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REVIEW published: 29 November 2017 doi: 10.3389/fpls.2017.02047

Structural and Functional Diversity of Plant Virus 30-Cap-Independent Translation Enhancers (30-CITEs)

Verónica Truniger*, Manuel Miras and Miguel A. Aranda

Plant Virology, Centro de Edafología y Biología Aplicada del Segura, Consejo Superior de Investigaciones Científicas, Murcia, Spain

Most of the positive-strand RNA plant viruses lack the 50-cap and/or the poly(A)-tail that act synergistically to stimulate canonical translation of cellular mRNAs. However, they have RNA elements in the 50- or 30-untranslated regions of their RNAs that are required for their cap-independent translation. Cap-independent translation enhancers (CITEs) have been identified in the genomic 30-end of viruses belonging to the family Tombusviridae and the genus Luteovirus. Seven classes of 30-CITEs have been described to date based on their different RNA structures. They generally control the efficient formation of the translation initiation complex by varying mechanisms. 0 Edited by: Some 3 -CITEs bind eukaryotic translation initiation factors, others ribosomal subunits, Encarna Martinez-Salas, bridging these to the 50-end by different mechanisms, often long-distance RNA–RNA Centro de Biología Molecular Severo 0 Ochoa (CSIC), Spain interactions. As previously proposed and recently found in one case in nature, 3 - Reviewed by: CITEs are functionally independent elements that are transferable through recombination Carmen Hernandez, between viral genomes, leading to potential advantages for virus multiplication. In this Instituto de Biología Molecular y review, the knowledge on 30-CITEs and their functioning is updated. We also suggest Celular de Plantas (CSIC), Spain 0 Aurelie Rakotondrafara, that there is local structural conservation in the regions interacting with eIF4E of 3 -CITEs University of Wisconsin-Madison, belonging to different classes. United States 0 *Correspondence: Keywords: plant virus, cap-independent translation, 3 -UTR, translation enhancer element, eukaryotic translation 0 0 Verónica Truniger initiation factor, eIF4F, recombination, 5 –3 RNA–RNA interaction [email protected]

Specialty section: INTRODUCTION This article was submitted to 0 0 0 Virology, The m7G(5 )ppp(5 )N cap-structure that is covalently linked to the 5 end and the poly(A) tail at a section of the journal the 30 end are critical cis-acting elements during canonical eukaryotic mRNA translation, which is Frontiers in Plant Science initiated with the recognition of the 50-cap by the eukaryotic translation initiation factor (eIF) 4E Received: 02 October 2017 and the poly(A) tail by the poly(A) binding protein (PABP). The scaffolding protein eIF4G binds to Accepted: 15 November 2017 both and thus links the 50- and 30-ends of the mRNA resulting in its circularization. In plants, eIF4E Published: 29 November 2017 and eIF4G shape the eIF4F complex, whereas eIF4A is probably loosely attached to the complex Citation: and does not co-purify (Hinnebusch and Lorsch, 2012). A peculiarity of plants is the existence of Truniger V, Miras M and Aranda MA eIF(iso)4F, which includes the isoforms eIF(iso)4E and eIF(iso)4G. The significance of the existence (2017) Structural and Functional of both complexes in plants is not fully understood; they seem to have overlapping functions but Diversity of Plant Virus 30-Cap-Independent Translation differential substrate preferences (Browning and Bailey-Serres, 2015) suggesting specialized roles. Enhancers (30-CITEs). Translation initiation is rate limiting and a highly regulated step (Aitken and Lorsch, 2012). 0 Front. Plant Sci. 8:2047. While most plant-encoded mRNAs contain a 5 -cap and a poly(A)-tail that act synergistically doi: 10.3389/fpls.2017.02047 to stimulate translation, ∼80% of known positive-strand RNA plant viruses lack one or both of

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these features in their genomic and subgenomic RNAs. Thus, cases the 30-CITEs are not functional in vitro in wheat germ non-canonical translation initiation occurs for most plant RNA extract (WGE). viruses and functions independently of a 50 cap or/and a poly(A) tail, using different cis-acting elements and mechanisms (Miras BTE et al., 2017a). It is widely accepted that alternative mechanisms The 30-CITE of Barley yellow dwarf virus (BYDV) is the evolved by viruses to recruit the host’s translational machinery best characterized and was named BYDV like-element (BTE) allow them to compete with the host mRNAs, avoiding host (Figure 1). It is present in all members of the genera defenses at the level of translation. Luteovirus (family Luteoviridae) and Necrovirus, Dianthovirus, Several viruses of the family Tombusviridae and the genus and Umbravirus (family Tombusviridae)(Wang et al., 2010; Luteovirus have been shown to contain cap-independent Simon and Miller, 2013). The BTE structures consist of a long translation enhancers in the 30-end of their genome basal helix that connects with the rest of the viral genome, from (30-CITEs) (Simon and Miller, 2013). Their mRNAs lack both the which two (necrovirus), three (luteovirus) or five (dianthovirus) 50-cap and 30-poly(A) elements. In general, 30-CITEs are able to helices radiate, giving it a cloverleaf-like structure (Wang recruit host translation initiation factors, which are subsequently et al., 2010). BTEs contain a highly conserved sequence stretch brought to the opposite end of the RNA. Based on their different of 17-nucleotides (nt), GGAUCCUGGgAaACAGG, where the RNA structures, these 30-CITEs can be classified into seven underlined bases pair to form the stem of stem-loop I (SL-I). The classes (Miras et al., 2017a). Their molecular mechanisms bases in lower case can vary, but the consensus GNRNA (N is any are also diverse, depending on different host translation base, R is a purine) in the apical loop is conserved (Koev et al., initiation factors and varying circularization mechanisms. 2002). This sequence is supposed to form a particular structure Some 30-CITEs have been shown to interact with eIF4G, others that adds stability to the RNA hairpin, similar as shown for the with eIF4E and others directly with ribosome subunits. For GNRA-loop first identified in λ boxB RNA (Heus and Pardi, 1991; some of the viral genomes carrying 30-CITEs, the translation Legault et al., 1998). Wang et al.(1997) identified in the conserved initiation complex formed at the 30-end is brought to the 17 nt of the BTE of several viruses a sequence stretch that was opposite end by 50–30-RNA interactions based on sequence complementary to the 18S rRNA and proposed a model where complementarity, in other cases by ribosome interactions with this complementarity results in direct binding of the ribosome to both ends. the BTE. Additionally, the unpaired bases at the hub of the helices 30-CITEs can remain functional when placed at the 50-end, and SL-II from BYDV BTE seemed to be critical, as mutations similar to the more abundant IRESes (internal ribosome entry reduced its activity (Guo et al., 2000; Kraft et al., 2013). sites), able to enhance cap-independent translation of many animal virus and some eukaryotic mRNAs (Balvay et al., 2009). TED But while IRESes are able to recruit the ribosome internally The first 30-CITE that was discovered was the one present (independent of the 50-terminus), for several 30-CITEs it has been in Satellite tobacco necrosis virus (STNV), a parasitic subviral shown that the ribosome has to scan from the 50-end of the RNA, RNA that encodes only its capsid protein; it was named as insertion of stable SL-structures at the 50-terminus (Guo et al., translation enhancer domain (TED) (Danthinne et al., 1993; 2001) or insertion of upstream out of frame AUGs abolished Timmer et al., 1993; Meulewaeter et al., 1998a). Its structure translation from the correct AUG (Rakotondrafara et al., 2006; has been predicted to form a long stem-loop with several Nicholson et al., 2010). internal bulges and a final loop (van Lipzig et al., 2002; Blanco- Construction and analysis of chimeric viruses have Pérez et al., 2016), but this structure has not been confirmed demonstrated that 30-CITEs can be exchanged between viruses, experimentally (Figure 2A). The STNV TED was shown to suggesting that they are functionally independent RNA elements. be functional in enhancing translation in vitro and in vivo, Since the publication of the last review on 30-CITEs (Simon and being capable of functionally replacing a 50-cap; also, it has Miller, 2013), a new class of 30-CITEs has been identified and been shown to confer cap-independent translation in vitro when the only known case of gain of a precise 30-CITE in nature has moved to the 50-untranslated region (UTR) of an uncapped been described (Miras et al., 2014). In this review, we update the reporter (Meulewaeter et al., 1998b). Recently, Pelargonium line knowledge on 30-CITEs and their functioning. pattern virus (PLPV), the recommended type member of the new Pelarspovirus genus belonging to the family Tombusviridae, was shown to contain a TED-like 30-CITE (Blanco-Pérez et al., 2016). 0 CLASSES OF 3 -CITEs BASED ON THEIR In silico and in vitro SHAPE analyses with the full-length PLPV SECONDARY STRUCTURES genome together with structural conservation analysis supported that the TED element (Figure 2A) was considerably larger (49 nt) To date, seven different classes of 30-CITEs have been identified. than previously proposed (Simon and Miller, 2013; Blanco-Pérez Their structures are very variable, therefore 30-CITE presence is et al., 2016). An enlarged structure of the STNV TED was also difficult to predict and identification has to be based on functional suggested on the basis of in silico predictions with the complete analysis. In most cases activity has been assayed by flanking STNV genome (Blanco-Pérez et al., 2016). Apart from STNV a reporter gene (for example luciferase) with viral 50- and/or and PLPV, TED-like 30-CITEs can be anticipated (although not 30-end sequences and analyzing the translation efficiency of yet experimentally verified) in the four members of the genus these constructs preferably in vivo in protoplasts, since in some Pelarspovirus, Pelargonium chlorotic ring pattern virus (PCRPV),

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FIGURE 1 | RNA secondary structures of BTEs, which are 30-CITEs that interact with eIF4F through its eIF4G subunit. Assayed RNA secondary structures of representative BTEs of BYDV, TNV-D, and RCNMV, based on data derived from Wang et al.(2010). The conserved BTE-specific 17 nt are indicted with arrows (start/end). Bases protected by eIF4G, apart from the 17 nt stretch, are encircled (Kraft et al., 2013). Sequences proved or proposed to be involved in 50–30 interaction through sequence complementarity are shown for BYDV and TNV-D BTEs.

Elderberry latent virus (ELV), Rosa rugosa latent virus (RrLDV) of a pseudoknot between the G-domain and the C-domain, and Pelargonium ringspot virus (PelRSV) (Blanco-Pérez et al., but complementary mutations that were meant to reestablish 2016) and additionally in Calibrachoa mottle virus (CbMV), a disrupted base-pairing between these regions were unable to member of the genus Carmovirus (Simon and Miller, 2013). restore the PEMV PTE cap-independent translation activity lost While in vitro translation experiments with the PLPV-TED in through the mutations in only one domain, suggesting that WGE were inconclusive (levels of newly synthesized p27 were maintenance of the sequence was also important (Wang et al., similar in wild-type and truncated viral genomic transcripts, thus 2009). the 30-CITE seemed not to be active) its translation enhancer activity could be confirmed for genomic (g) and subgenomic ISS (sg) RNAs in in vivo translation experiments, showing that it The shortest 30-CITEs known have the shape of an “I” is dependent on the presence of the genomic and subgenomic (I-shaped structure, ISS) and have been shown to be active 50-ends in cis (Blanco-Pérez et al., 2016). in the 30-UTRs of viruses belonging to different genera of the Tombusviridae family: the tombusvirus Maize necrotic spot virus PTE (MNeSV) and the carmovirus Melon necrotic spot virus (MNSV) Batten et al.(2006) described a 3 0-CITE in the 30-UTR of (Truniger et al., 2008; Nicholson et al., 2010; Miras et al., Panicum mosaic virus (PMV) (Panicovirus, Tombusviridae) that 2017b)(Figure 2C). The ISS consists of a stem-loop structure was later shown to be similar in structure to the one identified of about 60 nt with a 5–8 nt final loop and two internal bulges in the umbravirus Pea enation mosaic virus RNA 2 (PEMV2) protruding from the stem. The first structure of the MNeSV (Tombusviridae)(Wang et al., 2009)(Figure 2B). These 30-CITEs determined by SHAPE analysis (Nicholson et al., 2010) was later were named PTE (PMV or PEMV-like translational enhancer). further refined (Nicholson et al., 2013). Mutational analysis of PTEs are also found in several carmoviruses, as experimentally the MNeSV showed that only a few mutations were tolerated, identified in Saguaro cactus virus (SCV) (Chattopadhyay et al., more specifically the ones conserving base-pairing in the stem 2011) and proposed for Pelargonium flower break virus (PFBV), regions (Nicholson et al., 2010). These authors proposed ISSs Carnation mottle virus (CarMV), Honeysuckle ringspot virus for the two 30-CITEs that were previously identified in different (HnRSV), Pea stem necrosis virus (PSNV), Hibiscus chlorotic MNSV isolates, MNSV-Mα5 and MNSV-264 (Truniger et al., ringspot virus (HCRSV) and Galinsoga mosaic virus (GaMV). 2008), and the proposed 30-CITEs of three viruses belonging The other two known panicoviruses, Cocksfoot mild mosaic to the same family, the aureusviruses Maize white line mosaic virus (CMMV) and Thin paspalum asymptomatic virus (TPAV), virus (MWLMV) and Johnsongrass chlorotic stripe mosaic virus as well as one of the known aureusviruses, Pothos latent (JCSMV) and the tombusvirus Cucumber Bulgarian virus (CBV). virus (PoLV), also seem to contain a PTE (Simon and Miller, Although there is considerable sequence divergence between the 2013). PTEs consist of two short helical branches connected 30-UTRs of all known MNSV isolates and MNSV-264, the 30- by a C-rich or pyrimidine-rich, 3–6 nt bulge (C-domain) and CITE of MNSV-264 maintains the I-shape (Truniger et al., 2008). downstream a guanylate-rich bulge in the stem (G -domain) The structure of the MNSV-Mα5 ISS (Ma5TE), which is nearly (Wang et al., 2009). Wang et al.(2009) proposed the existence invariant in the MNSV 30-UTRs, has been solved by SHAPE

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FIGURE 2 | RNA secondary structures of TED, PTE, ISS, and YSS, which are 30-CITEs shown or proposed to interact with eIF4F through its eIF4E subunit. (A) Assayed (PLPV) and predicted (STNV) TED structures, based on data derived from Blanco-Pérez et al.(2016). (B) Assayed PTE structures of SCV, based on data derived from Chattopadhyay et al.(2011) and of PEMV and predicted PTE structure for PMV, based on data derived from Wang et al.(2009). The pseudoknot ( ψ) between C- and G-domain is indicated with a dashed line. (C) Assayed (MNSV and MNeSV) and predicted (MWLMV and MNSV-264) ISS (Nicholson et al., 2010; Miras et al., 2017b). (D) Predicted YSS of TBSV, as proposed by Fabian and White(2006) and CIRV, as proposed by Nicholson et al.(2013). (E) Assayed structures of eIF4E sensitivity elements (4E-SE) of Histone H4, based on data derived from Martin et al.(2011) and Cyclin D mRNAs, based on data derived from Culjkovic et al. (2006). Dashes point to conserved residues shown or predicted to be involved in eIF4E binding. The sequences proposed or shown to be involved in 50–30- interaction through sequence complementarity are shown. In the case of MNeSV the 50–30-interaction was proved with the 50-UTR of CIRV.

analysis (Miras et al., 2017b); from the ISS structure of MNSV- conserved Y-shaped structure (YSS) (Simon and Miller, 2013), Mα5 proposed by Nicholson et al.(2010) only the final SL as shown for the 30-CITE of Tomato bushy stunt virus (TBSV) (6 nt stem/7 nt loop) could be confirmed (Miras et al., 2017b). (Fabian and White, 2006)(Figure 2D). Another experimentally The Ma5TE, with its 45 nt, is the shortest ISS shown to be demonstrated YSS 30-CITE is the one from Carnation Italian functional. ringspot virus (CIRV, Tombusvirus, Tombusviridae)(Nicholson and White, 2008). The tombusviruses Cymbidium ringspot virus YSS (CymRSV), Cucumber necrosis virus (CNV), Artichoke mottled Nearly all viruses belonging to the genus Tombusvirus (family crinkle virus (AMCV), Pelargonium necrotic spot virus (PNSV), Tombusviridae) can be predicted to have 30-CITEs with a Grapevine Algerian latent virus (GALV), and Pelargonium leaf

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curl virus (PLCV) have been proposed to contain a YSS (Simon CXTE and Miller, 2013). The three helices forming these YSSs are all The newest 30-CITE, CXTE (CABYV-Xinjiang-like translation substantially longer than the three helices found in the PTE element), is also small (55 nt) and consists of two helices (Fabian and White, 2006). Mutations in the YSS of TBSV which protruding from a central hub (Miras et al., 2014). Since it altered the structure, thereby affecting stem or bulge formations, is different from the ones identified until now it belongs to 0 affected translation mediated by this 3 -CITE, while those that a new class of 30-CITEs. It has been identified in the new maintained the structure of the three stems had no substantial MNSV-N isolate as a second 30-CITE apart from its ISS Ma5TE effect on translation (Fabian and White, 2006), supporting the (Figure 3C). This translational enhancer was named CXTE, as it importance of the YSS for its activity. had been acquired by MNSV from an Asiatic isolate of Cucurbit aphid-borne yellows virus (CABYV Xinjiang, genus Polerovirus, TSS family Luteoviridae)(Miras et al., 2014). Thus, MNSV-N has two 0 This 3 -proximal translation enhancer, first discovered in Turnip 30-CITEs, the typical MNSV ISS and the CXTE. crinkle virus (TCV, Carmovirus, Tombusviridae)(McCormack It can be postulated that more types of 30-CITEs may exist, as et al., 2008) has a three-dimensional T-shaped structure (TSS), as the predicted secondary structures of the 30- RNA ends of several predicted by molecular modeling and confirmed by small-angle viruses of the family Tombusviridae do not resemble any of the X-ray scattering (SAXS)/NMR (Zuo et al., 2010). This is the first known 30-CITE classes (Simon and Miller, 2013). Thus, although 0 and only resolved 3D structure of a 3 -CITE. The TSS contains the final goal of 30-CITEs in cap-independent translation seems to three hairpins and two pseudoknots that fold into a structure be common, their sequences and structures are highly variable. similar to tRNAs (Zuo et al., 2010)(Figure 3A). A similar structure was proposed for the related carmovirus Cardamine chlorotic fleck virus (CCFV). Additionally, apart from its PTE HOST EUKARYOTIC TRANSLATION two functional TSSs have been identified in the PEMV2-30-UTR, one upstream of the PTE, the kl-TSS, and the other near the INITIATION FACTORS INVOLVED IN 0 30-end of the genomic RNA, the 30-TSS, with both predicted to DIFFERENT VIRAL 3 -CITE ACTIVITIES fold into structures similar to tRNAs (Gao et al., 2012, 2013) (Figure 3B). Although both are essential for virus accumulation BTE in vivo, mutations that disrupted the TSS near to the 30-end had The BTE has been shown to bind eIF4F through the eIF4G no effect on translation (Gao et al., 2013). More recently, the subunit of the heterodimer (Treder et al., 2008). In WGE depleted same authors have shown that the 30-TSS is also functional in of cap-interacting factors addition of the isoform eIFiso4F also translation in vitro when deleting the kl-TSS and the PTE (Gao allowed BYDV BTE-mediated translation to recover, but to a et al., 2014). lesser extent than addition of eIF4F. Interestingly, lower levels Unlike the tRNA-like structures present in tobamo-, tymo-, of eIF4F/eIFiso4F were required for efficient BTE-mediated and rubiviruses, which are aminoacylated and contribute to translation than for cap-dependent translation (Treder et al., translation together with the 50-cap, TCV and CCFV genomic 2008). Binding of eIF4G to the BTE occurs with high affinity RNAs are uncapped and their TSS are present in an internal (Kd = 177 nM) and is sufficient to facilitate translation alone. location, thus they cannot be aminoacylated (Dreher and Miller, On the other hand, eIF4E alone does not promote BTE-mediated 2006; Dreher, 2009). translation, but the presence of both subunits of eIF4F results The RNA-dependent RNA polymerase (RdRp) has been in a 25% increase in translation efficiency, while the affinity of 0 shown to bind the 3 -end of the TCV genome leading to a BTE binding to eIF4F is four-fivefold higher (Kd = 37 nM) conformational change of the TSS region (Yuan et al., 2009). The (Treder et al., 2008). This could be due to a conformational resulting conformation is suggested to be thermodynamically change in the eIF4G subunit induced by eIF4E (Zhao et al., favored, as the original TSS conformation is formed again 2017). Through footprinting experiments with the BYDV, TNV- by experimentally degrading the RdRp. This RdRp-mediated D and RCNMV BTEs it was shown that eIF4G protects the SL conformational switch is expected to prevent TSS function, formed by the 17-nt conserved sequence and several additional allowing replication to initiate at the 30-end (Yuan et al., 2010). residues as marked in Figure 1 (Kraft et al., 2013). Analysis of the Thus, it has been proposed that in TCV an integrative network minimal eIF4G fragment required for BYDV BTE binding and of interactions is required for virus accumulation, with different activity revealed that it had to include the eIF4A and eIF3 sites SLs involved in regulation of TCV translation and replication and an adjacent RNA-binding domain, but the eIF4E and PABP (Yuan et al., 2012). Very recently a new structural model for binding sites were not necessary (Kraft et al., 2013; Zhao et al., an extended TSS has been proposed that includes the A-rich 2017). Sharma et al.(2015) proposed that after eIF4F binds to region upstream adjacent to the TSS (in red in Figure 3A). the BTE also eIF4A, eIF4B, and ATP bind, forming the helicase It was shown that this region is implicated in RdRp binding, complex and recruiting the 40S ribosomal subunit. Although the inducing the conformational change of the TSS possibly by 40S ribosomal subunit can bind the BTE in the absence of the disruption of the H4a/ψ3 pseudoknot that leads to loss of the helicase complex, its presence improves binding affinity. eIF4A, H4b/H5 interaction (Le et al., 2017). This conformational switch eIF4B, and ATP are also able to enhance binding of eIF4G to is expected to interrupt translation through disruption of the TSS BTE of BYDV in the absence of the ribosome (Zhao et al., 2017). while promoting replication. The authors proposed that the binding of eIF4A/eIF4G/ATP

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FIGURE 3 | RNA secondary structures of T-shaped 30-CITEs and CXTE. (A) Assayed two- (McCormack et al., 2008) and three-dimensional structure Zuo et al. (2010) of the TCV TSS, based on data derived from McCormack and Zuo and collegues, respectively. Pseudoknots (ψ2and ψ3) are indicated with a dashed line. Hairpins are H4a/b and H5. Additional nucleotides present in the extended TSS model appear in red (Le et al., 2017). (B) Assayed RNA secondary structures of the two PEMV2 TSS, kl-TSS (Gao et al., 2012, 2013) and TSS (Gao et al., 2014), based on data derived from Gao and colleagues. 50-end sequence interacting by sequence complementarity with the kl-TSS and with kl-H is shown. (C) Assayed RNA secondary structure of CXTE, the newest 30-CITE, based on our data derived from Miras et al.(2014).

alters the BTE structure resulting in enhancement of the binding absence of eIF4G, with the binding affinity to eIF4F being very affinity between BTE and eIF4G followed by recruitment of similar (Wang et al., 2009). Mutational analysis of eIF4E allowed the 40S ribosome subunit directly to the BTE. However, in concluding that eIF4E binding to PTE of PEMV2 correlated with a previous model it was proposed that base-pairing between its cap-independent translation activity (Wang et al., 2009). In a sequence present in the conserved 17 nt of the BTE that spite of the strong binding of TED to eIF4E, this subunit alone was complementary to the 18S rRNA could result in direct was not able to enhance TED-mediated translation (Wang et al., ribosome binding to the BTE (Wang et al., 1997). The presence 2009). Thus, this 30-CITE also requires the eIF4F complex for of these complementary sequences partially in internally base- activity. Footprinting experiments with eIF4E showed protection paired stretches could explain the requirement of helicase activity in the region around the pseudoknot of the PEMV2 PTE (formed for increased ribosome binding to the BTE and thus for BTE- by G- and C-domains) (Wang et al., 2011). In the lateral bulge mediated translation (Sharma et al., 2015). containing the G-domain, one highly reactive G-residue (G17) has been proposed to protrude into the cap-binding pocket, being TED clamped by eIF4E (Wang et al., 2011)(Figure 2B). The 30-CITE of STNV, TED, has been shown to bind wheat As shown for STNV TED, mutation of both highly conserved eIF4F and eIFiso4F, but with preference for the first complex, tryptophans located in the cap-binding pocket of eIF4E was with a K of 30 and 50 nM, respectively (Gazo et al., 2004). shown to strongly reduce its binding to PEMV2 PTE (Wang et al., d 0 This interaction is required for TED activity, as in WGE depleted 2009), suggesting that also for this 3 -CITE an intact cap-binding in cap-associated eIFs, TED-mediated translation was strongly pocket is required. The much higher eIF4E binding affinity of reduced, but could be restored by the addition of eIF4F or PTEs may be achieved by binding the pseudoknot, which is 0 its isoform. The binding to the individual eIF4F subunits was absent in the ISS 3 -CITEs. studied in competition assays. While the eIF4G binding to TED was not affected by the addition of unlabeled TED in trans, ISS eIF4E binding was specifically inhibited (Gazo et al., 2004). Thus, The ISS of MNeSV has been shown to interact with wheat eIF4F although binding affinity of TED to eIF4E or eIFiso4E alone was in WGE (Nicholson et al., 2010). In this case, eIF4F binding found to be more than 10 times lower than to the respective occurred through its eIF4E subunit, but additions of purified eIF4F or eIFiso4F complexes, complex binding likely occurred recombinant eIF4E or eIF4G alone were not able to restore through interaction with the eIF4E subunit. Mutation of one translation in WGE depleted in cap-associated eIFs. The Kd of of the two highly conserved tryptophans of the cap-binding binding of eIF4F to the MNeSV ISS was estimated to be around pocket of eIF4E strongly reduced its binding affinity to STNV 190 nM. Interestingly, two G residues, shown to be highly flexible 0 TED (Wang et al., 2009), suggesting that this 3 -CITE requires in the structure, were found to be important for eIF4F binding, an intact cap-binding pocket as shown for PTE and ISS (see as deletion (G13) or mutation (G47C) strongly reduced its eIF4F below). binding capacity (Nicholson et al., 2010). These G residues can also be identified in bulges in the structures of the other ISSs PTE (assayed or predicted) (Figure 2C). In contrast to the previous 30-CITEs, the PTE of PEMV2 has been In the case of the Ma5TE ISS, genetic and biochemical shown to bind eIF4E with high affinity (Kd = 58 nM) even in the evidence for an interaction between Ma5TE and eIF4E exists

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(Nieto et al., 2006; Miras et al., 2017b). One amino acid change addition of eIF4F or eIFiso4F (Nicholson et al., 2013). In an RNA- in melon eIF4E causes loss of susceptibility to MNSV due to the affinity chromatography experiment, the CIRV YSS complexed strong reduction in the cap-independent translation efficiency with all four eIF4F components and the same result was obtained mediated by its Ma5TE (Nieto et al., 2006; Truniger et al., for the TBSV YSS (Nicholson et al., 2013), suggesting that this 2008). In agreement with the results obtained for MNeSV ISS, mechanism is general for these types of 30-CITEs. in vitro binding assays showed that Ma5TE also binds eIF4F: eIF4Fp20, formed by purified recombinant melon eIF4E with a CXTE 20 kDa truncated eIF4G fragment, was shown to be competent The eIFs required for activity of the newly identified third 0 0 for Ma5TE binding, with the Kd estimated to be 2 µM(Miras MNSV-3 -CITE (CXTE), with high homology to the 5 -end of et al., 2017b). The eIF4E residues that are important for Ma5TE the CABYV 30-UTR, are still unknown. The CXTE is functional translation activity were studied by mutational analysis: apart in melon plants silenced for eIF4E, thus it may be eIF4E- from the residue responsible for the loss of susceptibility, cap- independent (Miras et al., 2014). binding pocket residue tryptophan 82 and residues of eIF4E involved in its interaction with eIF4G through its canonical and TSS non-canonical binding domains were also shown to be required In contrast to the other 30-CITEs described above, no eIFs that for in vivo Ma5TE activity (Miras et al., 2017b,c). Thus, although bind to the TCV TSS have been described to date, although binding of eIF4F to Ma5TE seems to occur through eIF4E, the translation of reporter constructs flanked by TCV 50- and eIF4F complex is also required for efficient cap-independent 30-UTRs has been shown to be reduced in the absence of eIF4G in translation driven by Ma5TE. Again, as previously shown for an Arabidopsis eIF4G-deficient mutant (Yoshii et al., 1998, 2004). STNV TED and PEMV2 PTE (Wang et al., 2009), an intact cap- This structure has been shown to directly recruit and bind to binding pocket seems to be important for MNSV ISS activity the P-site of the 60S subunit (Stupina et al., 2008) and the 80S (Miras et al., 2017b). ribosome (Zuo et al., 2010). In the PEMV2 30-UTR the two TSSs Mapping of the eIF4F binding sites in Ma5TE by footprinting identified have been both shown to bind to the 60S subunit and analysis revealed that, in the presence of eIF4Fp20, the highly the 80S ribosome, but one of them, the kl-TSS, additionally binds accessible residue A32 was protected, whereas G17 and C28 to the 40S ribosomal subunit (Gao et al., 2013, 2014). In PEMV2, (Figure 2C), which were paired and thus not accessible without all three 30-CITEs, the PTE and the two TSSs, are required for eIF4F, became accessible. Mutations in these residues affected efficient translation (Gao et al., 2014). the in vivo cap-independent translation activity of Ma5TE, while no binding of eIF4Fp20 to Ma5TE A4109C could be observed in vitro (Miras et al., 2017b). These three residues (adenosine MECHANISMS FOR BRINGING THE eIFs and G/C pair) can be identified in similar positions in the other BOUND TO THE 30-CITE TO THE 50-END ISSs (Figure 2C). Interestingly, mutation of the corresponding adenosine residue in MNeSV 30-CITE (reducing its flexibility by As translation occurs in the 50–30 direction of the mRNA, the pairing it) also strongly affected the activity of this ISS (Nicholson eIFs bound to the viral 30-CITEs have to be brought to the et al., 2010). This suggests that this adenosine could be involved 50-end for translation initiation complex formation. In most in eIF4F binding in all ISS 30-CITEs. Similarly, the two unpaired viral RNAs with 30-CITE mediated cap-independent translation, guanosines shown to be involved in MNeSV ISS binding to eIF4F this has been proposed or shown to be achieved by a kissing- are conserved in all other ISSs, suggesting that these residues loop interaction based on sequence complementarity between the could be also involved in eIF4F binding through eIF4E (arrows 30-CITE and the 50-end (most often present in the 50-UTR, but in in Figure 2C). some cases in the beginning of the first ORF). In these cases, the Interestingly, the ISS of MNSV-264 also conserves these 30-CITE activity is 50-end dependent and mutations disrupting residues (Figure 2C), although it has been shown to be able to base-pairing affect translation, while compensatory mutations function in the absence of eIF4E. MNSV-264 is able to infect restore activity. The interacting sequences are usually unpaired, melon plants with reduced eIF4E expression due to its specific thus located in conserved apical loops of hairpins (Simon and silencing, in contrast to MNSV-Mα5 that is unable to infect Miller, 2013). (Rodríguez-Hernández et al., 2012). Thus, it can be hypothesized that MNSV-264 ISS may be active in cap-independent translation BTE with eIF4E but additionally is also able to function in its absence One example is the BTE-mediated translation of BYDV: the using eIFiso4E or without the 4E factor. Multiplication of several sequence in the loop of SL-III from the BTE of BYDV is potyviruses has been shown to be functional with both eIF4E and complementary to the loop of SL-D in the 50-UTR. This eIFiso4E (Ruffel et al., 2006; Hwang et al., 2009; Jenner et al., complementarity is required for efficient translation mediated 2010). by BYDV BTE (Guo et al., 2001; Wang et al., 2010)(Figure 1). This BTE was functional even if complementarity was achieved YSS by insertion of foreign sequences outside the BTE (from viral or Another 30-CITE that is functional with either eIF4F or eIFiso4F non-viral origin) (Rakotondrafara et al., 2006). The presence of is the YSS of CIRV. The strongly reduced YSS-mediated eIF4A, eIF4B and ATP was shown to enhance binding of eIF4G to translation activity in factor-depleted WGE could be restored by BTE, possibly due to BTE structure alteration (Zhao et al., 2017).

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Two alternative models for cap-independent translation by BTE occur in the absence of circularization (Meulewaeter et al., have been proposed (Miras et al., 2017a): either after binding 1998b). of eIF4F, eIF4A, and eIF4B to BTE the 40S ribosomal subunit is directly recruited to it through sequence complementarity PTE between the BTE and the 18S rRNA and delivered to the 50-end All viral genomes where PTEs have been identified, excluding through the 50–30 complementary interaction; or after binding of the PEMV2, contain sequence stretches in their 50-ends that are eIF4A and eIF4B to BTE followed by ATP hydrolysis, its affinity complementary to a loop in its structure (Simon and Miller, for eIF4F is increased but first the 50–30 interaction has to bring 2013)(Figure 2B). In the case of the carmovirus SCV the the formed complex close to the 50-end before it recruits the 43S functionally important 50 complementary sequence is located in preinitiation complex. In both cases ribosome scanning occurs the loop of a hairpin in the coding region of p26 (Chattopadhyay from the 50-end. The complementarity between BTE and 50-UTR et al., 2011), possibly because of its short 50-UTR, while in the is maintained among BTEs, suggesting that this interaction may panicovirus PMV the complementarity is with the first hairpin be required for translation enhancement activity by BTEs. of the 50-UTR (Batten et al., 2006). For this PTE it was shown The only exception is the BTE-like 30-CITE (named 30TE- that maintenance of its distance to the 50-UTR is important for DR1) of Red clover necrotic mosaic virus (RCNMV; genus its efficient activity (Chattopadhyay et al., 2014). For the other Dianthovirus, family Tombusviridae)(Figure 1), where no virus genomes with proposed PTEs, complementary sequences effect on translation was observed by mutating complementary between the PTE (Nicholson et al., 2010) and the 50-end have sequences between BTE and 50-UTR (Sarawaneeyaruk et al., been identified (Simon and Miller, 2013), suggesting that the 2009). In this case, in addition to its BTE, an A-rich sequence mechanism of circularization is common. As will be described (ARS) (60 nt upstream to it) with strong affinity for PABP was below, the PEMV2 PTE depends on another 50–30 interaction, identified in the RCNMV 30-UTR and both ARS and 30-CITE, the kl-TSS that helps to bring the eIF4F complex to the were shown to coordinately recruit eIF4F or eIFiso4F and the 50-end. 40S ribosomal subunit to the viral RNA (Iwakawa et al., 2012). The ARS is conserved in all dianthoviruses. Interestingly, while ISS cap-independent translation of the RNA1 of this bipartite virus As with the other 30-CITEs described above, 50–30-UTR is controlled by a BTE that depends on eIF4F for activity, no interactions based on sequence complementarity have been 30-CITE could be identified in its RNA2. Translation of this RNA2 identified between the apical loop of the only SL forming the has been shown to depend on eIFiso4F, suggesting that the eIFs ISS of MNeSV and the loop of the first SL of the 50-UTR required for cap-independent translation of the RNAs of this (Scheets and Redinbaugh, 2006) and shown to be required bipartite virus differ (Tajima et al., 2017). for virus genome translation and multiplication (Nicholson et al., 2010)(Figure 2C). This interaction was shown not TED to interfere with eIF4F binding, while ribosome scanning Again, the TED-mediated translation of STNV is dependent on was dependent on the ISS and occurred from the 50-end the presence of the 50-end in cis (Meulewaeter et al., 1998a). (Nicholson et al., 2010). Similarly, ISS-mediated translation The eIF4F or its isoform bound to TED was predicted to reach of MNSV was shown to be 50-UTR dependent (Truniger the ATG through a RNA interaction with the apical loop of et al., 2008) and complementarity between bases in the the 50 end based on sequence complementarity (Figure 2A). loops of the ISS and SL1 of the 50-UTR exists (Figure 2C). However, this could not be confirmed by mutational analysis, For the two aureusviruses MWLMV and JCSMV and the as disruption of this potential base-pairing reduced translation tombusvirus CBV as well, complementary sequence stretches only slightly, while restoring complementarity by mutations at between the 50-end and the loop of their predicted ISSs could the opposite end did not restore translation (Meulewaeter et al., be identified. Thus, in the ISS-mediated translation activity, the 1998a). Thus, the authors suggested that base-complementarity eIF4F bound to the 30-CITE reaches the 50-end through 50–30 was biologically relevant but not crucial for the constructs and interaction. assays used. But for the recently described TED of PLPV, a long distance interaction between TED and 50-end based on YSS sequence complementarity has been shown to occur (Figure 2A). The YSS-mediated translation efficiency of TBSV has also been The complementary sequence in the 50- end (involving 6–8 shown to depend on a 50–30-UTR interaction between its YSS bases) is located in the viral p27 ORF (Blanco-Pérez et al., and 50-UTR. Mutational analysis that disrupts and restores 2016), possibly because its 50-UTR is very short. This 50–30 complementarity confirmed this interaction, which is required interaction is required for efficient translation of the viral genome for protein synthesis and virus multiplication (Fabian and White, and thus for infectivity. Complementary sequence stretches are 2004, 2006)(Figure 2D). Moreover, the YSS of CIRV was also conserved in other viral genomes proposed to contain TED- shown to require a 50–30 interaction between its YSS and 50- like 30-CITEs, suggesting that 50–30 RNA-RNA interactions are UTR, based on sequence complementarity, in order to bring necessary in all TED-mediated translation mechanisms to bring the eIF4F (or isoform) complex bound to the YSS to the the eIF4F bound to the 30-UTR to the opposite end (Blanco- 50-end (Nicholson and White, 2008; Nicholson et al., 2013), Pérez et al., 2016). In line with this, moving of the TED suggesting that 50–30 interactions are common in YSS-mediated of STNV to the 50-end allowed TED-mediated translation to translations (Figure 2D). Additionally, for both TBSV and CIRV

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FIGURE 4 | Conservation of unpaired 30-CITE residues suggested to be involved in eIF4E binding. (A) Residues of the first bulge present downstream of the final stem-loop of ISS, PTE, TED, and YSS CITEs and of the histone H4 and cyclin D1 4E-sensitivity elements. (B) Residues of the second bulge present upstream of the final stem-loop of these 30-CITEs. Conserved adenosine [in (A), similar to A32 of MNSV ISS] and guanosine residues [similar in (A) to G47 of MNeSV ISS and in (B) to G13 of MNeSV ISS and the highly reactive G17 residue from the G-domain of PTE] are framed. (C) 3D structural alignment [Rclick server (Nguyen et al., 2017)] of the left arm of the YSS of CIRV and TBSV (colored in yellow and green, respectively) with the ISS of MNeSV (red colored), based on the predicted tertiary structures [RNAComposer server (Popenda et al., 2012)]. YSS of CIRV and TBSV and ISS of MNeSV are based on data derived from Nicholson et al.(2010, 2013), respectively. Below, alignment of sequence stretch of both structures aligning in the 3D prediction (Rclick server). Identity of residues is emphasized in bold. Adenosine and guanosine residues predicted to be important for eIF4E binding are shown in blue in the 3D structural alignment and in red in the sequence alignment.

YSS ribosome scanning has been shown to occur from the 50-end 30-UTR of MNSV-N two 30-CITEs, CXTE, and ISS Ma5TE, are (Fabian and White, 2006; Nicholson et al., 2013). present. In reporter constructs having only the CXTE sequence (not the entire 30-UTR) flanking the luciferase gene at the CXTE 30-end, its activity in translation was shown to depend also on In MNSV-N, the CXTE activity has also been shown to depend on the presence of the 50-UTR in cis (Miras et al., 2014). This the presence of the 50-UTR of MNSV in cis (Miras et al., 2014). result excludes the possibility that the CXTE takes advantage But in this case, although complementary sequence stretches of the possible 50–30 interaction occurring between Ma5TE and between the CXTE and the 50-UTR exist, mutational analysis did 50-UTR for its activity. Thus, the mechanism of the CXTE in not support its importance in CXTE-mediated translation. In the cap-independent translation is currently unknown.

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TSS Martin et al., 2011) also have A and G residues in the downstream In the case of TCV, no base-pairing between its TSS 30-CITE loop of its double SL structure (Figure 2E), similar to the ISS. and 50-UTR could be identified. Here, the 60S ribosomal subunit Toe-print analysis of the H4 4E-SE in the presence of eIF4E bound to its TSS is expected to reach opposite end through showed RT-stops at the A and the following C, suggesting that a protein bridge by binding to the 40S subunit bound to its eIF4E binding involves this loop (Martin et al., 2011). 50-UTR (Stupina et al., 2011). Thus, the 80S ribosome would Analysis of the residues present downstream the apical loop 0 simultaneously bind to both TCV UTRs and this would explain in bulge 1 of the assayed or predicted structures of TED 3 - why ribosomal binding to the 50-UTR was altered in the presence CITEs allows for the identification, as in all the ISSs, of A of the 30-UTR. and G residues (adjacent or separated by a maximum of 2 nt) As in the case of PEMV2 the PTE is not able to contact the conserved in all of them [Figures 2A,C (arrows), 4A]. Thus, 50-end by direct sequence complementarity, the eIF4F complex these two residues could be involved in eIF4E binding in these 0 bound to this 30-CITE is brought to the 50-end by a kissing- 3 -CITEs. In the case of the PTE structures, these two residues loop interaction occurring through the adjacent TSS-like 30-CITE are less conserved (Figures 2B, 4A). Additionally, one or two (kl-TSS) which binds the 60S ribosomal subunit (Gao et al., guanosine residues can also be identified in ISS and TED CITEs 2012, 2013)(Figure 3B). The hairpin at the 50-end involved in upstream of the apical loop in bulge 2 in a similar position as this interaction is located in the coding region of the first ORF. the G-domain of PTEs, suggesting their possible role in eIF4E This interaction was shown to be critical for virus translation binding (Figures 2A–C, 4B). Also, the paired G/C present in and multiplication, and its loss affected translation to a greater the final stem that became accessible in the presence of eIF4E in extent than deletion of the adjacent PTE. This suggested that the MNSV ISS can be identified in all TED and PTE CITEs as a G/C 50–30 interaction supported additional translation functions (or C/G) pair (Figures 2A–C). besides bringing the PTE bound eIF4E to the 50-end (Gao et al., The five residues proposed above to play a role in eIF4F 2012). The kl-TSS is able to simultaneously bind to the ribosome binding through eIF4E can also be identified in the YSSs, located and the 50-end (Gao et al., 2013). Additionally, the second TSS- in the left arm of the Y (Figures 2D, 4A,B). If 3D structural like 30-CITE in the 30-UTR of PEMV2 has been shown to require predictions of the left helix of the YSS of CIRV or TBSV a small hairpin upstream of the TSS named kl-H with an apical are superimposed on the prediction of the ISS from MNeSV loop that is able to interact by sequence complementarity with (Figure 4C), it can be observed that the G downstream of the the same 50-proximal hairpin that is utilized by kl-TSS (Gao et al., apical loop in bulge 2 and A and G in bulge 1 of MNeSV seem 2014). Recently, it has been shown that while efficient translation to be localized at similar positions in both YSSs. This prediction of the gRNA requires only two of the three PEMV2 30-CITEs, may suggest that binding of eIF4F to YSS also occurs through the the kl-TSS and the PTE (Du et al., 2017), efficient translation eIF4E subunit as shown for the ISS, PTE and TED. This, however, of the sgRNA additionally requires the second ribosome binding has to be further studied. TSS. Here the RNA–RNA interaction of the 50-end of the sgRNA with the kl-TSS is required (Gao and Simon, 2017). The requirement for different translation mechanisms could be a way 30-CITES ARE FUNCTIONALLY for regulating virus protein expression at the different stages INDEPENDENT ELEMENTS THAT CAN of the viral infectious cycle. In conclusion, all three PEMV2 BE TRANSFERRED AMONG VIRUSES BY 30-CITEs are required for efficient virus translation. RECOMBINATION WITH IMPORTANT ADAPTIVE CONSEQUENCES ARE RESIDUES INVOLVED IN eIF4E BINDING CONSERVED IN 30-CITEs? As previously observed by several authors and reviewed by Simon and Miller(2013), different 3 0-CITEs can be found in viruses Several residues of these 30-CITEs have been described to be belonging to the same family, the same genus, the same species protected in the presence of eIF4E and to be important for 30- and even the same isolate. For example, BTEs can be found in CITE activity: in the case of the PEMV2 PTE the G-domain viruses belonging to different families, such as in dianthoviruses residues, especially the first one (G17 in Figure 2B)(Wang from the Tombusviridae family and in luteoviruses from the et al., 2011); in the case of the MNeSV ISS two G residues Luteoviridae family. Also, in the genus Carmovirus four different were protected: the first (G13) downstream of the apical loop in 30-CITEs have been identified, TED, ISS, YSS, and TSS, and the second lateral bulge 2, resembling this PTE G-domain and perhaps more will be discovered, as the RNA structure prediction the second (G47), present upstream of the apical loop in the of the 30-end of several carmoviruses has not allowed for first bulge 1 (Figure 2C)(Nicholson et al., 2010); and finally an the identification of any of the known 30-CITEs. Additionally, unpaired adenosine adjacent to this second G residue (A32) that in isolates MNSV-N and PEMV2 two and three, respectively, was protected in MNSV ISS as well as the G/C pair (G16/C28) different 30-CITEs have been identified, belonging to different in the stem of the final stem-loop (Figure 2C)(Miras et al., classes (Nieto et al., 2011; Gao et al., 2014; Miras et al., 2014). 2017b). Interestingly, in the Cyclin D1 and Histone H4 mRNAs The diversity of 30-CITEs in closely related viruses suggested the eIF4E-sensitivity elements (4E-SE) shown to be involved in that these RNA elements may be interchanged between different eIF4E binding without the need of cap (Culjkovic et al., 2006; viruses. To test this hypothesis, Nicholson et al.(2013)

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FIGURE 5 | New 30-CITEs acquired by MNSV isolates through recombination. (A) Sequence alignment of the 50-end of the 30-UTR of MNSV isolates. GenBank accession numbers of MNSV sequences included in the alignment are Ma5-AY122286, HM-GU480022.1, Chiba-AB250684, Yamaguchi-AB250687, Nagasaki-AB250686, Al-DQ339157, Dutch-NC001504, ISR (Israel)-DQ922807, Kochi-AB250685, Kouchi-AB189943, Ma24-EU589616, Ma71-EU589619, Pa58-EU589620, Pa57-EU589621, 17A/01A-M29671, ABCA13-01-KR094068, USA-2016-KY124137, N- KF060715, GX-KY264755. The inserted sequence is marked in gray. Asterisks mark conserved nucleotides of all sequences or in gray of the insertions. (B) Proposed recombination event generated by the RDP4 software, a computer program for characterizing recombination events in sequence alignments using several different recombination analysis methods and tests for recombination hot-spots. The sequences included in this analysis are the 30-UTRs of MNSV [see part (A) of this figure] and CABYV isolates [GenBank accession numbers are CABYV-Xinjiang: EU636992; CABYV-Beijing: EU000535; CABYV esp (JF939814), CABYV-N (NC_003688), CABYV-K1 (LC082306), CABYV-FJ: GQ221223; CABYV-JAN: GQ221224; CABYV-R_TW82: JQ700306]. RDP4 colors similar sequences with similar colors. The statistical significance is very high, with a P-value < 0.001. (A,B) Of this Figure is based on our data derived from Miras et al.(2014). (C) Sequence alignment of the 30-CITEs of MNSV isolates. GenBank accession numbers as above, plus MNSV-264 (AY330700), USA12 (KY124136), USA14 (KY124135), based on our data derived from Truniger et al.(2008). In the middle, the upper line with asterisks marks conserved nucleotides between sequences above; the lower asterisks line marks conservation between upper and lower sequences and asterisks line at the bottom shows nucleotides conserved between MNSV-264 and USA-2012/4.

experimentally exchanged viral 30-CITEs. They replaced the YSS RNA recombination events appear to be responsible for the of the tombusvirus CIRV with the ISS from the tombusvirus acquisition of different 30-CITEs by MNSV field isolates. In MNeSV and the PTE from the aureusvirus Cucumber leaf one case, the 30-UTR of the isolate MNSV-264, different to spot virus (CLSV) and the BTE from Tobacco necrosis virus those from all other MNSVs, had probably been acquired by (TNV-D). While maintaining the sequence complementarity recombination from an unknown source (Nieto et al., 2011). between the 30-CITE and the 50-UTR, these chimeric viruses were In another case, an insertion through natural recombination able to translate and multiply efficiently, with the exception of of exactly a 30-CITE was found to have occurred in another the BTE-substitution, suggesting that 30-CITEs are functionally MNSV isolate, MNSV-N: here not the whole 30-UTR, but interchangeable elements. only 55 nt were acquired by recombination of MNSV with From an evolutionary perspective, the absence of correlation an Asiatic CABYV isolate (Figure 5). This stretch of 55 nt between virus taxonomy and the presence of different classes was shown to act itself as a 30-CITE in presence of the 50- of 30-CITEs (Simon and Miller, 2013) suggests independent UTR in cis (Miras et al., 2014). The transferred sequence 30-CITE evolutionary patterns and frequent exchange corresponds to the minimal 30-CITE version, as its activity through recombination. In line with this, independent was lost when shortened (Miras et al., 2014). Interestingly,

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three new MNSV isolates have been very recently reported been acquired by interfamilial recombination with CABYV, a that could possibly result from similar recombination processes: virus belonging to a different family (Luteoviridae); it is possible MNSV-GX, which contains an insertion in the 50-end of its that this sequence also acts as a 30-CITE in CABYV. Thus, some 30-UTR that is similar to the one from MNSV-N (Figures 5A,B) classes of 30-CITEs appear in viruses of different genera or even and MNSV-USA-2012 and 2014, that have low sequence identity families, although others are quite conserved within virus genera, with MNSV-264, although their 30-UTRs are highly similar like the YSSs that have been identified only in tombusviruses, (Figure 5C). All these MNSV variants suggest that acquisition or BTEs which have been identified in phylogenetically related of 30-CITEs through recombination might be frequent and/or viruses (Simon and Miller, 2013). This may reflect functional positively selected for. constraints specifically affecting 30-CITEs, or be a consequence In the case of MNSV-N and -264, the recombinant viruses of the biology of each viral species. In any case, this raises gained the ability to infect resistant melon (Truniger et al., 2008; questions on the spread of 30-CITEs and their origin: 30-CITEs Miras et al., 2014). Recessive resistance to MNSV in melon has as such have only been described in a few genera of plant been shown to be caused by a single amino acid change in viruses. But are they present in other plant virus genera and eIF4E, resulting in loss of susceptibility to this virus while the families, in viruses infecting other organisms, or are they even eIF4E resistance allele is functional for cap-dependent translation part of a conserved mechanism of translation of mRNAs of (Nieto et al., 2006). While the 30-CITE present in most MNSV an early origin and are spread among kingdoms? The presence isolates, Ma5TE, is eIF4E-dependent and not functional in the of 30-CITEs in different viral genomes leads to questioning presence of the eIF4E resistance allele, the one from MNSV-264 if cellular mRNAs could use similar strategies to initiate cap- and the CXTE are functional in eIF4E-silenced melons (Truniger independent translation, functional under stress conditions when et al., 2008; Rodríguez-Hernández et al., 2012; Miras et al., 2014). cap-dependent translation is inhibited. Weingarten-Gabbay et al. Additionally, in the case of MNSV-264, this isolate is able to infect (2016) used high-throughput approaches to identify thousands Nicotiana benthamiana, a non-host for MNSV, thanks to its new of sequences involved in cap-independent translation in both 30-CITE (Nieto et al., 2011). human and viral genomes. Data revealed that the human 50- or 30-UTRs of a set of mRNAs contained sequences involved in cap-independent translation and these were not CONCLUSION AND PROSPECTS mutually exclusive, but their mechanism remains unknown. The structural diversity of 30-CITEs prevents high-throughput Despite being considerably diverse in sequence and structure, 0 0 bioinformatics identification, but as 3 -CITE conformational most 3 -CITEs appear to facilitate translation through similar information progresses and new search algorithms are devised, mechanisms including recruitment of components of the cellular 0 0 the chances of identifying 3 -CITEs in mRNAs of varied species translational machinery to the 3 -CITE and delivery of these increase. through RNA–RNA interactions to the 50-end, where the ribosome enters onto the viral RNA. BTEs have been shown to interact with the eIF4G subunit of eIF4F, while TED, ISS, and AUTHOR CONTRIBUTIONS PTE 30-CITEs interact with eIF4E. But for these 30-CITEs for which factors involved have been identified, the order of binding VT wrote review. MM and MA edited and added specific and actions required for efficient cap-independent translation information to all sections. have still to be determined. Data obtained by high-resolution structures on how the eIFs bind the 30-CITE are also required to understand its translation enhancer activity. Still, for several 30- ACKNOWLEDGMENTS CITEs their mechanisms of action are still completely unknown. 30-CITES are modular, functional RNA elements that can be The research program on cap-independent translation of viral exchanged between viruses, and their acquisition may contribute mRNAs is supported in our laboratory by grants AGL2015-65838 to a gain of function resulting in a selective advantage, broadening and AGL2015-72804-EXP (MINECO, Spain) and 19252/PI/2014 the virus host range. Interestingly, in MNSV-N, the CXTE has (Fundación Seneca, Spain).

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Mechanism conducted in the absence of any commercial or financial relationships that could of plant eIF4E-mediated resistance against a Carmovirus (Tombusviridae): be construed as a potential conflict of interest. cap-independent translation of a viral RNA controlled in cis by an (a)virulence determinant. Plant J. 56, 716–727. doi: 10.1111/j.1365-313X.2008. Copyright © 2017 Truniger, Miras and Aranda. This is an open-access article 03630.x distributed under the terms of the Creative Commons Attribution License (CC BY). van Lipzig, R., Gultyaev, A. P., Pleij, C. W. A., van Montagu, M., Cornelissen, M., The use, distribution or reproduction in other forums is permitted, provided the and Meulewaeter, F. (2002). The 50 and 30 extremities of the satellite original author(s) or licensor are credited and that the original publication in this Tobacco necrosis virus translational enhancer domain contribute differentially journal is cited, in accordance with accepted academic practice. No use, distribution to stimulation of translation. RNA 8, 229–236. or reproduction is permitted which does not comply with these terms.

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