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FEBS Letters 580 (2006) 2591–2597

A translational enhancer element on the 30-proximal end of the Panicum mosaic virus genome

Jeffrey S. Batten1, Benedicte Desvoyes2, Yoshimi Yamamura, Karen-Beth G. Scholthof* Department of Plant Pathology and Microbiology, Texas A&M University College Station, TX 77843-2132, United States

Received 13 January 2006; revised 22 March 2006; accepted 3 April 2006

Available online 21 April 2006

Edited by Hans-Dieter Klenk

enhancer (TE) [7–9]. The BYDV 30-TE interacts with the Abstract Panicum mosaic virus (PMV) is a single-stranded po- 0 sitive-sense RNA virus in the family Tombusviridae. PMV geno- 5 -UTR to promote translation of uncapped and non-poly- mic RNA (gRNA) and subgenomic RNA (sgRNA) are not capped adenylated viral mRNAs [10]. In addition to BYDV, a similar or polyadenylated. We have determined that PMV uses a cap- strategy to translate virus proteins has been documented for independent mechanism of translation. A 116-nucleotide transla- viruses in the Tombusviridae, including Red clover necrotic tional enhancer (TE) region on the 30-untranslated region of both mosaic virus, Tobacco necrosis virus, Turnip crinkle virus, and the gRNA and sgRNA has been identified. The TE is required for Tomato bushy stunt virus (TBSV) [9,11–14]. (As recently dis- efficient translation of viral proteins in vitro. For mutants with a cussed by Miller and co-workers, BYDV might be a virus in compromised TE, addition of cap analog, or transposition of the the Tombusviridae, instead of the Luteoviridae [15].) Based on cis-active TE to another location, both restored translational previous work [5], and our results with PMV, it appears that competence of the 50-proximal sgRNA genes in vitro. cap-independent translation is a common, and defining fea- Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. ture, of small RNA plant viruses in the Tombusviridae. We have identified a cis-acting element on the 30-end of the Keywords: Cap-independent translation; Panicovirus; PMV RNA that is necessary for efficient translation of its gRNA 3-untranslated region; Translation strategies; RNA structure and sgRNA. Incorporation of a 50-cap structure to sgRNA tran- scripts partially restored translation of some PMV TE element mutants. Furthermore, the TE element is functionally active when placed mid-way on the PMV genome. Fabian and White 1. Introduction [12] suggested that the 50- and 30-ends of most Tombusviridae, might form structures similar to TBSV to facilitate translation. Panicum mosaic virus (PMV), a single-stranded, positive- We have determined from experimental evidence and RNA fold- sense RNA virus, is the type member of the Panicovirus genus ing analyses that manipulation or deletion of the PMV 30-TE af- in the family Tombusviridae [1]. PMV encodes two replicase- fects translation, likely via the destruction of a putative loop associated proteins, p48 and its read-through product p112, structure. We provide evidence that the maintenance of a Y- from the 50-proximal end of the 4326 nucleotide (nt) genomic shaped structure in the 30-TE is critical for functionality. We RNA (gRNA) [1–3] (Fig. 1A). Four proteins (p8, p6.6, p15, have further extended these observations in showing that the and the 26-kDa capsid protein) encoded from the single 30- TE is functional in the positive (but not negative) orientation coterminal subgenomic RNA (sgRNA) are implicated in virus when it is relocated elsewhere on the PMV gRNA and sgRNA. spread [4]. Following the strong start codon for p8 expression, p6.6 is expressed from a non-canonical start codon [1,4]. Inter- nal ribosome entry may be used to express the capsid protein 2. Materials and methods (CP) gene, following by leaking scanning to p15, a nested gene downstream of the CP start codon [1,4]. 2.1. Recombinant plasmids Like other members of the Tombusviridae, PMV gRNA and Standard molecular biology techniques were used to develop plas- sgRNA are not capped or polyadenylated, in contrast to cellu- mid constructs and for in vitro transcription-translation assays. The lar mRNAs. Therefore, PMV must employ other strategies for genomic and subgenomic mutants (Figs. 1A and 2A) were made either 0 translation of the gRNA and sgRNA in the host cell. Some by truncation from the 3 -end of the PMV cDNA constructs or by gen- erating internal deletions within the 30-UTR using restriction digestion plant viruses have discrete cis-acting RNA elements to facili- followed by plasmid re-ligation. The PMV sgRNA constructs were de- tate cap-independent translation [5,6]. This was first shown rived from an RT-PCR fragment (Sg1) encompassing the full-length for Barley yellow dwarf virus (BYDV) when a 100-nt RNA sgRNA plus an added 50-proximal T7 promoter sequence [1]. A set fragment on the 30-UTR was identified as a translational of constructs with genomic cDNA ending in CCC (PMV99) or the arti- ficially introduced 30-poly(A) tail (pPMV85) [1], were used for compar- ative translation and replication assays.

*Corresponding author. Fax: +1 979 845 6483. E-mail address: [email protected] (K.-B.G. Scholthof). 2.2. In vitro transcription and translation Transcripts of sgRNA and gRNA were produced as described pre- 1 Present address: G. C. Hawley Middle School, 2173 Brassfield Rd, viously [1,4]. In vitro wheat germ translation with [35S] methionine Creedmoor, NC 27522, United States. or [14C] leucine, SDS–PAGE, and autoradiography was performed 2 Present address: Centro de Biologı´a Mole´cular Severo Ochoa, CSIC- as described [1,4]. Relative band densities of the proteins were deter- UAM, Campus Cantoblanco, 28049 Madrid, Spain. mined with NIH Image Software .

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.04.006 2592 J.S. Batten et al. / FEBS Letters 580 (2006) 2591–2597

Fig. 1. Panicum mosaic virus (PMV) mutants. (A) The 4326 nucleotide PMV genomic RNA (gRNA). The replicase-associated proteins, p48 and p112, are expressed from the gRNA. The asterisk signifies the site of an amber read-through codon to enable the expression of the 112-kDa protein [1,3]. The 30-co-terminal subgenomic RNA (sgRNA) initiates at nucleotide 2851 (bent arrow) and encodes p8, p6.6, p15, and coat protein (CP). The 30-untranslated region of the gRNA is not to scale. (B) PMV replication and movement as co-infections with SPMV, a satellite virus. SPMV is used as a phenotypic marker; it enhances the symptoms on PMV-infected proso millet plants [27,28]. The control healthy plants did not accumulate PMV RNA (indicated by ‘‘na’’). Positive or negative results are indicated by plus (+) or minus (). (C) In vitro translation of PMV gRNA and its derivatives. The replicase-associated p48 and p112 proteins encoded from the gRNA transcripts are indicated. The p48C protein is derived from the 29-kDa C-terminal portion of p48, but its role in the biology of PMV is not known [2,3].

2.3. Replication assays in millet protoplasts and plants the experimental analyses were performed with in vitro analy- Foxtail millet (Setaria italica cv. German R) protoplasts or plants ses on various derivatives of the gRNA or sgRNA. were assayed for PMV replication at 40 hours or 14 days postinocula- We first examined the effects of mutations on the translation tion, respectively [1,4]. of sgRNA, rather than gRNA. For this purpose a set of Sg1-de- rived deletion constructs were used (Fig. 2A). In agreement 2.4. Secondary structure prediction The secondary structure predicted for the PMV 50- and 30-UTR at with the PMV gRNA results (gDSmaI; Fig. 1), the sgRNA mu- 30 °C were obtained using the Mfold program [16,17], version 3.0 on tant DSmaI(Fig. 2A), with a 30-UTR deletion, was competent the Burnet Institute website (http://mfold.burnet.edu.au/). for translation (Fig. 2B) although we detected a reduction in expression of p8 (83–36) and CP (52–29) (Fig. 2B). However, the ratios of p8:CP translated from Sg1 and DSmaI transcripts 3. Results and discussion were similar (1.6:1 and 1.2:1, respectively). Also in agreement with the results obtained with the gRNA, the mutation in To evaluate if the 30-UTR was required for PMV transla- DBstXI, strongly reduced translation from the sgRNA tion, several deletions were made. First, as a serendipitous con- (Fig. 2B). In fact, a deletion of only 4-nt at the BstXI site at sequence of our cloning strategy during the assembly of an nt 4172 (Fig. 1A; BstXI–) had the most pronounced effect when infectious PMV cDNA clone (pPMV85; Fig. 1A), we deter- compared to the results obtained with the DBstXI mutants. mined that the introduction of an extra 30-terminal poly(A) tail Internal deletions from BstXI to HpaI(DBstXI–HpaI) or HpaI of 30 nucleotides did not influence infectivity [1]. From this, to SmaI(DHpaI–SmaI) also virtually eliminated translation the artificial tail was deleted to create pPMV99, a full-length (Fig. 2A and B). These results suggested the presence of a construct with a 30-terminus of ‘‘CCC’’. Infectivity assays TE, that the BstXI site resides within this element, and that showed that PMV99 caused an infection that was indistin- its 30-border was in proximity to the HpaI site (nt 4201). guishable from that observed with PMV85 (Fig. 1B). Additional internal deletion mutants were generated to Transcripts from a 30-UTR truncation (nt 4243–4326; determine the 50-border of the putative TE. Introducing an gDSmaI) or an internal deletion spanning the ApaI sites from EcoRV site at nt 4012 (4 base changes) did not significantly af- nt 3142–3410 (pKB238) [3] did not disrupt translation of p48 fect translation from the sgRNA (Fig. 2B). Similarly, transla- or p112 from the gRNA (Fig. 1A and C). As expected, the tion was not compromised by a 73 nt deletion (nt 4012–4085) 30-proximal genes were not required for in vitro translation from EcoRV to Eco47III (DEcoV-47). However, a 116-nt dele- of the replicase proteins from KB238 transcripts (Fig. 1C). tion from Eco47III to HpaI(D47-HpaI) caused significant However, deletion of 154-nt from the 30-end of the PMV reduction of protein translation, reminiscent of the DBstXI cDNA (nt 4172–4326; gDBstXI) nearly abolished translation and BstXI-mutants (Fig. 2B). These results mapped the TE from the gRNA (Fig. 1A and C). These constructs were not to a 116-nt fragment from the Eco47III to the HpaI sites infectious in plants (data not shown), so the remainder of (Fig. 2A). J.S. Batten et al. / FEBS Letters 580 (2006) 2591–2597 2593

Fig. 2. The role of 30-UTR translation element for translation from the PMV subgenomic RNA (sgRNA). (A) Mutations on the sgRNA represent their location on the PMV cDNA, as shown in Fig. 1A. The proteins expressed from the PMV sgRNA are the 26-kDa coat protein (CP), p15, p8 and p6.6. (B and C) Transcripts from the subgenomic deletion mutants were either uncapped (panel B) or capped (panel C). (D) CAT accumulation from capped (+) or uncapped () transcripts. Relative optical density units from the p8 and CP in vitro translation products, and the ratios of the p8:CP are indicated below the gels. The molecular weight markers (M) are indicated in kilodaltons (kDa). 2594 J.S. Batten et al. / FEBS Letters 580 (2006) 2591–2597

In vitro translation of BYDV and TBSV mutants lacking stem-loop domain. However, the 4-nt change to the BstXI site the 30-TE was restored by addition of cap analog [18–20]. adds an extra dimension to the prediction by Fabian and Since PMV is in the same virus family, it seemed likely that White [12], in that the loop interaction between the 50–30 structure(s) on the PMV 30-UTR might mimic cap function. UTRs (Fig. 3A) is not the sole crucial component for PMV Because PMV sgRNA encodes four ORFs, and contains the translation. We suggest that the entire Y-shaped domain con- 30UTR, it was possible to examine the translational effect of tributes to robust translation (Fig. 3B). When the Y-shaped the presence of a cap analog on the 30-end mutants. The domain of the 30-TE was perturbed, by disruption of D2 objective was to test the hypothesis that PMV uses a cap-inde- and/or D3, translation decreased significantly (DBstXI and pendent translation strategy. In vitro transcripts of the DHpaI–SmaI). Based on the predicted structure, this suggests sgRNA 30-UTR mutants with a 50-m7GpppN cap were evalu- that maintenance of the Y-shaped domain, although on a ated for translation (Fig. 2C). Capped and uncapped chloram- shorter stem, was sufficient for translation to occur. However, phenicol acetyl transferase (CAT) gene transcripts were complete disruption of the Y-shaped domain abolished trans- generated as control template. This permitted verification that lation from DBstXI–HpaI and BstXI– (Figs. 2B and 3). This in our experimental conditions, cap analog was efficiently suggests that D2 and D3 are both critical for viral translation. incorporated, as determined by increased translational effi- Presumably, D2 (GC loop) can be thought of as being static, ciency of CAT [21] (Fig. 2D). For PMV sgRNA DHpaI–SmaI with two hairpin loops including D1 to prevent changes in transcripts, the addition of 50-cap analog essentially restored the Y-shaped conformation. D3, the long stem loop, is also translation of p8 and CP. For the longer 30-UTR truncations, necessary for long-distance interactions to the 50-UTR. These DBstXI and the internal deletion of Eco47III to HpaI(D47- domains may help the gRNA or sgRNA mRNA circularize by HpaI), the translation of p8 protein also was slightly enhanced 30–50 base-pairing. Since a similar Y-shaped domain (termed by the addition of cap during the transcription reaction R3.5) was predicted in TBSV and is essential for 30 cap-inde- (Fig. 2C). For the other mutants, cap had less prominent pendent translational enhancer [12], the 50–30 RNA-RNA restorative effects on p8 translation and no increase in CP interaction mechanism of PMV might mimic those reported was observed. Similar effects of cap were found for the for TBSV. PMV gRNA mutants (data not shown). That the RNA tran- Having established that the PMV 30-TE is a critical region scripts were intact indicated that loss of translational activity that may operate through a Y-shaped structural domain, one experiments was not due to RNA decay (data not shown). would predict that repositioning of the TE would not necessar- The relatively stronger response of p8 to cap addition and ily disrupt function. To test whether the physical (cis) location possibly p6.6 (compared to CP) suggest that they utilize the of the PMV TE affects translation, a 116-nt Eco47III to HpaI 30-TE to facilitate translation. In contrast, expression of the blunt-end fragment encompassing the Y-shaped TE element 30-proximal genes (p15 and CP) on the sgRNA may require was inserted at the PflMI site (nt 3129) within the p6.6 ORF additional unknown features or mechanisms for efficient (Figs. 1A and 4A). Two sets of constructions were made, as translation [4], to load host-encoded translational initiation shown in Fig. 4A. First, the 116-nt fragment was deleted from factors. the 30-UTR and transferred into the PflMI site (D series), and Our results suggest that PMV uses the 30-UTR for cap-inde- second, an additional element was inserted into the wild type pendent translation, although the effects are less straightfor- PMV gRNA or sgRNA cDNAs. We also tested whether mu- ward for the expression of the 30-proximal CP gene. tants with TE inserted in the negative orientation (one or Interestingly, we had previously found that abolishing the two copies) placed in the PflMI site affected translation. As expression of PMV CP (by introduction of premature stop co- predicted, the translation of p6.6 was disrupted when the dons or weakening the start codon context) did not affect 116-nt element was inserted at the PflMI site (Fig. 4B). expression of p8 in vitro [4]. However, in protoplasts and The Sg1-constructs lacking the 30-TE region, derived from plants it was clear that the reduction or lack of full-length the D47-HpaI mutant (a deletion from Eco47III to HpaIon CP expression also reduced or abolished p8 expression [4]. the PMV cDNA) were used to clone the D constructs From this, it is possible that in vivo, the PMV CP is an acces- (Fig. 4A). From this, the addition of one (D1+) or two sory factor for translation, either as a regulator or via interac- (D2+) copies of the ectopic TE in the positive orientation into tions with host factors. the PflMI site restored p8 accumulation to near wild-type lev- As reported for BYDV and TBSV, the 50–30 RNA–RNA els (Fig. 4B), but CP expression was scant. This is clearly re- interaction is required for efficient cap-independent translation flected by the skewed ratios of p8:CP in these mutants (4:1) of the viral RNA [5,10,12]. In a similar fashion, on the PMV compared to Sg1 (1.6:1). The addition of the 116-nt fragment, genome, nt 10–15 (50-UTR) and nt 4150–4155 (30-UTR) may while retaining the wild-type 30-UTR (1+), was competent for interact (Fig. 3A), as proposed by Fabian and White [12]. expression of p8 protein (Fig. 4B) when compared to Sg1 tran- Using Mfold [16,17], we modeled this putative interaction scripts. However, the accumulation of CP was again decreased (Fig. 3). From the folding program and our in vitro and and the ratio of p8:CP increased. in vivo results, the 30-TE we identified may be significant for From our previous experiments, mutants upstream of the the biology of PMV. The TE folds into Y-shaped structure CP ORF, including those on p6.6, did not negatively effect with two conserved terminal loops (regions D1 and D2) on a in vitro translation of CP or p15 [4]. From this, inspection of long stem structure (D3) (Fig. 3B). In this conformation the the sequence of the 116-nt fragment raises several other possi- TE promotes translation, as illustrated in Fig. 3. From this bilities to explain why CP expression declined. For TE inserts it follows that the TE structure likely recruits translational in the positive orientation at the PflMI site, the 116-nt RNA machinery, such as translation factors, as shown for satellite includes a start codon with a strong surrounding nucleotide tobacco necrosis virus [22]. The mutants (DSmaI and DE- context for translation (gacAUGa) of a 3.5-kDa open reading coV-47) were efficiently translated, and had the Y-shaped long frame. (BLAST analyses of this ORF returned no hits other J.S. Batten et al. / FEBS Letters 580 (2006) 2591–2597 2595

Fig. 3. Schematic representations of the proposed 50–30 RNA–RNA interaction and the putative structure of the 30-TE of PMV. (A) The RNA secondary structures for the 50 and 30-UTR are shown using Mfold [16,17]. The free energy of the structures at 30 °C is indicated. Nucleotides in 50 and 30-UTR that are predicted to interact are in white on black circles and the dashed lines indicated the putative interaction. Numbers denote the nucleotide positions on the genomic RNA of PMV. (B) Comparison of the predicted stem-loop structures of 30-TE in 30-UTR mutants from nt 4111 to 4200. The structure of the 30-TE in the minus orientation ()30-TE is also shown. The open regions with dashed outline represent deletions on the 30-UTR. The plus (+) or minus () at the bottom of the figure indicates if the transcripts were translation competent in vitro. than PMV.) If the non-canonical start codon of p6.6 (cua- – the ratio of p8:CP increased about 2-fold (Fig. 4B). Yet, a GUGg) is accounted for, a putative fusion protein, with the 4.9-kDa protein was not detected, suggesting that the regula- 116-nt insert resulting in a 4.9-kDa-encoding ORF, could be tion may be structural. expressed. Based on Kozak’s ribosome scanning models Placement of the ectopic 116-nt TE in the negative orienta- [23,24], the accumulation of the PMV CP from the sgRNA tion of the PflMI site did not restore translation of p8, and should be greatly reduced compared to wild-type (or as an in- may have been inhibitory (Fig. 4A and B, D2–). Additional creased ratio of p8:CP). And, in fact, this is what was observed copies of the TE in either orientation did not greatly alter 2596 J.S. Batten et al. / FEBS Letters 580 (2006) 2591–2597

sgRNA translation (Fig. 4B) when added to sgRNA con- structs possessing the native 30-TE. As with the capping exper- iments, the ratios of p8:CP always favor translation from the 50-proximal gene. The (cis) location of Y-shaped structure of the TE on the wild-type 30-UTR appears to be essential for proper expression of the upstream sgRNA proteins (CP and p15). Manipulation of the PMV gRNA (using cDNA mutants and in vitro transcripts) showed that deletion of the 116-nt element abolished translation of p48 and p112 (Fig. 4C). A single ecto- pic copy of the 116-nt TE (inserted at the PflMI site at nt 3129 in the p6.6 ORF) in the positive orientation in a gRNA con- struct lacking the native 30-TE restored translation of p48, albeit not to wild-type levels (Fig. 4C, gD vs. gD1+). Further- more, a negative-sense 116-nt element in the background of the native PMV cDNA or two negative-sense copies of TE to the PflMI site had minimal ability to restore translation (Fig. 4C; g1– and gD2–). The negative-sense copy at the PflMI site might be deleterious due to complementary binding to the wild-type 30-TE or by directly interfering with cap-independent transla- tion. On the sgRNA the 116-nt TE increased translation of the 50-proximal gene (p8) when in the (+) orientation. These data may suggest slight differences in the way the TE functions for gRNA and sgRNA translation. One obvious difference is the distance between the TE and the respective 50-ends of the gRNA and sgRNA. The PflMI-TE is only 278 nt downstream of the 50-end of the sgRNA, but more than 3000 nt from the 50- end of the gRNA. Alternatively, or in addition, in the context of the gRNA, the ectopic PflMI-TE may fold improperly or is less accessible for ribosome recruitment. The genomic RNA mutants were tested for replication in millet protoplasts (data not shown). The deletion or insertion mutants did not replicate even those for which translation ap- peared to be near wild-type levels (Figs. 1 and 3). A single exception was gEcoRV (4 nt changed to introduce the EcoRV site) that replicated poorly in protoplasts. Changes in this mu- tant were located in the non-coding region at the beginning of the 30-UTR (Fig. 1A) and bordering the 116-nt element. The gEcoRV mutant was tested on proso and foxtail millet plants, but accumulation was not detected on either inoculated or upper leaves after multiple greenhouse trials, suggesting that it may be part of a cis-acting element for replication and/or movement. Further, the results suggest that the 30-TE is particularly important for translation of p48 and p112 from the gRNA and p8 and p6.6 from the sgRNA. This leaves open the possi- bility that the translation the 30-terminal CP and p15 genes Fig. 4. In vitro translation of PMV sgRNA and gRNA derivatives with the 116-nt RNA distal to the 30-UTR. (A) The sgRNA (Sg1) may use a combination of leaky scanning with as yet unidenti- deletion and insertion mutants. An 116-nt Eco47III to HpaI fragment fied translation strategies, such as RNA editing or some type on the 30-end of the PMV cDNA was cloned into the PflMI site as of internal regulatory expression sequence [25,26]. Using the single (1) or double copy (2) either in the positive (+) or negative () 116-nt cis-acting TE, it will be possible to evaluate cooperative 0 orientation. The 3 -TE is indicated by the solid or striped Y-shaped interactions with host proteins that contribute to both replica- structure, representing (+) or () orientation, respectively. Some constructs have the 116-nt element deleted (D) from the 30 UTR. tion and translation of this monocot infecting member of the Parallel mutants were constructed to test expression from the PMV Tombusviridae. However, the presence of a 30-TE does appear gRNA. (B) Sg1 assays show optical density units of the p8 and CP to be a feature that is common for many mRNAs including in vitro translation products, and the ratios of p8:CP. The lane labeled those of single-stranded (+)-sense viruses in dicot and monocot ‘D’ refers to the D47-HpaI sgRNA with a deletion from Eco47III to HpaI. (C) In vitro translation products from of each mutant were host plants. compared to those from wild type gRNA (PMV85). Construct gEcoRV created an EcoRV site at nt 4012 (Fig. 1A) and allowed for Acknowledgments: This work was funded by USDA-NRI (99-3503- generation of gEcoV-47, representing a deletion from nt 4012 to 4085 7974) and THECB-ATP (000517-0043-2003) grants awarded to (EcoRV to Eco47III). J.S. Batten et al. / FEBS Letters 580 (2006) 2591–2597 2597

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