line represents sequences upstream of
ORF 5b in the plasmids p5b N, p5ab N, pZ5ab ~, p5b TM, p5ab TM and
pZ5ab TM.
Methods
Construction of recombinant plasmids and in vitro RNA synthesis. To
construct recombinant plasmids corresponding to the unique region of
MHV mRNA 5, the cDNA insert of pJMS 1010 (Ebner et al., 1988) was
isolated by excision with PstI, digested with Ddel and, after treatment
with T4 DNA polymerase, a 630 bp fragment was cloned into Smallinearized pGEM1. In the resulting construct, p5ab, the initiation
codon of ORF 5a lies 37 bp downstream of the cloning site. For the
construction of a plasmid corresponding to ORF 5b alone, pJMS1010
DNA was digested with PstI and the isolated cDNA insert was further
digested with TaqI and RsaI. The fragment containing the coding
region of ORF 5b was treated with T4 DNA polymerase and cloned
into SmaI-linearized pGEM1 to produce the construct p5b.
In order to place an ORF upstream of ORF 5a, PCR was carried out
with oligonucleotides OLV 25 (5' CACAGGAGCTCACCAATGGCCATGATTACGAATT 3') OLV 26 (5' GGGATCCGAGCTCTTAGTTAGTTAATCCTGCACCATCGTCTG 3') and SmaI-linearized
pROS DNA (Ellinger et al., 1989). The PCR product contains a SacI
restriction site upstream of the Kozak consensus sequence ACCATGG,
which is in frame with a fragment of the fl-galactosidase gene from
nucleotides 6 to 1125, a stop codon that terminates this ORF and a
second SacI restriction site. After digestion with SacI this fragment was
cloned into Sacl-linearized pGEM 1 and p5ab. The resulting constructs
were designated pZ and pZ5ab, respectively.
To enlarge ORF 5b, a cDNA fragment of pHCV-N (J. Ziebuhr,
unpublished results), which contains the N gene of the human
coronavirus (HCV) strain 229E, was cloned into the ORF 5b coding
region. The 718 bp EcoRl fragment of pHCV-N was treated with the
Klenow fragment of DNA polymerase I and ligated to BamHI linkers.
The BamHI restriction sites ofp5b, p5ab and pZ5ab were eliminated by
linearization, treatment with the Klenow fragment and religation. The
plasmids were then linearized with BstEII, treated with Klenow
fragment and alkaline phosphatase and ligated to BamHI linkers. The
plasmids and the cDNA fragment were then digested with BamHI and
ligated together to produce the constructs p5b N, p5ab N and pZ5ab N.
The enlarged ORF 5b, designated ORF 5b z~, contains 27 codons from
the 5' end of ORF 5b, 241 codons from the HCV N protein gene, 63
codons from the 3' end of the ORF 5b and a total of six artefactual
codons.
To increase the methionine content of the ORF 5b product, eight
A U G codons were inserted into the ORF 5b coding region. First, in
vivo recombination-PCR mutagenesis (Yao et al., 1992) was used to
generate a Bpu 1102I restrition site 10 nucleotides upstream of the ORF
5b termination codon in p5b, p5ab and pZ5ab. Oligonucleotides OLV
9 (5' CAGCAATAAACCAGCCAGAAG 3'), OLV 10 (5' CGGCTGGCTGGTTTATTGCTG 3'), OLV 28 (5' TATAGGCTTAGCATTATGAGTAGTACCACTCAGGGA 3') and OLV 29 (5' ATAATGCTAAGCTTATATTATCATCCACCTCTAACG 3') were used for this
purpose. The constructs p5b TM, p5ab 1° and pZ5ab I° were then generated
by exchanging the Bpul 1021-BamHI fragment of the derived plasmids
with a synthetic double-stranded DNA fragment comprised of
the annealed oligonucleotides OLV 30 (5' TTAGCAAGAATGCTT(ATG)TACATTATGACTAGTGGG 3') and OLV 31 (5' GATCCCCACTAGTCATAAAGT(CAT)vAAGCATTCTTGC 3').
The nucleotide sequences of all the plasmids described above were
confirmed by dideoxynucleotide chain-termination sequencing. Fig. 1
summarizes the structure of these plasmids and indicates the size of
potential translation products encoded in mRNAs derived from them.
For in vitro transcription, plasmid DNAs were linearized with
restriction enzymes as shown in Fig. 1 and RNA was synthesized with
T7 RNA polymerase in the presence of the synthetic cap structure
m7G(5')ppp(5')G (Pharmacia) as described previously (Contreras et
al., 1982; Melton et al., 1984).
Preparation of L cell ribosomal wash factors. L929S cells (1 x 109;
European Collection of Animal Cell Cultures) were used to prepare
ribosomal wash factors as described by Schreier & Staehelin (1973).
The crude initiation factors (IF) fraction, adjusted to 300 A260/ml, was
used without further purification.
Preparation of an L cell lysate and in vitro translation. The L cell
lysate was prepared from L929S cells as previously described (Siddell,
1983). Prior to translation, the lysate was treated with micrococcal
nuclease. A 340 lal incubation mixture containing 20 mM-Hepes pH 7.0,
85 mM-KCI, 1.2 mM-MgClz, 2 mM-dithiothreitol, 1 mM-ATP, 0.2 mMGTP, 50 mM-spermine, 250 mM-spermidine, 12 mM-creatine phosphate,
0.1 mg creatine kinase, 0.2 mM of each amino acid except methionine,
1 mM-CaCI~, 5.5 units of micrococcal nuclease (11000 units/mg), 20 ~tl
of L cell ribosomal wash factors and 200 p.1 of the L cell lysate was
incubated at 20 °C for 10 min. After adding EGTA pH 7.0 to a final
concentration of 2 mM, the mixture was incubated on ice for 5 min.
Translation reactions consisted of 20 111of the nuclease-treated L cell
lysate supplemented with 3 gl of [35S]methionine (15 mCi/ml), 1 gl calf
liver tRNA (10mg/ml) and either 1 gl of water, 1 gl of water
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M H V m R N A 5 translation
f
(a)
,o, .,,¢# + # J
(c)
(b)
200K97K69K
..........
46K
Z
:
,
!
~i~i~~i~~
30K ~
3043
v. . . . . . . . . . . .
-Z
• 5bN
-X
,,o i
21.5K14K~
12.5K
6.5K
m
i
•
- - - - "
_
3a
~
i~
~'*°
_
_
.
.
......
D
-5a
!
__
~_
"-5a
Z5b 10
Mll
2 3 4 5 6 7 M2
MI l 2 3 4 5 6 7 M2
M1 1 2 3 4 5 6 7 M2
Fig. 2. In vitro translation of mRNAs derived from transcription plasmids. The translation products of cell-free protein synthesis were
electrophoresed in 17 % SDS polyacrylamide gels and detected by autoradiography. The Mr markers (Amersham) were; aprotinin,
6.5K; cytochrome C, 12.5K; lysozyme, 14-3K; trypsin inhibitor, 21.5K; carbonic anhydrase, 30K; ovalbumin, 46K; BSA, 69K;
phosphorylase b, 97.4K and myosin, 200K. These are contained in lanes M 1 (low Mr markers) and lanes M2 (high Mr markers). Lanes
2 contain poly(A)-RNA from MHV-infected cells. Other lanes contain the synthetic mRNAs indicated•
containing 2.5 pmol (0.4 to 1.8 ~g) of synthetic mRNA or 1 gl of water
containing 0-5 gg of polyadenylated RNA from MHV-infected cells.
Incubations were carried out at 34 °C for 2 h and 15 lal aliquots of the
translation reaction were electrophoresed on discontinuous, 17%
SDS-polyacrylamide gels as described by Laemmli (1970). The
radioactivityincorporated into the translation products was determined
using a PhosphorImager (model 400E; Molecular Dynamics). Cytoplasmic, poly(A) RNA from MHV-infected cells was prepared as
previously described (Siddell, 1983).
Results
Identification o f M H V m R N A 5 translation products
In order to identify the translation products of the M H V
O R F s 5a and 5b, m R N A s were synthesized from BstEIIlinearized p5ab ( m R N A 5a) and BamHI-linearized p5b
( m R N A 5b). In vitro translation of m R N A 5a directed
the synthesis of a polypeptide with an apparent M r of
12000 (Fig. 2a, b and c, lane 3). The predicted Mr of the
O R F 5a product (107 amino acids) is 12 500. The in vitro
translation of m R N A 5b directed the synthesis of a
polypeptide with an apparent M r of 14000 (Fig. 2a, lane
4). The predicted M r of the O R F 5b product (88 amino
acids) is 10200.
The discrepancy between the expected and observed
electrophoretic mobility of the O R F 5b product was
surprising. We therefore performed two further experiments to confirm it's identity. The in vitro translation of
m R N A derived from BamHI-linearized p5b TM ( m R N A
5b TM) directed the synthesis of a polypeptide with an
apparent M r of 11000 (Fig. 2 b, lane 4). The predicted M r
of the O R F 5b 1° product (99 amino acids) is 11 500.
Thus, despite the incorporation of 11 additional amino
acids in the O R F 5b translation product, its electrophoretic mobility had increased. In the translation
products directed by m R N A 5b 1°, we also observed a
protein of 33 000 apparent M r (indicated as X in Fig. 2b,
lane 4). We assume that this is an aggregated form of the
O R F 5b a° product.
Secondly, the identity of the O R F 5b product was also
confirmed by the translation of m R N A derived from the
HindIII-linearized plasmid p5b N. The in vitro translation
of m R N A 5b z~ (Fig. 2 c, lane 4) directed the synthesis of
a polypeptide with an apparent Mr of 36000. The
predicted M r of the O R F 5b N product (337 amino acids)
is 37200.
In vitro translation of the bicistronic m R N A 5ab
The in vitro translation of m R N A derived from BamHIlinearized p5ab ( m R N A 5ab) directed the synthesis of
both the O R F 5a and O R F 5b products (Fig. 2a, lane 5).
This result is consistent with the idea that the M H V
m R N A 5 is functionally bicistronic (Skinner et al., 1984;
Budzilowicz & Weiss, 1987; Leibowitz et al., 1988). In
the experiment shown, the ratio of O R F 5a to O R F 5b
products translated from m R N A 5ab was about 8:1,
showing that, at least in this system, O R F 5a is more
efficiently translated than O R F 5b.
To strengthen the conclusion that the m R N A 5ab is
functionally bicistronic we carried out two further
experiments. Firstly, we translated m R N A derived from
BamHI-linearized p5ab TM ( m R N A 5abl°). In this case,
the detection of the O R F 5b 1° product shouid be
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3044
V. Thiel and S. G. Siddell
enhanced by the incorporation of additional radioactivity and, indeed, this result can be clearly seen in Fig.
2(b), lane 5. Secondly, we translated mRNA derived
from HindIII-linearized p5ab N, (mRNA 5abN). In this
experiment, the ORF 5b ~ product with an apparent M r
of 36000 was also clearly detected (Fig. 2c, lane 5).
In vitro translation o f O R F 5b but not O R F 5a f r o m a
tricistronic m R N A containing an additional upstream
ORF
To test whether ORF 5b can be expressed independently
of ribosomes that enter from the 5' end of the mRNA, we
translated mRNA derived from BamHI-linearized pZ5ab
(the tricistronic mRNA Z5ab). The result is shown in
Fig. 2 (a), lane 6. As expected the upstream ORF Z was
expressed, resulting in the synthesis of a polypeptide with
an apparent Mr of 51000. The predicted M,. of the ORF
Z product (376 amino acids) is 41 500. Importantly, no
ORF 5a product was detected in the translation reaction.
This indicates that very few, if any, ribosomes scan
through the upstream ORF Z and initiate the synthesis
of an ORF 5a polypeptide. In contrast, the ORF 5b
product was readily detected. The amount of ORF 5b
product synthesized from the tricistronic mRNA Z5ab
was similar to the amount of ORF 5b product expressed
from an equimolar concentration of the bicistronic
mRNA 5ab (compare Fig. 2a lanes 5 and 6).
To rule out the possibility that a polypeptide of 14000
apparent Mr can be synthesized by the aberrant
translation of ORF Z (for example, premature termination or internal initiation), we translated mRNA
derived from BamHI-linearized pZ (mRNA Z). As
expected this mRNA directed the synthesis of the ORF
Z gene product and no polypeptide with an apparent M r
of 14000 was detected (Fig. 2a, b and c, lane 7).
To strengthen the conclusion that ORF 5b, but not
ORF 5a, is translated from a tricistronic mRNA
containing an additional upstream ORF, we carried out
two further experiments. Firstly, we translated mRNA
derived from BamHI-linearized pZ5ab TM (mRNA
Z5abl°). The result is shown in Fig. 2(b), lane 6. In this
translation reaction, the ORF Z product and the ORF
5b TM product were easily identified. Again, using equimolar concentrations of mRNA, approximately equal
amounts of ORF 5b TM products were expressed from the
bicistronic and tricistronic mRNAs, mRNA 5ab TM and
mRNA Z5ab TM (compare Fig. 2b lanes 5 and 6). The
ORF 5a product was not expressed from the tricistronic
mRNA Z5ab 1°. Secondly, we translated mRNA derived
from HindIII-linearized pZ5ab ~ (mRNA Z5ab-~). The
result is shown in Fig. 2(c), lane 6. Again, the ORF Z
product and the ORF 5b ~ product were easily identified
but a translation product for ORF 5a was not detected.
Discussion
The results presented in this paper lead us to the
conclusion that, in the context of the MHV mRNA 5
unique region, the initiation of protein synthesis on ORF
5b can occur independently of ribosomes that enter from
the 5' end of the mRNA. This has been shown by the
translation of the tricistronic mRNAs, Z5ab, Z5ab 1° and
Z5ab N, where the 5'-proximal and 5'-distal ORFs, ORF
Z and ORF 5b/bl°/b N, are translated, whilst the internal
ORF, ORF 5a, is translationally inactive. Clearly, the
upstream ORF Z, provides an effective barrier to
scanning ribosomes but does not prevent the initiation of
ORF 5b translation.
Our conclusions are based upon the translation of
synthetic mRNAs in vitro. As always, it can be argued
that the results are due to peculiarities of the in vitro
system or, for example, the degradation of mRNA
during the translation reaction. However, we have taken
care to use a translation system derived from a murine
cell line and, at least in the case of the tricistronic
mRNAs Z5ab, Z5ab TM and Z5ab N, the mRNA degradation interpretation would require a ribonuclease
activity that specifically renders the ORF 5b initiation
codon accessible to scanning ribosomes.
Furthermore, our results are not the first evidence for
the internal entry of ribosomes on a coronavirus mRNA.
Liu & Inglis (1992), using an approach very similar to
that described here, have concluded that the tricistronic
mRNA 3 of IBV encodes three proteins, 3a, 3b and 3c,
and that the translation of the most distal ORF 3c is
mediated by a cap-independent mechanism involving
internal initiation. Taken together, these data strongly
suggest that the translation of the coronavirus sM
proteins, i.e. the ORF 3c product of IBV and the ORF 5b
product of MHV (Cavanagh et al., 1994), involves the
internal entry of ribosomes on a polycistronic mRNA.
Clearly, in vivo studies using reporter gene constructs and
the naturally occurring mRNAs will be required to
strengthen this conclusion.
The initiation of protein synthesis by internal ribosome
entry has been most extensively studied by picornavirus
RNAs (for a recent review see Meerovitch & Sonenberg,
1993). In this case, ribosome entry is mediated by a
region in the 5' untranslated region of the genomic RNA,
the so-called 'internal ribosome entry site' (IRES) or
'ribosome landing pad' (RLP). The function of the
IRES/RLP is dependent upon conserved primary sequence, for example a polypyrimidine tract (Pestova et
al., 1991), as well as base-pairing interactions that give
rise to extensive RNA structures able to bind transacting factors and, directly or indirectly, components of
the translational machinery (Jang & Wimmer, 1990;
Meerovitch et al., 1993).
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M H V m R N A 5 translation
Recently, Le et al. (1992, 1993) have proposed a
general model of conserved tertiary structural elements
in picornavirus IRES/RLPs. This model includes RNA
stem-loops that can be modelled into compact superstructures involving pseudoknots together with possible
base-pairing interactions between the picornavirus IRES/
RLP and 18S rRNA. Most interestingly, they have
recognized similar structural elements in the unique
region of the IBV mRNA 3 (Le et al., 1994) and suggest
that these features may be the hallmark of elements that
mediate the internal initiation of cap-independent translation.
An obvious question is whether or not the same
features can be identified in the unique regions of the
MHV mRNA 5. This question can be approached
theoretically with software capable of predicting tertiary
RNA structures and experimentally using chemical and
enzymatic probing in combination with covariant nucleotide mutation analysis. We are currently using both
approaches. Furthermore, it will be of interest to examine
interactions between the putative MHV mRNA 5
IRES/RLP element and cellular proteins. The in vitro
translation system we have used is prepared from a
murine cell line and it may be possible to search for
proteins that specifically interact with the MHV mRNA
5 unique region directly in this system.
In the long term, we are interested in the biological
relevance of internal ribosome entry on the MHV mRNA
5. The MHV ORF 5b product is believed to be an
essential structural protein of the virus, but its functional
role(s) in the replication cycle is still unknown. Why, in
contrast to all other MHV subgenomic mRNAs, does
the unique region o f m R N A 5 encode two proteins? And
why is the initiation of ORF 5b translation mediated by
a complex mechanism such as internal ribosome entry?
Also, if the putative IRES/RLP element of mRNA 5
encompasses ORF 5a, it is difficult to envisage translation of both proteins from the same mRNA, at least at
the same time. If there are two forms of the MHV
mRNA 5 in the infected cell, then how are they
distinguished? The answers to these and other questions
must await further experiments.
3045
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This work was supported by the Deutsche Forschungsgemeinschaft
Conserved tertiary structural elements in the 5' nontranslated region
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