Journal of General Virology (1994), 75, 2141-2149. Printedin Great Britain
Review
2141
article
Plant viruses as model systems for the study of non-canonical translation
mechanisms in higher plants
Wolfgang Rohde,* Andrea Gramstat, Jiirgen Schmitz, Eckhard Tacke and Dirk Priifer
Max-Planck-Institut fiir Ziichtungsforschung, Carl-von-Linnd-Weg 10, D-50829 K6ln, Germany
During protein synthesis the translational apparatus of
the cell performs three basic steps, (i) initiation by the
recognition of translational start codons, (ii) elongation
of the nascent polypeptide chain by the sequential
decoding of triplets within an open reading frame (ORF)
and (iii) termination at appropriate stop codons. This
canonical sequence of events is controlled firstly by the
primary structure of mRNAs and the interactions of
coding triplets with the anticodons of tRNAs. In
addition, for prokaryotes ample evidence has
accumulated to demonstrate that ribosomes, both by
themselves and through interaction with the elongation
factor EF-Tu, play an important role in the initial
selection and proofreading of the appropriate aminoacyltRNA (Maden, 1993; Powers & Noller, 1994). Lastly,
translational efficiency and specificity is modulated by 5'
or 3' untranslated mRNA regions, in that primary
(enhancer sequences), secondary (stable stem-loops) and
tertiary (pseudoknots) structures or small ORFs in the
untranslated region influence the expression of the major
ORF(s) (for a recent review on post-transcriptional
regulation of plant gene expression see Gallic, 1993).
Also proteins have a part to play in modulating
translational efficiencies, as in the case of the cauliflower
mosaic virus (CaMV) trans-activator protein TAV (de
Tapia et al., 1993). This possibly occurs by direct
interaction of mRNA with the protein and/or rRNA on
the small and large ribosomal subnnits, as postulated for
EF-Tu in the prokaryotic system (Powers & Noller,
1994).
With reference to the primary structure of mRNA,
translational mechanisms that allow an alternative
reading of the genetic code have been studied in great
detail in prokaryotic and animal systems as well as in
yeast (for a recent review see Farabaugh, 1993). This
article focuses on higher plants and plant virus model
systems that have allowed the dissection of some of the
aspects contributing to the non-canonical translation of
mRNA. By combining both recently published data and
unpublished observations we hope it will stimulate
further investigations in this area.
0001-2428 © 1994SGM
Initiation
Initiation of protein synthesis on eukaryotic mRNAs is
generally dependent on the presence of a cap structure,
with translation starting at the first 5' AUG triplet
displaying an optimum context for initiation. The
mechanism by which this selection process occurs has
been postulated to proceed by the recognition of the
RNA 5' end through a 'recognition' complex and
subsequent linear scanning along the RNA. This complex
was originally proposed to consist of the 40S ribosomal
subunit and various initiation factors (for reviews see
Kozak, 1989a, 1992). Variations on the theme have been
deduced from studies on picornaviruses in animal cells
and on the plant virus, cowpea mosaic virus (CPMV).
These have indicated that complexes of initiation factors
of the elF4 (Sonenberg, 1991) or the eIF2 groups
(Thomas et al., 1991, 1992), rather than the 40S subunit
itself, perform the scanning process, with ribosomes
entering at a later stage.
One alternative to this distal entry of part of the
translational machinery and its linear migration is the
internal entry of ribosomes guided b y ' internal ribosome
(a)
28K
1~'~ 2a I
70K
(b)
67K
] 2b
23K 56K
I 1143lI 5
17K
12K
25KI"fl
223:
12K
i
o
[ ] 19K Poly(A)
1000 2000 3000 4000 5000 6000 7000 8000
I
I
I
I
I
I
I
I
Nucleotides
Fig. 1. Schematic structure of the PLRV (a) and PVM (b) RNA
genomes. Numbers within boxed areas indicate the various ORFs.
Numbers outside the boxed areas show the Mr values of the
corresponding geneproducts.
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2142
W. R o h d e and others
entry sites' (IRES; Herman, 1989). A second alternative
to continuous ribosome migration has been recently
proposed from studies on CaMV gene expression
(Ffitterer et al., 1993). In this 'shunt' mechanism the
initial scanning complex (possibly a complex of
ribosomes and initiation factors) starts linear migration
at the CaMV 35S RNA 5' end, and continues until a cisacting element in the viral leader sequence forces the
complex to bypass more than 300 nucleotides of sequence
before scanning is resumed.
Initiation at non-A UG codons
Translation initiation at codons other than AUG has
been known to occur for some time in bacteria and has
been recognized more recently in eukaryotes. For
example, in mammalian systems ACG and other triplets
may be used for the initiation of protein biosynthesis
(Beccera et al., 1985; Curran & Kolakofsky, 1988;
Peabody, 1987, 1989; Mehdi et al., 1990) and insect cells
make very efficient use of AUU, as demonstrated in the
baculovirus system (Beames et al., 1991). Several protooncogenes start translation at a CUG triplet or at several
triplets as seen, for example, with the mouse pim-1
oncogene. Two serine-threonine protein kinases are
encoded by this gene as a result of alternative translation
initiation at CUG and AUC (Saris et al., 1991).
The first, somewhat indirect, evidence for non-AUG
initiation in plants was obtained by Hohn and
co-workers (Schultze et al., 1990), and this initial
observation was followed up by studies on the translation
regulation of CaMV 35S RNA using transient expression
of reporter gene fusions in protoplasts (Gordon et al.,
1992). Among nine triplets that differ from AUG by a
single base substitution, ACG, CUG and GUG were the
most efficient codons in initiating translation, although
their relative strength varied from 15 to 30 % of AUG
activity. The same group reported non-AUG initiation in
the expression of ORF I of the pararetrovirus rice tungro
bacilliform virus (RTBV). Evidence has been obtained
by transient expression of appropriate reporter gene
fusions that ORF I synthesis initiates at an AUU codon,
although the efficiency of translation is only 10 % when
compared to constructs in which AUG replaces the
AUU codon (Rothnie et al., 1994).
The AUU codon has also been identified in another
plant virus sequence, as an initiator triplet both in vitro
and in vivo, but is apparently much more efficient
compared with RTBV AUU initiation or in competition
with the wild-type AUG codon(s) (Tackle et al., 1994). In
these experiments the tobacco mosaic virus (TMV; U1
strain) 5' leader (omega) sequence of 68 nucleotides,
which enhances translation in both prokaryotes and
eukaryotes (Gallic & Walbot, 1992), was fused to the
gene for the 17K protein of potato leafroll luteovirus
(PLRV) (Fig. l a), which is the putative movement
protein (Tacke et al., 1993). In protein extracts from
transgenic potato lines expressing this transgene, anti17K sera detected 17K protein (initiation at the 17K
AUG) as well as a second immunoreactive protein some
3K larger that accumulated in the plant to approximately
the same amounts. Rearrangement of the transgene
during stable transformation was excluded at least for
one transgenic potato line. The two integrated transgene
copies of line P17-1 were recloned and their coding
region was shown to be identical by sequence analysis.
Mutational analysis of the omega sequence identified one
of two in-frame AUU triplets serving as an efficient
translational start codon and causing synthesis of the
N-terminally extended 17K protein.
The translation-enhancing activity of the omega
sequence is promoted by the recently identified core
element of the 25 nucleotide (CAA)n region, together
with at least one copy of an eight-base direct repeat
(Galtie & Walbot, 1992). Its direct interaction with
ribosomes has been shown by RNA protection experiments, which identified the first AUU as the most distal
T-located triplet within the RNA ribosome complex
(Konarska et al., 1981; Tyc et al., 1984). The TMV
enhancer in the chimeric omega-17K construct also
promotes efficient non-AUG translation initiation by
decoding the second of two AUU triplets in frame with
the 17K ORF. It remains to be seen whether, in the
natural TMV context (in which the AUU(s) are in frame
with the 126K ORF1 protein), the omega sequence
performs the same function.
The expression in transgenic plants of two proteins
from the PLRV 17K ORF differing in their aminotermini as a result of translation initiation at AUG and
non-AUG codons is reminiscent of the situation in
mammals and insects, with a single gene encoding two
polypeptides that differ only at their N termini (Saris et
al., 1991; Beames et al., 1991). Expression of such
modified proteins may have functional consequences, as
in the case of 17K mutant and wild-type protein
expression in the same cell of the transgenic plant. The
PLRV 17K protein, the putative movement protein of
the virus, is a single-stranded nucleic acid-binding protein
(Tacke et al., 1991). The domain responsible is located in
the basic C-terminal half, and the acidic N terminus
exhibits the capacity for homodimer formation via an
amphipathic a-helix (Tackle et al., 1993). Mutant and
wild-type 17K proteins can form heterodimers that are
apparently non-functional, as transgenic plants expressing both proteins are resistant to virus infection (Tacke
et al., 1994). Engineered virus resistance by the ex-
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Review." plant virus translation mechanisms
pression of a non-functional movement protein has also
been reported in a transgenic tobacco plant using a
mutant TMV 30K movement protein (Lapidot et al.,
1993; Malyshenko et al., 1993).
The example of an N-terminally modified PLRV 17K
protein demonstrates how biological functions can be
modulated as a result of non-canonical translation.
Another example of non-AUG initiation in the
expression of viral genes appears to be the synthesis of
a 28K protein from RNA2 of soil-borne wheat mosaic
virus (SBWMV), the type member of the furovirus group
(Shirako & Wilson, 1993). Translation initiates at the
AUG of the gene encoding the 19K capsid protein (CP)
and at an upstream in-frame non-AUG codon. This
initiator triplet has been tentatively assigned to an inframe GUG codon located some 30 codons upstream of
the 19K CP AUG. Although the in vivo role of this
modified CP has not been elucidated, its mode of
synthesis is reminiscent of the N-terminal extension of
the PLRV 17K protein in transgenic plants.
Internal initiation
Initiation of protein synthesis at internally located
translational start.codons has been shown to occur by
leaky scanning, the guiding of internal ribosome entry by
IRES or the shunt mechanism. Leaky scanning has been
postulated to operate in the expression of various viral
genes. One example is the translation of plum pox
potyvirus genomic RNA into the polypeptide precursor,
which initiates at nucleotide position 147 despite the
presence of an upstream AUG triplet in the same frame
at position 36 (Riechmann et at., 1991). A more detailed
analysis has identified the internal initiation mechanisms
in CPMV M gene RNA translation. Both leaky scanning
as well as internal entry govern the synthesis of a 95K
protein from CPMV M RNA (Verver et al., 1991;
Thomas et al., 1991). M RNA encodes two C-coterminal
proteins of t05K and 95K, which are translated from inframe AUGs at positions 161 and 512 respectively
(Holness et al., 1989; Verver et al., 1991). The sequence
between positions 16t and 512 mediates internal
ribosome entry during in vitro translation of a bicistronic
mRNA in a wheatgerm or reticulocyte system, although
this view has been challenged on the basis of in vivo
experiments in an animal system (Belsham &
Lomonossoff, 1991).
The genomes of luteoviruses contain several other
examples of internal initiation, for example in PLRV
(Tacke et al., 1990; Prfifer, 1989), beet western yellows
virus (Veidt et al., 1988) and barley yellow dwarf virus
(BYDV; Dinesh-Kumar et aI., 1992; Dinesh-Kumar &
2143
Miller, 1993). The 3' gene cluster is separated by a small
intergenic region from the 5' genes, as depicted in
Fig. 1(a) for PLRV, and genes are translated from a
subgenomic RNA (sgRNA1). Both PLRV and BYDV
encode a 17K protein (ORF4; see above), which is
encoded within the luteovirus CP (ORF3). Transient
expression assays with /%glucuronidase (GUS) gene
fusions in tobacco and potato protoplasts showed that
translational initiation efficiency at the PLRV 17K AUG
codon is sevenfold higher than initiation for CP synthesis
(Prfifer, 1989; Tacke et al., 1990). These results corroborate the recent demonstration of the accumulation
of 17K protein in PLRV-infected cells (Tacke et al.,
1993). With BYDV, similar in vivo experiments resulted
in only a two- to threefold preference for 17K synthesis.
Mutations of the AUG context for both CP and 17K
suggested that efficient initiation at the 17K AUG is a
prerequisite for optimum CP synthesis. With reference to
the known pausing of the 80S ribosomal complex in the
course of formation, the authors propose that this
ribosome stalling at the 17K AUG allows the unwinding
of the upstream stable stem-loop structure, which in
BYDV (and PLRV) RNA contains the CP AUG codon
(Dinesh-Kumar & Miller, 1993).
The pararetrovirus RTBV represents one example of a
combination of both transcript processing and alternative translation initiation. Its ORF IV product is
synthesized from a processed mRNA that results from
the removal of a 6.3 kb intron sequence (Ffitterer et al.,
1994). During splicing the 5' end of ORF IV is arranged
in frame with a small ORF in the RTBV leader sequence
and, in a protoplast test system, some 90 % of the overall
protein synthesis initiates at the small ORF AUG,
whereas only 10% of protein synthesis starts at the
internal ORF IV AUG codon. In the light of this finding
the authors discuss the possibility that RTBV synthesizes
two ORF IV proteins with differing N-terminal
sequences.
The extremely large differences in translational
efficiencies observed for various systems throw some
doubt on the general validity of in vivo as well as in vitro
data with respect to the actual situation during virus
replication in the plant. In vitro translation systems may
be devoid of, or suboptimal for, important translation
factors, and they are dramatically influenced by salt
conditions (Kozak, 1989b; Jackson, 1991). Also, in vivo
data have to be interpreted with care. Reporter gene
fusions used for in vivo transient or stable expression
experiments may also yield erroneous results if transactivating proteins, the translational product(s) itself or
3'-located sequences that are not present on the
constructs for transient expression or stable transformation regulate translational efficiencies. In addition,
reporter gene expression in transgenic plants may be
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2144
W. R o h d e and others
(a)
UCU UUU
Arg
UCU UUU
Lys
Arg
I
Peptidyl
Lys
I
Peptidyl
(b)
~
~
~
-~--AAU AGA AAA UGA--~
Ill Ill
UCU UUU
A~g
aP~gtirdaYnls~ran~ifer
"~
f
~
~
~~+GTP
"~--AAUAGA AAA UGA---~
Ill
UUU
Lys
-~
r
~
!
Arg
I
A~g
I
Peptidyl
~
"~-AA UAG AAA AUG A--~
III Ill
UUU UAC
Lys
Lys
Peptidyl
~
Met
Peptidyl
Fig. 2. Models for - 1 ribosomal frameshifting at the PVM frameshift site. (a) Simultaneous slippage of peptidyl- and aminoacyltRNAs according to Jacks et al. (1988). This model cannot apply, as interaction of peptidyl-tRNAArgwith a UAG stop codon does
not take place. The boxed area represents the string of four adenosine nucleotidesand the shaded triplet is the translational start codon
for the synthesis of the 12K protein. (b) P site slippageproposed for the CP-12K - 1 ribosomal frameshift. Peptidyl-tRNA~'S slips on
the adenosine string into the - 1 frame and allows methionyl-tRNA~I~tdecoding of the AUG codon now present at the A site. E, Exit
site; RF, release factors.
subject to translational control depending on age, growth
conditions or tissues of the plants (Hensgens et al., 1992).
Ribosomal
frameshifting
Ribosomal frameshifting represents a further strategy
for the regulation of gene expression during translation,
in that two or more proteins are produced from a single
coding sequence. Two components have been identified
that contribute to the efficient function of the signal in
frameshifting. One is a ' shifty' heptanucleotide sequence
of the general structure X XXY YYZ, which has been
proposed to allow the simultaneous slippage of peptidyland aminoacyl-tRNAs at the P and A sites of the
ribosome (Jacks et al., 1988). Secondly, specific RNA
structures, generally located immediately downstream of
the shifty sequence, have been proposed or identified that
promote efficient frameshifting, as for example a
pseudoknot in the O R F l a - O R F l b region of avian
infectious bronchitis coronavirus (Brierley et al., 1989).
RNA pseudoknots or stem-loop structures apparently
facilitate frameshifting by impeding ribosomal movement (Tu et al., 1992). Although pseudoknot participation in efficient frameshifting (as well as in amber stop
codon suppression) has been proposed for a large variety
of viral systems (Ten Dam et al., 1990), in some cases the
slippery heptameric sequence alone appears to be
sufficient for triggering the frameshift event, for example
in human immunodeficiency virus type 1 (Wilson et al.,
1988).
Ten Dam et al. (1990) proposed the sequence U U U A
AAU in the ORF2a-ORF2b overlap region of PLRV as
a potential slippery sequence for mediating ribosomal
frameshifting, and this proposal was verified by mutation
analysis of this region (Prfifer et al., 1992; Kujawa et al.,
1993). For the German isolate, participation of a
pseudoknot structure was excluded (Prtifer et al., 1992).
Instead a stable stemqoop structure was identified as the
second component of the frameshift signal. In BWYV
however, a G GGA AAC sequence operates in conjunction with a pseudoknot (Garcia et al., 1993). For the
BYDV PAV isolate evidence for a pseudoknot in
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Review: plant virus translation mechanisms
addition to a G GGU UUU slippery sequence has not
been documented (Brault & Miller, 1992). The position
at which ribosomal frameshifting in the 582 nucleotide
overlap of PLRV ORF2a-ORF2b takes place is noteworthy. The frameshift occurs some 125 nucleotides
downstream from the ORF2b start, and the ORF2b
sequence in the stem-loop following the heptanucleotide
signal codes for a cluster of basic amino acids. This
region is a domain for nucleic acid binding and it may
represent the site on the viral replicase for the binding of
the PLRV RNA template during replication (Prtifer et
al., 1992).
Another example of a - 1 ribosomal frameshifting
event in the synthesis of a putative RNA polymerase is
the translation of the bipartite dianthovirus red clover
necrotic mosaic virus RNA1 (Xiong et al., 1993 ; Kim &
Lommel, 1994). The 88K polymerase is encoded by
RNA1 as a trans-frame protein by - 1 ribosomal
frameshifting from the N-terminal 27K ORF to a 57K
ORF. Frameshifting occurs on the shifty heptanucleotide
G GAU UUU that is followed by the 27K UAG stop
codon. Mutation analysis of a pseudoknot that was
predicted to form within the 110 nucleotides downstream
of the frameshift site had no effect on frameshifting
efficiency or virus infectivity, but destabilization of a
stable stem-loop structure within this region resulted in
the loss of frameshifting and infectivity (S. Lommel,
personal communication).
An unusual mechanism of - 1 ribosomal frameshifting
appears to operate in the expression of the carlavirus
potato virus M (PVM) 12K nucleic acid-binding protein
(Fig. lb); Gramstat et al., 1990, 1993; A. Gramstat,
D. Prtifer & W. Rohde, unpublished results). It is
presumably translated from a dicistronic mRNA that is
used for the synthesis of both the 5'-located gene coding
for the 34K PVM CP (ORF5) and the 12K protein. The
CP ORF terminates in UGA, which is part of the 12K
AUG initiation codon (Fig. 2). Internal initiation at this
(and the second) AUG in vitro results in the formation of
the 12K polypeptide. In addition, the protein is produced
as a CP-12K trans-frame protein by - 1 ribosomal
frameshifting. Mutational analysis identified a homopolymeric string of four A nucleotides (Fig. 2a), which
together with the CP stop codon were required for
efficient frameshifting. The stop codon was indispensable
for frameshifting, as substitution of UGA by the sense
codon UGG, but not by the stop codons UAA or UAG,
abolished trans-frame protein formation. Single base
substitutions in the A string dramatically reduced
frameshift efficiencies, whereas replacement of the string
by four U residues had a stimulatory effect. The amber
stop codon preceding the frameshift signal in the - 1
frame and thereby providing a very narrow window for
frameshifting in the wild-type sequence (Fig. 2 a) did not
2145
participate in the frameshift, and pseudoknots or stable
stem-loop structures were not involved either. Thus the
slippery PVM sequence AAAAUGA is the first frameshift signal with a shifty stop codon to be analysed in a
eukaryotic system.
This unusual signal requires a novel mechanism of
frameshifting. Ribosomal frameshifting by - 1 is generally accepted to proceed by the simultaneous slippage
of peptidyl- and aminoacyl-tRNAs at the frameshift
signal (Fig. 2 a) thereby allowing the decoding of a new
set of codons in the - 1 frame (Jacks et al., 1988). This
model cannot apply in the case of CP-12K trans-frame
protein synthesis, as a simultaneous slippage in the - 1
frame will place the peptidyl-tRNA onto the UAG stop
codon (Fig. 2a), but mutations of this triplet did not
interfere with frameshifting. Instead we propose that
after peptidyl transfer and translocation to the P site in
the 0 frame, the complex awaits termination by release
factors because of the presence of the CP UGA stop
codon in the A site. However, a small proportion of
peptidyl-tRNAs slip on the A string into the - 1 frame,
with the conservation of codon-anticodon interaction,
and this allows the entry of methionyl-tRNA~et for the
decoding of the AUG codon now present at the A site
(Fig. 2b).
The above examples illustrate the - 1 ribosomal
frameshifting event and some of the components of the
frameshift signal in selected plant systems. However,
during polypeptide elongation ribosomes may shift in
different directions and across different distances, as
shown by detailed studies in bacteria (Weiss et al., 1990).
It remains to be seen whether plant ribosomes can
perform similar saltatory movements.
Stop codon suppression
Stop codon suppression in plants has been documented,
with examples for both viral and plant genes. Readthrough of a leaky amber stop codon in TMV RNA
results in the synthesis of a 183K protein (Bruening et al.,
1976). Beier et al. (1984a, b) were able to demonstrate
that two naturally occurring tyrosine-specific suppressor
tRNAs located in the cytoplasm of tobacco cells direct
the in vitro translation of the readthrough protein. The
pseudouridine residue in the GWA anticodon of these
major tRNA Tyr molecules is one structural requirement
for UAG (and UAA) suppression, as tRNA Tyr with a
GUA anticodon did not stimulate readthrough protein
synthesis (Zerfass & Beier, 1992a). These results extend
previous observations on suppressor activity and
anticodon modification in that modification of the
anticodon G residue into QWA prevents UAG suppression (Beier et al., 1984b). In addition, sequences
flanking the TMV UAG stop codon influence sup-
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W. Rohde and others
pression by tobacco tRNA Tyr (Skuzeski et al., 1991;
Zerfass & Beier, 1992a). Most notably, mutation analysis
of the two downstream codons CAA and UUA demonstrated that the 3' context, in the form of UAG CAR
YYA, confers leakiness and represents part of the signal
for TMV UAG suppression.
In luteoviruses like PLRV (Fig. 1a) a UAG amber
stop codon separates the ORFs for CP (ORF3) and an
in-frame ORF5 (56K) that, on the basis of protein
protein interaction analysis using insect proteins, is
probably involved in virus transmission by aphids (van
den Heuvel et al., 1993). In relation to the termination of
capsid protein synthesis, PLRV UAG suppression occurs
at an efficiency of 1% in vivo, determined by transient
expression in tobacco and potato protoplasts (Tacke et
al., 1990; Priifer, 1989). Formation of the readthrough
protein by in vitro translation in reticulocyte lysates has
been reported for BYDV (Dinesh-Kumar et al., 1992),
but purified tRNA fractions from potato as well as
tobacco did not support in vitro suppression of the
PLRV UAG triplet, indicating that this suppressor
tRNA is present in only minor amounts in uninfected
plants (Prtifer, 1992). The UAG context in luteoviruses
is not compatible with the requirements for the TMVspecific suppressor tRNA, as the TMV UAGsuppressing tRNA Tyr from tobacco does not allow
readthrough. However, in the presence of the glutaminespecific tRNAs from Tetrahymena thermophila which,
owing to their UmUA and CUA anticodons, are able to
non-specifically suppress UAA and U A G but not UGA
stop codons, suppression was observed (Prtifer, 1992).
In situ localization of GUS in leaves of transgenic
potato and tobacco plants, expressing gene fusions
containing the U A G codon, in the PLRV context and
in-frame with the GUS reporter gene, identified GUS
mainly in the phloem, with some activity in the cell rays
connecting the adaxial and abaxial phloem of the midrib
(Prtifer, 1992). Replacement of U A G by the GCC sense
codon resulted in GUS activity in all tissues, as expected
for CaMV 35S promoter-driven gene expression in these
plant species. These results can be explained by the
tissue-specific expression or modification of the PLRV
UAG suppressor tRNA, or on the basis of its overall low
but preferential expression in the phloem. In any event,
the phloem-specific luteoviruses have to make use of the
phloem during replication in order to ensure aphid
transmissibility of progeny virus particles by the synthesis
of the CP-56K readthrough protein, the putative aphid
transmission factor (Fig. 1 a).
Zerfass & Beier (1992b) characterized the first UGA
suppressor tRNAs in plants. As a model system for in
vitro U G A suppression they used the translation of
tobacco rattle tobravirus (TRV) RNA1, which contains
an ORF for a 134K protein terminating in UGA and
followed by an in-frame ORF for a putative 194K
readthrough protein (Hamilton et al., 1987). In a
wheatgerm system depleted of endogenous mRNAs and
tRNAs and supplemented with tRNA fractions from
uninfected tobacco, two tryptophan-specific tRNAs with
CmCA anticodons were active as TRV RNA1 UGAsuppressor tRNAs, one nuclear tRNA from the cytoplasm and one encoded by the chloroplast genome. The
chloroplast tRNA Trp is most efficient at TRV U G A
suppression (Zerfass & Beier, 1992b) and the codon
context for efficient U G A suppression is much less
stringent than in the case of TMV RNA U A G
suppression (H. Beier, personal communication). In view
of the high sequence identity between chloroplast and
mitochondrial tRNAs (Sprinzl et al., 1991 ; Mardchal et
al., 1985), the analogous mitochondrial tRNA ~rp is a
potential candidate for in vivo UGA suppression during
TRV replication (Zerfass & Beier, 1992b). The
assumption of plastid participation in TRV replication
is supported by the cytopathic effects occurring with
mitochondria of TRV-infected tobacco plants as well as
by the observation that in cells infected with the
tobravirus, pepper ringspot virus, almost all of the
RNAl-containing particles are lined up at the outer
membrane of mitochondria (Harrison et al., 1976).
U G A stop codon suppression is also involved in the
synthesis of an 84K readthrough protein by the decoding
of the CP ORF stop codon on SBWMV RNA2 (Shirako
& Wilson, 1993). Although the suppressor tRNA(s)
involved in this UGA readthrough have not been
identified, the UGA context is identical to that of TRV
and suggests that, in monocotyledonous plants like
wheat, tryptophan-specific suppressor tRNAs also exist.
One further aspect of SBWMV CP gene translation is
noteworthy; the core sequence of the 19K SBWMV
capsid protein is not only part of the 84K readthrough
protein, but also of the 28K protein synthesized by nonA U G initiation (see above). This example, therefore,
very clearly underlines the flexibility of the translational
machinery to maximize the exploitation of limited genetic
information.
In prokaryotes as well as in eukaryotic systems, UGA
stop codon suppression has been attributed to a specific
UGA-decoding tRNA s~(' that directs the incorporation
of selenocysteine into the nascent polypeptide chain
(Hatfield et al., 1992a, b; Hatfield & Diamond, 1993).
Additional requirements for UGA decoding by tRNA s~e
are a specific EF-Tu translation factor and a special
mRNA structure (Farabaugh, 1993, and references cited
therein). In higher plants an/-cysteinyl-tRNA synthetase
from mungbean was shown to utilize selenocysteine for
the charging of acceptor tRNA(s) (Shrift et al., 1976),
and a selenocysteinyl-accepting tRNA was identified in
Beta vulgaris, although it has not been characterized as
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Review." plant virus translation mechanisms
tRNA s~ (Hatfield et al., 1992b). Plant proteins containing selenocysteine residues have been described
(Brown & Shrift, 1980), but incorporation of this amino
acid may not necessarily depend on the presence of a
UGA stop codon, as deduced from the sequence of a
plant cDNA clone with homology to the selenocysteinecontaining animal glutathione peroxidases (in which
selenocysteine is encoded by UGA; Criqui et al., 1992).
Stop codon suppression has also been implicated in
the expression of plant genes such as the synthesis of
storage proteins in barley (C-hordeins; Entwistle et al.,
1991) and maize (c~-zeins; Liu & Rubenstein, 1993). The
C-hordein ORF in the genomic clone 2-horl-14 is
interrupted by an amber stop codon within the coding
sequence for the octapeptide repeats. Evidence for
readthrough was obtained both in vitro by translation in
the wheatgerm system and in vivo by transient expression
of reporter gene fusions in barley endosperm tissue after
particle gun bombardment. Although the authors
demonstrated that the promoter of this particular gene is
even more active than that of a control wild-type Chordein gene, the absence of both full-length or truncated
proteins after in vitro translation of barley endosperm
mRNA showed that they represent a minor component
of the storage proteins in barley endosperm (Entwistle et
al., 1991). The sequencing of genes belonging to a maize
c~-zein subfamily 4 (SF4) gene cluster provided evidence
that four of the genes designated type 2 (T2) contained
one or two early in-frame stop codons (Liu &
Rubenstein, 1993). In addition, cDNAs that had retained
these stop codons were isolated for one of the SF4 T2
genes, supporting the transcriptional activity of this gene
in vivo and excluding the possibility of RNA editing
(Covello & Gray, 1993). The physiological importance of
the putative readthrough proteins is not known, but they
might well represent regulatory intermediates in the
biosynthesis of maize storage proteins.
Prospects
As discussed above, for several selected systems alternative strategies for plant gene expression at the
translational level for the synthesis of two or more
proteins from a coding region operate during initiation,
elongation and termination. Although the importance of
translational control by ribosomal frameshifting and
stop codon suppression has been recognized for some
time, one intention of this review is to attract more
attention to altei"native initiation strategies, either by
non-AUG or internal initiation, as a third option in the
alternative reading of the genetic code to enable the
synthesis of more than one polypeptide from a sequence.
The mechanisms controlling initiation at triplets other
than A U G remain to be unravelled, but the association
2147
of CaMV TAV with ribosomes (T. Hohn, personal
communication) and its translational trans-activation
capacity would represent one example of the possibility
that viral proteins or host factors, modified in the course
of virus infection, may influence the choice of triplets for
translation initiation and thus help to decompress the
limited genetic information of viral genomes.
The use of in vivo plant systems (protoplasts,
transgenic plants) in the study of translational
mechanisms operating in the expression of plant or plant
viral genes has come of age and enhanced our knowledge
of alternative readings of the genetic code. The identification, for example, of tryptophan-specific tRNAs from
tobacco that decode UGA stop codons (Zerfass & Beier,
1992b) has increased the complexity of this triplet
beyond its function in protein synthesis termination or in
encoding selenocysteine (Hatfield et al., 1992b). One
additional functional aspect is its capacity to act as a
shifty stop codon and promote ribosomal frameshifting,
as in the expression of the PVM CP-12K trans-frame
protein (Gramstat et al., 1993). Further insight into the
in vivo activity and specificity of UAA-, UAG- or UGAdecoding plant tRNAs will be gained by the expression
of appropriate gene fusions with reporter genes by
transient expression in protoplasts or after stable
transformation and regeneration of transgenic plants
(Tacke et al., 1990; Priifer, 1992; Carneiro et al., 1993).
The identification of suppressor tRNA activity in
transgenic plants by in situ localization of a reporter
enzyme provides a unique opportunity to study the
regulation of suppressor activity during plant development, its specificity to cell lines, tissues and organs, or
its modulation in the course of virus infection. The
action of proteins activating translation can be studied in
crosses of transgenic plants. Plants transgenic for
reporter gene fusions that respond to translational
activators have been used to monitor virus spread from
primary sites of infection (Zijlstra & Hohn, 1992). It has
to be emphasized, however, that in vivo systems like
transient expression in protoplasts or the establishment
of transgenic plants may not necessarily reflect the actual
situation during virus replication, but provide only a
simplified insight into molecular mechanisms underlying
the translation of plant or plant viral genes by alternative
readings of the genetic code. Nevertheless, in view of the
established examples of non-canonical translation, genes
cannot be discarded as non-functional pseudogenes
because of the absence of an A U G codon or the presence
of in-frame stop codons, and the absence of open reading
frames should not prevent the presence of an open
reading mind.
The authors wish to acknowledgethe support of their colleaguesby
their providingof reprints and preprints of manuscripts. Specialthanks
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2148
W . R o h d e and others
go to Dr H. Beier for her critical reading of the manuscript. Part of this
work was supported by grant Ro 330/6-2 of the Deutsche
Forschungsgemeinschaft.
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