Institute of Biology, Leiden University, Gorlaeus Laboratories, PO Box 9502, 2300 RA Leiden, The Netherlands
Correspondence
John Bol
j.bol{at}chem.leidenuniv.nl
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ABSTRACT |
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INTRODUCTION |
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The mRNAs of many animal and plant RNA viruses lack a cap structure, a poly(A) tail or both, yet they efficiently compete with host mRNAs for the translational machinery. RNAs of animal picornaviruses and plant potyviruses lack a 5' cap structure and translation initiates at an internal ribosome entry site (IRES) in the 5' untranslated region (UTR). An interaction between eIF4G bound to the IRES and PABP bound to the poly(A) tail of picorna- and potyviruses appears to be required for efficient translation of the viral RNAs (Svitkin et al., 2001; Gallie, 2001
). Several genera of plant viruses contain a 3'-terminal tRNA-like structure (TLS) instead of a poly(A) tail. The TLS of Brome mosaic virus (BMV; genus Bromovirus) and Tobacco mosaic virus (TMV; genus Tobamovirus) can be aminoacylated with tyrosine and histidine, respectively, and the 3' UTRs of these viruses stimulate translation of a reporter gene by mimicking the function of a poly(A) tail (Gallie & Kobayashi, 1994
). Plant viruses from the genera Luteovirus (family Luteoviridae) and Necrovirus (family Tombusviridae) contain neither a cap structure nor a poly(A) tail. Translation of the RNAs of the luteovirus Barley yellow dwarf virus requires base pairing between a stemloop in the 5' UTR and a stemloop in a 100 nucleotide (nt) translation element that is present in the 3' UTR, presumably to deliver translation factors and/or ribosomes to the 5' end (Guo et al., 2001
).
We have studied the translation strategy of Alfalfa mosaic virus (AMV; genus Alfamovirus). Within the family Bromoviridae, the tripartite plus-strand RNA genomes of viruses from the genera Bromovirus and Cucumovirus are infectious as such, whereas initiation of infection by viruses from the genera Alfamovirus and Ilarvirus requires addition of coat protein (CP) to a mixture of the genomic RNAs (reviewed by Bol, 1999, 2003
; Jaspars, 1999
). AMV RNAs 1 and 2 encode the replicase proteins P1 and P2; RNA 3 encodes the viral movement protein (P3) and CP, which is translated from a subgenomic messenger, RNA 4. This RNA 4 can replace CP in the inoculum to initiate infection. At their 3' termini, AMV RNAs contain a sequence of 145 nt with a high level of sequence similarity. The 3'-terminal 112 nt of this sequence can adopt two alternative conformations: a linear array of hairpins separated by the sequence AUGC with a high affinity for CP, or a structure that resembles the TLS of bromo- and cucumoviruses and is required for minus-strand promoter activity (Olsthoorn et al., 1999
). Binding of CP to AMV RNAs blocked minus-strand promoter activity in vitro and enhanced translation of viral RNA in tobacco protoplasts 50- to 100-fold (Olsthoorn et al., 1999
; Neeleman et al., 2001
). We have proposed that after infection of plants with AMV RNAs 1 to 4, initially RNA 4 is poorly translated into CP but then this CP stimulates translation of its own messenger and finally stimulates the translation of RNAs 1 and 2 into the replicase proteins. After targeting of the genomic RNAs to membrane bound replication complexes and dissociation of CP from the 3' end of the RNAs, the TLS conformer can be formed and the replicase proteins initiate viral minus-strand RNA synthesis (Neeleman et al., 2001
; Bol, 2003
).
Fig. 1(A) shows the structure of the 3' UTR of AMV RNAs 3 and 4 with the CP-binding conformer at the 3' end. The sequence contains at least two CP binding sites: CPB1 in the sequence that RNAs 1, 2 and 3 have in common, and CPB2 which is unique to RNA 3 (Houser-Scott et al., 1994
; Reusken et al., 1994
). The TLS conformer is generated by a pseudoknot interaction between nt 5 to 8 and 90 to 93 from the 3' end (Olsthoorn et al., 1999
). In this study we addressed the following questions: (i) is CBP1 and/or CBP2 involved in stimulation of translation, (ii) is translation affected when the pseudoknot interaction is disrupted by mutations in the RNA rather than by CP binding, and (iii) is translation stimulated by the binding of CP monomers or dimers?
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METHODS |
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Transcription with T7 RNA polymerase and inoculation of protoplasts.
Transcription with T7 RNA polymerase was done as described previously (Neeleman et al., 2001). Samples of 200 000 tobacco protoplasts were inoculated with 10 µg of transcripts of cDNA 4 derivatives, or with a mixture of transcripts of cDNAs 1, 2, 3 and 4 (2 µg of each transcript) and incubated for 18 h as described (Neeleman & Bol, 1999
).
In vitro translation and protein analysis.
Translation of RNA 4 in a rabbit reticulocyte lysate (Promega) was done according to the manufacturer's instructions. Proteins synthesized in vitro or in vivo were analysed by Western blotting (Towbin et al., 1979) using antiserum against AMV CP or P3. The protein extracted from 100 000 protoplasts was loaded per slot (Neeleman & Bol, 1999
). The relative accumulation of CP in protoplasts transfected with RNA 4 derivatives was estimated, taking the amount of CP in controls transfected with WT RNA 4 as 100 %. To this goal, the sample from the control protoplasts was mixed with extracts from non-transfected protoplasts to obtain 50, 30, 20, 10 and 5 % dilutions and these mixtures were loaded on the blots for comparison.
Analysis of CPRNA interactions.
A rabbit reticulocyte lysate was programmed with RNA 4 transcripts (20 µg per ml lysate) in the absence or presence of 3' UTR transcripts (100 µg per ml lysate). After incubation of the lysate for 90 min at 25 °C, 1 µl was withdrawn for protein analysis by Western blotting and 1 µl was withdrawn for RNA analysis by Northern blotting. To analyse RNAprotein complexes formed in the lysate, 4 µl of the lysate was mixed with 200 µl IP buffer (20 mM Tris pH 7·5, 150 mM NaCl, 1 mM EDTA), and 5 µg carrier RNA (total RNA extracted from tobacco) and 0·5 µl antiserum against CP was added. After incubation for 60 min at 4 °C, 50 µl of a 50 % suspension in IP buffer of protein ASepharose CL-4B (Amersham Pharmacia) was added. After incubation for another 60 min at 4 °C under continuous rotation, the beads were washed four times with 1 ml of IP buffer. RNA bound to the beads was extracted with Trizol (Invitrogen) in the presence of 1 µg carrier RNA. DIG-labelled minus-strand RNA 3 was used as probe to detect RNA 4 and 3' UTR transcripts on Northern blots (Neeleman & Bol, 1999).
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RESULTS |
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Role of the pseudoknot in translation of RNA 4
Under physiological conditions the TLS conformer of the 3' 112 nt of AMV RNAs is predicted to be more stable than the CP binding conformer (Olsthoorn et al., 1999). Interaction of the 3' termini of the RNAs with CP disrupts the pseudoknot interaction between nt 5 to 8 and 93 to 90 to allow the formation of hpA (Olsthoorn et al., 1999
). To see whether disruption of the pseudoknot interaction is sufficient to enhance translation of RNA 4, the sequence of nt 93 to 90 (5'-UCCU-3') was mutated to 5'-UGGG-3' in WT RNA 4 (mutant WT-
PK) and in RNA 4 with the R17A mutation (mutant R17A-
PK). The same mutation has been shown to block minus-strand promoter activity in vitro (Olsthoorn et al., 1999
). Disruption of the pseudoknot interaction neither affected translation of RNA 4 encoding WT CP (Fig. 5
, lane 2) nor did it stimulate translation of the R17A mutant to detectable levels (Fig. 5
, lane 4). This indicates that merely changing the structure of the 3' 112 nt of RNA 4 from the TLS conformer to the CP binding conformer is not sufficient to enhance translation efficiency.
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C-terminally truncated CP binds to viral RNA as a monomer
The defect of mutant N199 to stimulate translation and to initiate infection could be due to a defect in binding of the truncated CP to the viral RNA. Alternatively, the binding of CP monomers to viral RNA could be ineffective in performing these functions. A novel binding assay was developed to measure binding of CP to the 3' UTR of AMV RNAs. WT or mutant RNA 4 was translated in a rabbit reticulocyte lysate in the presence of a transcript corresponding to the 3' UTR of RNA 4. Subsequently, CP-antiserum and protein ASepharose beads were added to the translation mixture and CPRNA complexes bound to the beads were analysed. Fig. 8(A) shows an analysis of RNA 4 and 3' UTR transcripts present in a total RNA preparation extracted from the translation mixture whereas Fig. 8(B)
shows an analysis of RNAs that were bound to the Sepharose beads. In the cell-free system, WT and mutant RNA 4 transcripts were translated into CP with similar efficiencies (Fig. 8C
). When RNA 4 was omitted from the translation mixture, no binding of 3' UTR transcripts to the beads was observed (Fig. 8B
, lane 8). Translation of RNA 4 into WT CP resulted in binding of both RNA 4 and 3' UTR transcripts to the beads (Fig. 8B
, lane 2). Translation of WT CP from RNA 4 with its 3' UTR replaced by a plasmid-derived sequence (mutant WT-
T) resulted in a similar binding of 3' UTR transcripts to the beads but binding of mutant RNA 4 was significantly reduced (Fig. 8B
, lane 7). The residual binding of mutant RNA 4 may reflect the interaction of CP with binding sites in the CP gene (see Discussion). CP with the R17A or
N16 mutation did not bind to the 3' UTR in this assay (Fig. 8B
, lanes 4 and 6), in agreement with the results of other types of binding assays (Ansel-McKinney & Gehrke, 1998
; Tenllado & Bol, 2000
). However, CP with the N199 or
N10 mutations did bind the 3' UTR transcripts as efficiently as the WT CP did (Fig. 8B
, lanes 3 and 5). From these results we conclude that CP-N199 binds as monomers to the 3' UTR of AMV RNAs. Apparently, this binding is insufficient to stimulate translation and to initiate infection.
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DISCUSSION |
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The TLS conformer and hpE are the key elements in the 3' UTR required for minus-strand promoter activity of AMV RNAs (Olsthoorn et al., 1999; Olsthoorn & Bol, 2002
). The results with pseudoknot mutant WT-
PK (Fig. 5
) and mutant
E (Fig. 4A
) demonstrate that neither of these elements plays a role in translation of RNA 4. The sequence of CPB2 (Fig. 1A
) is outside the 3' region of 145 nt that shows sequence similarity in RNAs 1, 2 and 3 and this sequence is not found in RNAs 1 and 2. Deletion
FG removes the major part of this CP binding site but the deletion does not affect translation of RNA 4 (Fig. 4A
). Moreover, mutant
AC contains an intact CPB2 sequence but is untranslatable (Fig. 4A
). Thus, CPB2 plays no role in translation of RNA 4 and its significance in the AMV replication cycle is presently unclear. In contrast to our data, studies by Hann et al. (1997)
indicated that AUGC-motif 5 was part of a translation determinant.
The deletion analysis presented in Fig. 4(A) shows that elements in the 3'-terminal 112 nt of RNA 4 govern translation efficiency of this RNA. Deletion of one of the four hairpins in this region (hairpins A, B, C or D) reduced translation 4- to 5-fold whereas deletion of two of these hairpins (hairpins A and C) reduced translation to undetectable levels. The region of the 3' 39 nt (hairpins A and B, and AUGC-motifs 1, 2 and 3) is the minimal sequence that is sufficient for binding of CP in vitro (Reusken & Bol, 1996
; Houser-Scott et al., 1997
). This sequence is present in mutants
C and
D, which translate with an efficiency of about 20 % (Fig. 4A
). Apparently, the CPB1 sequence is not sufficient for WT levels of translation of RNA 4. AUGC-motifs 1, 2 and 3 are essential for binding of CP to CPB1 (Reusken & Bol, 1996
) and mutation of these motifs affected translation efficiency of RNA 4 (Fig. 4B
). This supports the notion that the CP binding activity of CPB1 is required for translation. Possibly, sequences required for CP binding to AMV RNAs in vivo are longer than the minimal sequence of 39 nt required in vitro and extend into the 3'-terminal 112 nt. Alternatively, efficient translation of RNA 4 may require a CP binding site of 39 nt and an upstream element of unknown function between nt 39 and 112 from the 3' end.
Baer et al. (1994) reported that a peptide consisting of the N-terminal 25 or 38 amino acids of AMV CP was sufficient to bind the 3'-terminal binding site in AMV RNAs and to initiate virus replication in protoplasts. In later experiments, it became clear that N-terminal peptides were much less active in initiating infection than full-length CP (Ansel-McKinney et al., 1996
; unpublished data quoted in Choi et al., 2003
). Here, we showed that RNA 4 transcripts encoding peptides corresponding to the N-terminal 63, 85 or 199 amino acids of CP were untranslatable in vivo and were unable to initiate infection in protoplasts (Fig. 7
). Similar to our results with mutant N199 (which lacks the C-terminal 21 amino acids of CP), Choi et al. (2003)
recently reported that RNA 4 transcripts encoding CP with C-terminal deletions of 18 or 19 amino acids (mutants CP
C18 and CP
C19) were largely defective in initiating AMV replication in protoplasts. Mutant N199 was found to be defective in dimer formation by using the yeast two-hybrid system (Tenllado & Bol, 2000
) whereas mutants CP
C18 and CP
C19 did not form dimers in an assay involving glutaraldehyde cross-linking (Choi & Loesch-Fries, 1999
). Our results presented in Fig. 8(B)
and band-shift assays done by Choi et al. (2003)
demonstrate that the C-terminally truncated CPs interact with the 3' UTR as monomers. Apparently, binding of monomers of N199 to viral RNA neither stimulated translation nor did it stimulate initiation of infection at significant levels.
The stimulation of translation of RNA 4 by its own translation product from undetectable to WT levels could be simply explained by the hypothesis that binding of CP stabilizes the RNA by protecting it from degradation. The results with mutant R17A and R17A-BMV (Fig. 2) showed that replacement of the 3' UTR of AMV RNA 4 by the 3' UTR of BMV resulted in efficient translation of the chimeric RNA independently of CP binding. Thus, the hypothesis would imply that the AMV 3' UTR stabilizes the RNA by binding of CP whereas the BMV 3' UTR stabilizes the RNA independently of CP, possibly by the binding of host factors. However, there is evidence that the role of the 3' UTR of AMV and BMV in translation is more complex than mere protection of the RNAs against non-specific degradation. RNA 4 transfected into protoplasts survived an incubation period of 18 h at similar levels whether or not the RNA was translated into CP (Neeleman et al., 2001
). By inoculation of protoplasts with AMV RNAs at different time-points, Houwing & Jaspars (2000)
showed that the messenger and replicon functions of these RNAs are preserved for several hours after inoculation. This argues against an exceptional sensitivity of these RNAs to degradation in the absence of CP. The observation that extension of the 3' termini of AMV genomic RNAs with a poly(A) tail obviated the requirement for CP in the inoculum to initiate infection led us to suggest that binding of CP to the 3' termini of AMV RNAs could mimic the function of the binding of PABP to the 3' poly(A) tail of cellular messengers (Neeleman et al., 2001
). Recently, it was shown that translation of the non-polyadenylated rotavirus mRNAs requires simultaneous interaction of the nonstructural protein NSP3 with initiation factor eIF4G and the mRNA 3' end (Vende et al., 2000
). By a similar mechanism, AMV CP could stabilize the complex of viral RNAs and initiation factors and promote the recruitment of 40S ribosomal subunits through the formation of a closed loop structure. Possibly, dimer formation is required for the putative interaction of CP with translation initiation factors. The 3' UTR of BMV has been proposed to act as a poly(A) mimic (Gallie & Kobayashi, 1994
). If in the family Bromoviridae the 3' UTR of the RNAs of viruses from the genera Bromovirus and Cucumovirus acts as a poly(A) mimic while CP of viruses from the genera Alfamovirus and Ilarvirus acts as a PABP mimic, this would explain the requirement of CP to initiate infection by the latter two genera.
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Received 19 August 2003;
accepted 2 October 2003.