tRNA-Like Structure Regulates Translation of Brome Mosaic Virus RNA

For various groups of plant viruses, the genomic RNAs end witha tRNA-like structure (TLS) instead of the 3' poly(A) tail ofcommon mRNAs. The actual function of these TLSs has long beenenigmatic. Recently, however, it became clear that for turnipyellow mosaic virus, a tymovirus, the valylated TLS^TYMV of thesingle genomic RNA functions as a bait for host ribosomes anddirects them to the internal initiation site of translation(with N-terminal valine) of the second open reading frame forthe polyprotein. This discovery prompted us to investigate whetherthe much larger TLSs of a different genus of viruses have acomparable function in translation. Brome mosaic virus (BMV),a bromovirus, has a tripartite RNA genome with a subgenomicRNA4 for coat protein expression. All four RNAs carry a highlyconserved and bulky 3' TLS^BMV (about 200 nucleotides) with determinantsfor tyrosylation. We discovered TLS^BMV-catalyzed self-tyrosylationof the tyrosyl-tRNA synthetase but could not clearly detecttyrosine incorporation into any virus-encoded protein. We establishedthat BMV proteins do not need TLS^BMV tyrosylation for theirinitiation. However, disruption of the TLSs strongly reducedthe translation of genomic RNA1, RNA2, and less strongly, RNA3,whereas coat protein expression from RNA4 remained unaffected.This aberrant translation could be partially restored by providingthe TLS^BMV in trans. Intriguingly, a subdomain of the TLS^BMVcould even almost fully restore translation to the originalpattern. We discuss here a model with a central and dominantrole for the TLS^BMV during the BMV infection cycle.

INTRODUCTION

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Introduction

Thirty years ago, the exciting discovery was made that the RNAsof certain plant-infecting viruses can be aminoacylated. Forthe genera Tymovirus, Bromovirus, Tobamovirus, and Furovirusof the family Bromoviridae, the 3' ends of the viral RNAs canbe charged with either valine, tyrosine, or histidine in a waythat is comparable to the aminoacylation of the correspondingcanonical tRNAs (for reviews, see references 8, 10, and 22).On the basis of chemical and enzymatic probing experiments andfunctional assays, these 3' untranslated regions (UTRs) canall be folded into structures more or less resembling thoseof canonical tRNAs. The full functional meaning of these tRNA-likestructures (TLSs) has remained enigmatic.

For the tymovirus turnip yellow mosaic virus (TYMV), it wasrecently discovered, however, that valylated TLS^TYMV entrapsribosomes and directs them to the second open reading frame(ORF) of the single genomic RNA for synthesis of the replicasedomain-containing polyprotein (3). Removal of the TLS^TYMV fromthe native TYMV RNA completely abolished polyprotein synthesis,whereas translation of the first ORF into movement protein andthat of the subgenomic RNA (sgRNA) into coat protein were unchanged.The 3'-linked valine was found to become incorporated at theN terminus of the polyprotein in a cap-independent and initiator-tRNA-independentfashion. Polyprotein synthesis could also start, however, bythe action of a nonaminoacylated or 3'-truncated TLS^TYMV. Thisdiscovery prompted us to investigate whether the much largerTLSs of a different genus of viruses have a comparable functionin translation, and in the present study we focus on the TLS^BMVof Brome mosaic virus (BMV), the type species of the genus Bromovirus.

The TLS^TYMV-mediated initiation of TYMV polyprotein synthesisis exceptional, since eukaryotic translation normally startswith initiation-factor-dependent recognition of the 5' m⁷G(5')pppNcap and recruitment of the 40S ribosomal subunit. Then, by numerousevents that are catalyzed and regulated by at least 12 initiationfactors, the 40S ribosomal subunit with initiator tRNA is preparedfor scanning towards the first AUG as a start codon. After 60Ssubunit association, the ribosome can enter the elongation phaseof the translation cycle. Efficient translation depends on 5'-to 3'-end communication of the mRNA, established by a 3' poly(A)stretch that communicates via the poly(A) binding protein (PABP)and eIF4G with eIF4E bound to the capped 5' end. This circularform is believed to assess the mRNA integrity and to recycleribosomes for multiple rounds of translation. Indeed, the 5'cap binding factors and PABP synergistically stimulate translation(33).

As intracellular parasites, viruses need the host translationapparatus for their reproduction, and their RNAs compete withthe host mRNAs for ribosomes to start translation. Remarkably,viral RNAs do not always contain the usual 5' cap and 3' poly(A)entities, although they are efficiently translated. For TYMVRNA, this circularization appears to be established by 3'-TLS^TYMVcommunication with the start of ORF2 (3). For BMV, with a differentgenomic organization, the 3' TLS^BMV may also contribute to communicationwith the 5' end in a somehow related way.

BMV has a tripartite genome (Fig. 1A): RNA1 codes for a proteinwith domains homologous to methyltransferases and helicases(Me/He), RNA2 codes for an RNA-dependent RNA polymerase (RdRp),and RNA3 codes for the viral movement protein (MP) and the coatprotein (CP). Because of the downstream location of the CP cistron,the latter is exclusively expressed from an sgRNA synthesizedfrom RNA3. All of these RNAs carry a 5' cap and a highly conserved3' TLS^BMV, a bulky structure of about 200 nucleotides (Fig.1B). Though not strongly mimicking a canonical tRNA^Tyr, theTLS is a substrate for tyrosylation both in vivo and in vitro(9, 11, 16, 21), and it functions as a promoter for minus-strandsynthesis (25, 31). It also functions as a nucleation site forcoat protein assembly and encapsidation of the BMV RNAs intovirions (7).

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FIG. 1. Genomic organization of BMV. (A) Schematic view of genomic RNAs 1 to 3 with ORFs for the following proteins (molecular masses are shown in parentheses in the figure): methyltransferase/helicase (Me/He), RdRp, MP, and CP, which is only translated from the sgRNA. All of the RNAs have the same 5' cap (•) and 3' TLS^BMV. (B) Secondary structure of the TLS^BMV with nucleotide numbering in the 3' $->$ 5' direction. The nucleotides in the shaded boxes were hybridized to two DNA oligonucleotides for TLS^BMV disruption by RNase H. The various stem-loop structures are indicated (A to E).

It is not self-evident to see an analogy between the TLS^BMVand the TLS^TYMV with respect to the latter's functioning asan initiator of translation of a second and overlapping ORF,since the genomic organization of BMV is quite different, withoutoverlapping ORFs. Our experimental strategy was as follows.We first studied the effect of TLS^BMV disruption on the translationof the BMV RNAs in an in vitro wheat germ system and discoveredclear effects on genomic RNAs 1 to 3, but no effect on the sgRNA.We looked for TLS^BMV-mediated tyrosine incorporation in BMVtranslation products but did not get a final answer due to thediscovery of an unexpectedly high level of self-tyrosylationof the tyrosyl-tRNA synthetase (TyrRS) induced by the TLS^BMV.Finally, we did complementation studies with the TLS^BMV addedin trans to BMV RNAs with 3'-disrupted TLSs and localized thecomplementation activity to a specific TLS^BMV subdomain. Onthe basis of our results, we discuss a model with a prime rolefor the TLS^BMV during the BMV infection cycle.

MATERIALS AND METHODS

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Materials and Methods

RNA preparations. Native BMV RNA was purchased from Promega (Madison, Wis.). InfectiousBMV clones were kind gifts from P. Ahlquist (17). For the synthesisof 5'-capped T7 transcripts of BMV RNA1, -2, and -3, a T7 RiboMAXtranscription kit (Promega) was used, with the following adaptationsto the protocol. The reaction was initiated in the presenceof 1 mM cap analogue, m⁷G(5')ppp(5')G (New England Biolabs,Beverly, Mass.), together with a 5 mM concentration (each) ofATP, CTP, and UTP. After a 15-min initial incubation at 37°C,the mixture was supplemented with 1 mM GTP to allow furtherchain elongation. This was repeated every 15 min until the transcriptionmixture contained 5 mM GTP. The total incubation time was 210min. ³²P-labeled transcripts were obtained by internal incorporationof [ ${alpha}$ -³²P]CTP (3,000 Ci/mmol). The T7 transcripts of tRNA^Tyrfrom Saccharomyces cerevisiae and of the different TLS^BMV specieswere synthesized without m⁷G(5')ppp(5')G addition. Prior torunoff transcription, the plasmids for the transcription ofBMV RNAs 1 to 3 were linearized with EcoRI, and the plasmidsfor (mutant) TLS^BMV and tRNA^Tyr were linearized with BstNI (11).Small transcripts were purified by 8% (wt/vol) polyacrylamidegel electrophoresis under denaturing conditions (PAGE/DC), followedby electroelution and ethanol precipitation (11), and largetranscripts were purified by phenol extraction and ethanol precipitation.The resulting RNA pellets were dissolved in 50 µl of H₂Oand applied to a Micro Bio-Spin (P-30) column (Bio-Rad, Hercules,Calif.) in order to fully remove unincorporated nucleotidesand low-molecular-weight contaminants. This step was repeatedonce. The integrity of the RNAs was checked by gel electrophoresisin agarose.

TLS disruption. For the disruption of TLS^BMV, two different DNA oligonucleotides(Invitrogen Europe) were designed, with one being complementaryto stem-loop structure C and part of structure D and the otherbeing complementary to stem-loop structures B3 and E (Fig. 1B).These regions were chosen because of their 100% conservationbetween the different BMV RNAs. A 30-fold molar excess of eachDNA oligonucleotide compared to BMV RNA was allowed to annealwith the RNA, and the TLS^BMV was disrupted by digestion withRNase H (New England Biolabs) (3). Treated samples underwentphenol extraction and further purification in Micro Bio-Spin(P-30) columns. Although the DNA oligonucleotides did not encompassthe whole TLS^BMV, this procedure allowed the complete removalof the TLS^BMV. The latter was checked in a reverse transcriptionassay with SuperScript II reverse transcriptase (using a protocolfrom Invitrogen) by using the same DNA oligonucleotides as 5'³²P-end-labeled primers, followed by PAGE/DC analysis, and nomore products were observed. The specificity of the RNase Hcleavage procedure was also checked with [ ${alpha}$ -³²P]CTP-labeled T7transcripts of BMV RNA, and no additional cleavage sites upstreamof the TLS^BMV were detected (not illustrated). This specificand restricted cleavage also appeared from the almost full complementationof the disturbed BMV RNA (-TLS) translation by the additionin trans of TLS^BMV(-A, -B1, -B3, -E), as shown in Fig. 5.

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FIG. 5. Translational complementation in trans with shortened TLS^BMV variants and with yeast tRNA^Tyr as a reference molecule. The effect of increasing concentrations of TLS^BMV(-B2) and TLS^BMV(-C) (left gel) and of TLS^BMV(-A, -B1, -B3, -E) and yeast tRNA^Tyr (right gel) on the translation of 3'-truncated BMV RNAs 1 to 3 was monitored. Above each gel, the schematic RNA structures of the TLS^BMV variants are shown, with lacking parts indicated in gray. Relative band intensities were calculated from three independent experiments. See the legend for Fig. 2 for further information.

Translation. All in vitro translation assays were carried out in a wheatgerm system (Promega) as described previously (3), with theincorporation of in vitro translation-grade [³⁵S]Met (37 TBq/mmol)(Amersham Pharmacia Biotech) for autoradiographic analysis ona PhosphorImager (Bio-Rad) after sodium dodecyl sulfate-PAGEseparation.

For measurements of the half-lives of the different BMV RNAsunder these translation conditions, samples programmed with[ ${alpha}$ -³²P]CTP-labeled BMV RNAs were taken at different time pointsand mixed with 2 volumes of stop solution (98% [vol/vol] formamide,10 mM EDTA, 0.025% [wt/vol] bromophenol blue) for subsequentPAGE/DC analysis. Band intensities were scanned on a PhosphorImager.

For measurements of the 5' cap dependencies of translation ofthe different BMV RNAs, wheat germ translation mixtures withincreasing concentrations of the cap analogue m⁷G(5')ppp(5')G(New England Biolabs) were preincubated for 5 min before theaddition of the BMV RNAs. The preincubation mixtures were complementedwith additional MgCl₂ (0.8 mol/mol of cap analogue) in orderto compensate for the chelating effect of the cap analogue (2).

Tyrosylation. All tyrosylation reactions, with 1 µM TyrRS from yeastand 4 µM [3,5-³H]Tyr (1.81 TBq/mmol) (Amersham PharmaciaBiotech), were essentially performed as described by Fechteret al. (11). [³H]Tyr incorporation was analyzed by liquid scintillationcounting or by PAGE as described by Barends et al. (3).

RESULTS

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Results

TLS^BMV regulates BMV RNA translation. In order to establish whether the TLS^BMV might have any functionin protein synthesis, we disrupted the TLS^BMV of the populationof native BMV RNAs isolated from virions by adding an excessof a mixture of two different DNA oligonucleotides complementaryto two conserved parts of the TLS^BMV (Fig. 1B) and by site-directedcleavage of the heteroduplex with RNase H. As a result (Fig.2), the translation of RNA1 and RNA2 decreased about 10-fold,whereas the translation of RNA3 into MP decreased about 2- to5-fold, depending on the other RNA concentrations. The translationof sgRNA into CP remained completely unaffected. This effecton translation was independent of the amount of oligonucleotidesadded in the range of a 20- to 600-fold excess over BMV RNAduring the TLS^BMV disruption procedure (not illustrated). Apparently,the TLS^BMV does regulate translation of the genomic RNAs. Onecould argue that the TLS^BMV functionally mimics a poly(A) tailand that its removal would thus simply reduce translation byan increased susceptibility of the 3' end and upstream regionsto degradation. However, sgRNA translation into CP (Fig. 2)was completely unaffected by the removal of the TLS^BMV, whichargues against such an explanation. As shown below, degradationcannot possibly be the explanation, because the addition ofthe TLS^BMV in trans effectively restored translation of thegenomic RNAs. Just as for the population of native RNAs, thesame pattern of reduced translation activity was observed afterthe removal of the TLS^BMV from the T7 transcripts of RNAs 1to 3, and Fig. 2 illustrates the effects for the separate transcripts.The removal of the TLS^BMV caused the enhanced appearance ofa protein band of about 50 kDa (see asterisk in Fig. 2) forthe mixture of native RNAs. The product apparently originatesfrom RNA2 (-TLS) and may be due to a change in RNA folding whichcauses ribosomal pausing.

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FIG. 2. Effects of TLS^BMV disruption on BMV RNA translation in a wheat germ system. The separate genomic RNAs 1, 2, and 3 (15 nM for each T7 transcript), their mixture (RNA1-3) (25 nM for each T7 transcript, i.e., 75 nM for the total population), and a native BMV RNA population mixture (8.6 nM for the total population) were subjected to TLS^BMV disruption (-) and used for in vitro translation. The translation patterns of [³⁵S]Met-labeled protein bands are compared to those for intact RNAs (+). The positions of molecular weight markers and the BMV proteins are indicated on the left and the right, respectively. The asterisk indicates an incomplete product of enhanced intensity after TLS^BMV disruption. The bottom panels show the scanned relative intensities (%) of full-length protein bands together with their incomplete products, taking the intensities produced by the intact (+) RNAs as 100% (average values from at least two independent experiments).

In order to check the translational activities of the 5'-cappedgenomic BMV T7 transcripts in our wheat germ system in termsof RNA stability and cap dependency of translation, we determinedtheir half-lives during translation and their responses to increasingconcentrations of the cap analogue m⁷GpppG as a competitiveinhibitor of translation initiation (the inhibitor concentrationsat half-maximum inhibition [IC₅₀ values] are presented). Table1 shows that both RNA stability and cap dependency are verysimilar for the three different RNAs. Also, for the native BMVRNAs, we found comparable IC₅₀ values of m⁷GpppG for RNA1 to-3 and sgRNA, as already reported by Ali et al. (2). The similarityof the two sets of IC₅₀ values, with all at about 20 µMm⁷GpppG, underscores the efficiency of the in vitro cappingprocedure for T7 transcripts described above. Because of thesimilar stabilities and cap dependencies of all the BMV RNAsat rate-limiting concentrations, we think that the differenteffects on their translational activities caused by TLS^BMV removalmay be related to a difference in initiation efficiencies, asituation that was previously found for the genomic and subgenomicTYMV RNAs (3).

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TABLE 1. The stabilities of BMV RNAs 1 to 3 in a wheat germ translation system and the dependence of their translational activities on a 5' cap structure^a

Is tyrosylation of TLS^BMV important for translation of BMV RNA? In a way that is analogous to that for the Val-TLS^TYMV of TYMVRNA, the tyrosine residue of the Tyr-TLS^BMV might become incorporatedas the N-terminal amino acid of BMV proteins. For the isolationof tyrosylated BMV RNAs and free TLS^BMV, we tried several approachesbut only obtained unexpectedly low recoveries of tyrosylatedRNAs after a standard acidic phenol extraction (phenol solutionssaturated with 0.2 M sodium acetate at pHs 4.5, 5.0, 5.5, and7.5 were tested). Only <1% [³H]Tyr-TLS^BMV could be retrievedfrom a TLS^BMV fraction after phenol extraction (as measuredby liquid scintillation counting of trichloroacetic acid (TCA)-precipitatedaliquots before and after phenol extraction), whereas typically60% [³H]Tyr-tRNA^Tyr could be retrieved by the same procedure.

To study Tyr-TLS^BMV behavior during BMV RNA translation, wethen tried a coupled reaction system by preincubating a TLS^BMV[³H]tyrosylation mixture (with 1 µM TLS^BMV, with or withouttRNA^Tyr as controls) for 60 min and adding it to a standardtranslation reaction mixture with a 1,500-fold excess of unlabeledTyr and with ribosomes preprogrammed with 8.6 nM native BMVRNA mixture or with 100 nM native TYMV RNA (as a control). Thesubsequent incubation step of the coupled tyrosylation-translationreaction took 90 min. No BMV-encoded proteins with TLS^BMV-derived[³H]Tyr were detected by PAGE and autoradiography. However,two protein bands, of about 45 and 70 kDa, did appear in eithercase (Fig. 3A, lanes 1 and 4). The same band pattern was alsoobserved when the preincubated tyrosylation mixtures were analyzedwithout the addition of the wheat germ system (not illustrated).Surprisingly, these [³H]Tyr-containing proteins appeared tocorrespond to the yeast TyrRS and bovine serum albumin (BSA),which were present at 1 and 5 µM, respectively, in ourstandard tyrosylation reactions. During the 60-min preincubationstep in the absence of translation, the TLS^BMV must have stronglyinduced Tyr-AMP formation in an idling reaction of the TyrRS,resulting in the subsequent tyrosylation of TyrRS itself andof BSA. In contrast, complex formation between TyrRS and yeasttRNA^Tyr only led to background levels of idling Tyr-AMP formationand protein modification, comparable to the levels without tRNA^Tyradded (Fig. 3A, lanes 2 and 5 and lanes 3 and 6, respectively).In a kinetic tyrosylation assay with the TLS^BMV, one set oftime points was stopped in the usual way by TCA precipitationto reveal the accumulated amounts of [³H]tyrosylated RNAs andproteins, and another set was stopped by the addition of a 150-foldmolar excess of RNase A prior to TCA precipitation in orderto identify exclusively the amount of [³H]tyrosylated proteins(Fig. 3B). After 60 min, up to 50% of the total counts of TCA-precipitable[³H]Tyr appeared to originate from the side reaction with TyrRSand BSA. In a parallel experiment with the tRNA^Tyr aminoacylationreaction, only background levels of protein-linked [³H]Tyr wereretained after RNase A treatment (Fig. 3C). For unknown reasons,the remaining 50% of the [³H]Tyr linked to the TLS^BMV (Fig.3B) could not be recovered in significant amounts by a standardacidic phenol extraction (see above), whereas for Tyr-tRNA^Tyra significant level of recovery was obtained. Due to these complicationsand to the high background levels of [³H]Tyr modification ofTyrRS and BSA in coupled BMV RNA [³H]tyrosylation and translationexperiments, we could not answer the question about a putativerole of the Tyr-TLS^BMV as a ribosomal substrate for the N-terminaldonation of Tyr to BMV proteins.

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FIG. 3. [³H]Tyr-labeled proteins and RNAs in a coupled tyrosylation-translation system. (A) Autoradiogram of a protein gel after sodium dodecyl sulfate-PAGE of coupled tyrosylation-translation reaction mixtures. The premix compositions of the various samples are indicated above the gel, molecular weight markers are shown on the left, and reference positions of BSA and TyrRS are shown on the right. For further details, see the text. (B and C) Tyrosylation kinetics were monitored for the TLS^BMV (B) and for yeast tRNA^Tyr (C). During the 60-min incubation of the [³H]tyrosylation mixture, samples were either directly quenched in TCA ( ${circ}$ ) or incubated in the presence of a 150-fold molar excess of RNase A for 2 min at 37°C prior to TCA precipitation, filtration, and liquid scintillation counting (•). All data are representative of three independent experiments.

TLS^BMV complementation in trans. To examine whether the TLS^BMV could also function in trans duringtranslation, just like the TLS^TYMV, we analyzed translationin mixtures of the 3'-truncated RNAs 1 to 3 in the presenceof increasing concentrations of free TLS^BMV, and indeed, theTLS^BMV partially restored the translation of the 3'-truncatedRNAs (Fig. 4). An optimum for this complementation mechanismwas observed with about an eightfold excess of TLS^BMV over BMVRNA (Fig. 4). A further increase of added TLS^BMV reduced translation.Importantly, the addition of excess amounts of yeast tRNA^Tyrdid not have significant effects on the translation efficiencyof the 3'-truncated BMV templates (Fig. 5).

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FIG. 4. Translational complementation in trans by the free and complete TLS^BMV. The defective translation of T7 transcripts of BMV RNAs 1 to 3 with a disrupted 3' TLS^BMV (-) was complemented with increasing concentrations of free TLS^BMV (indicated in molar excess over the 3'-truncated RNAs) and was compared to the translation pattern of the intact RNAs (+). Optimal complementation was observed at about an eightfold molar excess of free TLS^BMV. Relative band intensities were calculated from three independent experiments. See the legend for Fig. 2 for further information.

In order to further localize the complementation activity, wetested TLS^BMV variants that lacked certain substructures andhad been studied previously for their substrate activities withTyrRS (11). The TLS^BMV variant without hairpin structure B2,TLS^BMV(-B2), restored translation even better than the completeTLS^BMV (Fig. 5). Again, this complementation showed an optimum,at about a 2.5-fold molar excess of TLS^BMV over the 3'-truncatedBMV RNAs. In contrast, TLS^BMV(-C), which lacks hairpin C, couldnot complement much for translation in trans, and already ata 2.5-fold molar excess of TLS^BMV(-C) or higher, the translationof the 3'-truncated BMV RNAs was inhibited even further, untilthere was no translation with a 12.5-fold excess. When increasingconcentrations of TLS^BMV(-C) were added to a translation mixtureprogrammed with an intact and native population of BMV RNAs,all translation, including that of sgRNA, was inhibited, withhalf-maximum inhibition at a 50-fold excess of TLS^BMV(-C) (notillustrated). TLS^BMV(-C) possibly inhibits translation by bindingand inactivating an essential factor. Intriguingly, TLS^BMV(-A,-B1, -B3, -E), which actually only harbors stem-loop structuresB2, C, and D, was surprisingly efficient at complementation(Fig. 5) and could restore translation almost completely (70to 80%) to the original pattern (a result also showing the absenceof significant upstream RNA cleavage after 3'-TLS^BMV removal).Apparently, a combination of stem-loop structures B2, C, andD is not only sufficient for the initiation of translation fromthe 5' ends of the genomic RNAs, but can initiate even moreefficiently than the complete free TLS^BMV.

DISCUSSION

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Discussion

This study was aimed at investigating to what extent the TLSof BMV RNA is involved in the translation of viral protein,as recently reported for TYMV (3). In contrast to the singlegenomic TYMV RNA with its overlapping ORFs 1 and 2, the genomeof BMV is located on three different genomic RNAs (Fig. 1) withoutany overlapping ORFs. Although the TLS^BMV does have functionalproperties resembling those of a canonical tRNA^Tyr, its structuraltRNA mimicry is not as obvious as that of the TLS^TYMV for tRNA^Val.

Effects of TLS^BMV removal. In our wheat germ system, disruption of the 3' TLS^BMV part ofthe BMV RNAs strongly reduced the translation of RNA1 and -2into Me/He and RdRp (needed early in infection), whereas thetranslation of RNA3 into movement protein was less dependenton an intact TLS^BMV. Coat protein expression from sgRNA wasnot significantly affected by TLS^BMV disruption (MP and CP bothfunction later in infection). Interestingly, this resemblesthe effect observed when the TLS^TYMV of TYMV RNA is disrupted:the expression of polyprotein (with domains for Me/He and RdRp)is blocked, whereas that of MP and CP is not affected (3).

The 3' UTR of the BMV RNAs, with three pseudoknot structuresjust upstream of the TLS^BMV, is known to stimulate translation(as a 3' fusion construct with reporter genes in carrot protoplasts)by functionally substituting for a poly(A) tail, as was alsoobserved for a comparable 3' UTR of tobacco mosaic virus (TMV)(13). Work by others (19) indicated the importance of the RNAcontext of 3' UTRs; results from experiments in vivo (barleyprotoplasts) and in vitro (wheat germ extract) with a seriesof partial UTR deletions confined to only BMV RNA3 and sgRNAshowed no UTR stimulation of translation but an essential stimulationof RNA replication for RNA3. For the single genomic TMV RNA,the 3'-UTR pseudoknot domain (also called UPD) just upstreamof the 3' TLS^TMV appeared to specifically bind eIF1A (34). Thesingle genomic TYMV RNA does not contain such a prominent pseudoknotdomain, and its polyprotein translation in a wheat germ systemis fully dependent on the presence of the TLS^TYMV (note thatits translation of MP from the genomic RNA and of CP from thesgRNA is TLS^TYMV independent [3]). Whereas this TLS^TYMV-mediatedpolyprotein translation of TYMV RNA was found to be cap independent(but not that of MP and CP [3]), translation of the three BMVRNA transcripts was fully cap dependent in a wheat germ system(Table 1). The same is true for the subgenomic BMV RNA4 andhas also been reported for translation of the native BMV RNAsin a rabbit reticulocyte lysate (2). Apparently, both the pseudoknotdomain and the TLS^BMV regulate BMV translation. Since our BMVRNAs with a deleted TLS^BMV still have an intact 3' pseudoknotdomain, the TLS^BMV may play a dominant role for the differentialstimulation of translation.

Novel TyrRS reaction induced by TLS^BMV. During our attempts to purify substantial amounts of [³H]Tyr-TLS^BMV,the TLS^BMV-triggered protein tyrosylation by TyrRS was an unexpecteddiscovery. Self-aminoacylation was already known for AspRS fromyeast, and this activity has been shown to be abolished by thepresence of tRNA^Asp (18). Therefore, the specific stimulationby the TLS^BMV of TyrRS-catalyzed protein tyrosylation and thecontrasting absence of such a stimulation by the cognate tRNA^Tyrare now interesting. The protein tyrosylation rate of the enzymewas much slower, however, than the initial TLS^BMV aminoacylationrate and did not significantly affect the k_cat and K_m valuesreported earlier for the TLS^BMV (11; data not shown). We didnot succeed in detecting a signal of TLS^BMV-donated N-terminalTyr to specific viral translation products in the high backgroundnoise of TyrRS-mediated tyrosylation of other proteins. On accountof our complementation studies with TLS^BMV subfragments, however,we concluded that the presence of a 3'-linked Tyr is not strictlyessential for the start of RNA1 and -2 translation and thattheir translation products are not necessarily initiated withTyr. This finding is parallel to observations for TYMV RNA (3).It is worthwhile to mention that in the latter study, no sidereactions of ValRS-mediated [³H]valylation of proteins werefound and the N-terminal [³H]Val incorporation took place withpurified [³H]Val-TLS^TYMV.

Complementation in trans by TLS^BMV fragments. In our wheat germ system, the defective translation of BMV RNAs1 to 3 caused by the removal of their 3' TLSs can be partiallycomplemented by the addition in trans of a small RNA fragmentcorresponding to a free TLS^BMV. Strikingly, even a subfragmentwith only the hairpin conformation of the structures B2, C,and D and without an acceptor-arm-like structure is sufficientfor restoring this translation and complements even better thanthe complete TLS^BMV fragment. Apparently, just as for the TLS^TYMV,a 3'-terminal structure upstream of the final 3' acceptor armis crucial for translation. This also implies that the completeTLS^BMV may represent a compromise for efficient functioningin both translation and replication (6), whereas the foldingof the shortened fragment may be more favorable for translation.For the distantly related Alfalfa mosaic virus (AMV), an Alfamovirusalso belonging to the family Bromoviridae, a structural switchof the 3' UTR from a hairpin array into a sort of TLS regulatesthe functional transition from translation to replication (24,27). For this TLS^AMV, only recognition by a CCA-adding enzymewas detected, but no aminoacylation occurred; the TLS conformationwas found to be required for the initiation of replication.The translationally active hairpin conformation that is requiredduring the first steps of the AMV infection and that no longercontains an acceptor-arm-like structure, is essentially inducedby coat protein binding to this region, which prevents the initiationof replication. Accordingly, for efficient translation at theearly stage of virus infection, a few molecules of coat proteinare required in the inoculum. Either the coat protein or theexposed loop sequences of the hairpin structures bind translationfactors and establish 5'- to 3'-end communication of the viralRNA (5). For BMV, a similar structural transition has been suggested(1, 29), but the presence of coat protein in the inoculum isnot required for an infection. Interestingly, a mutant AMV witha mutant coat protein with a lost affinity for the 3' UTR, hencedisplaying a lost infectivity for protoplasts, can be rescuedby replacing the 3' UTR of AMV with that of BMV (24a).

Stem-loop structure C has been extensively investigated andis known to be an essential docking site for the replicase complexin order to start minus-strand synthesis (31). Especially itsterminal triloop is important for the association of the replicase,either directly or via an as yet unidentified host factor. Hairpinstructure B2 has been suggested to structurally mimic the anticodonstem-loop structure of canonical tRNA^Tyr. Although B2 interactswith the TyrRS enzyme, extensive mutational analysis revealedthat the sequence of the loop structure can be changed withoutany loss of tyrosylation efficiency (11). In the current three-dimensionalmodel of the TLS^BMV, hairpin D is positioned such that it maymimic the T ${Psi}$ C hairpin of canonical tRNAs, and indeed the baseof this hairpin structure makes strong contacts with the TyrRSenzyme. However, no functional role for this hairpin has beenreported thus far (11, 12), and it is absent from the relatedTyr-accepting TLS^BBMV of broad bean mottle virus (6).

Model for a role of TLS^BMV in RNA 5'- to 3'-end communication. Assuming a 5'- to 3'-end communication in the case of BMV RNAs,one can envisage an intramolecular RNA-RNA interaction suchas that found for barley yellow dwarf virus. In the latter case,a stem-loop structure in the 3' UTR specifically base pairsvia a kissing-loop interaction with the loop sequence of a hairpinstructure in the 5' UTR (15). Could BMV RNA1 and -2 interactthrough a hypothetical kissing-loop interaction between 3' TLS^BMVstructures B2, C, and/or D and a structure in the 5' UTR, whereasthe 5' UTRs of RNA3 and sgRNA interact less or not at all onaccount of a totally different sequence and folding? If so,this may explain why TLS^BMV disruption does not have such pronouncedeffects on the translation of RNA3 and sgRNA as it does forRNA1 and RNA2. Otherwise, a 5'- to 3'-end communication forefficient eukaryotic translation can also be established byprotein-protein interactions other than those between initiationfactors and PABP, such as those shown for AMV RNA and for variouscellular mRNAs (14, 20, 23, 24, 30, 33). Recently, the translationof BMV RNAs 1 to 3 (but not that of sgRNA or cellular mRNAs)was shown to depend on host protein factors that are normallyinvolved in deadenylation-dependent mRNA decapping (26).

Apparently the TLS of BMV plays a remarkable and central rolein the life cycle of the virus. Upon its entry into the hostcell, the first step during infection is to start the synthesisof viral enzymes for RNA replication. Here we have shown that(parts of) the TLS^BMV plays a crucial role in the expressionof those enzymes. When the replicase has initiated minus-strandRNA synthesis and is moving on the plus strand from the 3' TLS^BMVin the 5' direction, it could clash with ribosomes that aretranslating this plus strand from 5' to 3'. However, the dualfunctioning of the TLS^BMV, both as a trigger for translationinitiation and as a promoter for minus-strand RNA synthesis,may prevent the simultaneous start of protein and RNA synthesison the same RNA molecule. TLS^BMV binding to the ribosome wouldprevent TLS^BMV binding by the replicase and vice versa. Thisdual regulation is absent for the sgRNA; its translation intocoat protein is not regulated by the TLS^BMV and it does notserve as a template for minus-strand synthesis. Perhaps additionalhost factors function in this dual regulation and serve as adocking site for either the ribosome or the replicase. Quadtet al. (28) reported that a subunit of translation initiationfactor 3 from wheat germ as well as from rabbit reticulocytesspecifically binds to the RdRp of BMV. At the end of the infectioncycle, the virus needs to encapsidate its genomic RNAs for theproduction of mature virions. Again, the TLS of BMV is a targetfor regulation, as a nucleation site for coat protein assemblyand RNA encapsidation (7). Coat protein binding to the TLS^BMVwould then prevent alternative interactions with ribosome orreplicase. For AMV, there is a different situation, in whichdeletion of the 3' UTRs of RNAs 1 and 2 does not interfere withtheir encapsidation by the coat protein originating from RNA3(32). For TYMV, the initiation signal for RNA encapsidationappears to be localized on a hairpin structure with characteristicC-C and C-A mismatches located in the 5' UTR close to the polyproteinstart codon, the contact area for the 3' TLS^TYMV (4).

The central role of the TLS^BMV in the temporal regulation ofthe consecutive processes of RNA translation, RNA transcription,and RNA encapsidation in BMV may be of comparable importancefor the infection cycles of other TLS-harboring viruses.

ACKNOWLEDGMENTS

We are grateful to P. Ahlquist and M. Prins for their kind giftsof infectious BMV clones. We thank J. F. Bol for valuable discussionsand Agnès Clénet for technical assistance.

This work was supported by a short-term FEBS fellowship to S.B.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, Leiden Institute of Chemistry, Leiden University, Einsteinweg 55, P.O. Box 9502, 2300 RA Leiden, The Netherlands. Phone: (31) 71 527 4770. Fax: (31) 71 527 4340. E-mail: b.kraal{at}chem.leidenuniv.nl.

REFERENCES

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