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Virology 344 (2006) 185 – 197 www.elsevier.com/locate/yviro

Translational control in positive strand RNA plant

Theo W. Dreher a,*,WR Allen Miller b,c

a Department of Microbiology and Center for Gene Research and Biotechnology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331, USA b Plant Pathology Department, 351 Bessey Hall, Iowa State University, Ames, IA 50011, USA c Biochemistry, Biophysics and Molecular Biology Department, 351 Bessey Hall, Iowa State University, Ames, IA 50011, USA

Received 6 September 2005; accepted 10 September 2005

Abstract

The great variety of genome organizations means that most plant positive strand viral differ from the standard 5V-cap/3V-poly(A) structure of eukaryotic mRNAs. The cap and poly(A) tail recruit initiation factors that support the formation of a closed loop mRNA conformation, the state in which translation initiation is most efficient. We review the diverse array of cis-acting sequences present in viral mRNAs that compensate for the absence of a cap, poly(A) tail, or both. We also discuss the cis-acting sequences that control translation strategies that both amplify the coding potential of a genome and regulate the accumulations of viral gene products. Such strategies include leaky scanning initiation of translation of overlapping open reading frames, stop codon readthrough, and ribosomal frameshifting. Finally, future directions for research on the translation of plant positive strand viruses are discussed. D 2005 Elsevier Inc. All rights reserved.

Contents

Introduction ...... 185 Closed loop formation for viral RNAs lacking a cap or poly(A) tail ...... 187 Viral mRNAs with capped 5V-ends and non-polyadenylated 3V-termini ...... 187 Viruses with genome-linked ...... 188 Viral genomes with unmodified 5V- and 3V-ends ...... 189 Strategic consequences of genome compression: leaky scanning, readthrough and frameshifting ...... 191 Overlapping open reading frames and leaky scanning ...... 191 Translational recoding: readthrough and frameshifting ...... 192 Future perspectives...... 193 Acknowledgments ...... 194 References ...... 194

Introduction control. In the realm of translation, conceptual changes include (i) the description of internal ribosome entry sites (IRES), (ii) Our understanding of post-transcriptional gene regulation the realization that the poly(A) tail participates directly in has changed vastly over the last sesquidecade and firmly translation initiation, and (iii) a better understanding and established the downstream steps of the gene expression prediction of unusual translation ‘‘recoding’’ events. Viral cascade as essential and varied contributors to gene expression RNAs have been at the center of many of the groundbreaking studies. * Corresponding author. Efficient translation of eukaryotic mRNAs is thought to E-mail addresses: [email protected] (T.W. Dreher), occur in a closed loop format (Fig. 1A), in which the 5V- and 3V- [email protected] (W.A. Miller). termini are brought into close proximity through the mediation

0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.09.031 186 T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 of interactions involving translation initiation factors (Hentze, 5V-cap and poly(A) tail (van Regenmortel et al., 2000). The 1997; Sachs et al., 1997). The key interactions involve poly(A) majority lacks one or both of these features. In understanding binding (PABP) bound to the 3V-poly(A) tail and the translation of these RNAs, research has focused on how initiation factor 4E (eIF4E) bound to the 5V cap, and the these non-canonical RNAs interact with the translation interaction of both proteins with eIF4G (the variants eIFiso4G initiation factors. The evidence indicates that the closed loop and eIFiso4E found in plants participate in similar interac- model of the mRNA is applicable in these cases, with a variety tions). These simultaneous interactions mutually increase of novel interactions substituting for the molecular bridging binding affinities (Le et al., 1997; Wei et al., 1998), stabilizing contacts that occur in normal cellular mRNAs (Fig. 1). the closed loop. The 40S small ribosomal subunit associates Another active area of research has been the deciphering of with the 5V-end of the mRNA via its interaction with eIF3, translational expression of viral genomes. Because of genome which simultaneously binds eIF4G, and then initiates scanning size constraints and the essentially monocistronic nature of towards the 3V-end (Pestova et al., 2001). Members of less than eukaryotic translation, positive strand RNA plant viruses 20% of plant positive strand RNA viral genera have genomic exhibit a range of devices that expand the expressible gene and subgenomic mRNAs structured like host mRNAs, with a content. Alteration of ribosomal behavior by ‘‘recoding’’ T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 187 permits readthrough and frameshifting, and leaky scanning and Dreher, 2004) RNAs supports the notion that these viral allows use of alternative translation start sites. These phenom- RNAs are translated in the closed loop format, although the ena are outlined in Fig. 2 and discussed in the second part of expected 5V–3V molecular bridges have not yet been identified this review. We lack space in this review for comprehensive (Figs. 1C and D). In the case of AMV RNA, the viral coat coverage of all translational control mechanisms in all positive protein (CP) has recently been shown to serve a bridging role strand RNA plant viruses. Instead, we provide examples that analogous to that of PABP in normal translation. AMV CP illustrate concepts and translational regulation strategies that binds with high affinity near the 3V-terminus to AUGC repeats we anticipate are widely applicable to these viruses. separated by hairpins (Bol, 2005; Guogas et al., 2004)andis also able to interact with eIF4G or eIFiso4G (Krab et al., 2005) Closed loop formation for viral RNAs lacking a cap or (Fig. 1B). This is analogous to the situation with rotavirus poly(A) tail mRNAs (Varani and Allain, 2002). AMV RNA has the distinctive property of requiring the presence of a few Viral mRNAs with capped 5V-ends and non-polyadenylated molecules of coat protein to successfully launch an infection 3V-termini (the phenomenon has been termed ‘‘coat protein activation’’). This long-standing puzzle has now been explained by the Viruses in more than a third of the genera of positive strand realization that the 3V-terminal region of each of the AMV RNA plant viruses have genomes with a 5V-cap but no poly(A) RNAs serves as a translational enhancer when bound by CP tail. In transfection experiments delivering reporter mRNAs (Neeleman et al., 2004). This CP-RNA interaction replaces the into plant protoplasts, the poly(A) tail has been shown to serve PABP-poly(A) interaction as indicated by the observation that as a potent translational enhancer that acts in synergy with the presence of an artificial poly(A) tail on AMV RNA obviates 5V-cap (Gallie, 1991). Its major effect on protein expression is the need for CP for translation (Neeleman et al., 2001). The to influence translational efficiency, with a smaller role in translation of ilarvirus RNAs is likely to be similarly controlled stabilizing the RNA (Gallie, 1991). Similar functions are by CP binding. provided by the 3V-UTRs of Tobacco mosaic (TMV) Translational enhancement has also emerged as a major (Gallie and Walbot, 1990), Alfalfa mosaic virus (AMV) (Krab role of the aminoacylatable tRNA-like structure (TLS) found et al., 2005), and Turnip yellow mosaic virus (TYMV) at the 3V-end of TYMV RNA. Full enhancing activity relies (Matsuda and Dreher, 2004) RNAs, and it appears reasonable on the remarkable tRNA mimicry of the TLS, particularly to expect that such a function is an important role of the 3V- its ability to be aminoacylated, and therefore requires a UTRs of all positive strand viral genomic RNAs. These cis- precise –CCA 3V-terminus (Matsuda and Dreher, 2004). This acting elements can be detected in reporter assays, in which explains the inability of an earlier study to detect translational they enhance protein expression when added to mRNAs with enhancement associated with the TYMV 3V-UTR (Gallie and appropriate negative control 3V-UTRs. Translation enhancing Kobayashi, 1994). The TYMV 3V-translational enhancer (TE) activity may be evident only under appropriate circumstances, is active when aminoacylatable with the natural valine or however, such as in the presence of coat protein (for AMV) or when discrete mutations have switched the specificity to upon aminoacylation (for TYMV) (Fig. 1). methionine (Matsuda and Dreher, 2004). The common The observation of synergy between the 5V-cap and the 3V- property of these aminoacylated variants is the ability to UTR for TMV (Gallie and Walbot, 1990) and TYMV (Matsuda tightly bind eEF1AIGTP (Dreher et al., 1999), implicating this

Fig. 1. The closed loop scheme for initiation of translation: thematic variations used by plant positive strand RNA viruses. Panel A shows in simplified terms (not all factors are shown; not to scale) the circularized format in which efficiently translated cellular mRNAs are believed to exist. The key responsible cis-acting features are the 5V m7GpppN cap (purple dot) and 3V-poly(A) tail, which synergistically enhance expression. Bridging interactions through eIF4E or eIF-iso4E (the cap-binding proteins), eIF4G or eIF-iso4G, and the poly(A) binding protein (PABP) bring the 5V- and 3V-termini into close proximity, and the interactions are mutually stabilizing. The first stage of translation initiation, the recruitment of the 40S small ribosome subunit to the 5V-end, depends on simultaneous interaction of eIF3 (a complex of multiple proteins) with eIF4G or eIF-iso4G and the 40S subunit. This step is depicted in step (1) and is followed by ribosome scanning (arrow) along the 5V-UTR (black line) in search of the AUG initiation codon. In response to base-pairing with the initiator tRNA (not shown) located in the ribosomal P site, the 60S large ribosome subunit joins (step 2) to initiate the elongation phase of translation. During peptide elongation (step 3), the codons in the ORF (thick red line) are read by tRNAs entering the ribosomal A site, until a termination codon, such as UAA, is reached (step 4), triggering termination of protein synthesis and subunit dissociation and release (step 5). Because of the closed loop format, ribosomes are near the 5V-end upon termination, facilitating new initiation. The boxed diagrams (B–G) illustrate variations of the initial 40S subunit recruitment step for viral mRNAs that lack a cap, poly(A) tail, or both, and which are discussed in the text. Red question marks indicate unknown or uncertain details. AMV, TYMV, and TMV are examples of viruses whose RNAs have a cap but no poly(A) tail. For AMV, a coat protein (CP) dimer binds to the 3V-terminal region and to eIF4G/iso4G. For TYMV RNA, aminoacylation (indicated by Val in the diagram) of the 3V-terminal tRNA-like structure (TLS) is needed for full 3V-translational enhancement, and it has been postulated that eEF1A binding is involved in closed loop formation. TMV RNA also has a 3V TLS capable of aminoacylation (His) and eEF1A binding, but 3V-translational enhancement relies on an upstream pseudoknot (UPSK). Intriguingly, this feature can also bind eEF1A, which may be involved in closed loop formation, apparently in a way that predominates over a TYMV-like interaction involving the TLS. TEV RNA has a poly(A) tail, but no 5V-cap. The 5V-end is covalently linked to VPg, which is not needed for translation but does interact with eIF4E and eIF- iso4E; it is not known whether this interaction influences translation. 40S ribosome subunits are recruited to the 5V-UTR through an IRES element whose function requires eIF4G and that may involve direct base-pairing to 18S ribosomal RNA. BYDV and STNV RNAs lack both canonical terminal elements and possess translational enhancer elements (BTE, TED) in an internal position (not at the 3V-terminus) of the 3V-UTR. These elements recruit translation initiation factors that are normally recruited to the 5V-end by the cap. In BYDV RNA, ribosome delivery to the 5V-end is accomplished through direct RNA base-pairing between elements in the 5V- and 3V-UTRs, while in STNV RNA, base-pairing between the 5V-UTR and rRNA may be involved. See text for details. 188 T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 factor in TE function (Fig. 1C). Mutations that interfere with expression from capped mRNAs on the order of 10-fold in eEF1A interaction support this conclusion (Matsuda and plant and animal systems in vitro and in vivo (Gallie, 2002). It Dreher, 2004), but direct evidence is needed to implicate is an unstructured sequence almost devoid of guanosine eEF1A in a molecular bridge between the 5V- and 3V-ends. If residues, featuring variations on a CAA repeat motif. The eEF1A were indeed involved, this would be an unprecedented translational enhancement activity of V likely derives from two link between the elongation and initiation components of the sources: (i) the lack of secondary structure reducing the translation machinery. Translational enhancement in vivo by dependence on initiation factors such as the helicase eIF4A the TYMV TLS does not depend on 5V TYMV sequences and and (ii) its ability to bind heat shock protein HSP101 (Wells et occurs by a mechanism that involves synergy with the cap al., 1998). By unknown means, the presence of HSP101 (Matsuda et al., 2004a; Matsuda and Dreher, 2004). These recruits eIF4G (Gallie, 2002)(Fig. 1D). The presence of a studies refute a mechanism in which the TLS was proposed to potent translational enhancer in the 5V-UTR may facilitate co- direct cap-independent initiation from one of the tandem translational disassembly, in which ribosome traffic drives initiation sites at the 5V-end of TYMV RNA (Barends et al., RNA uncoating in the 5V to 3V direction (Gallie et al., 1987b). It 2003). That provocative scheme appears to have been based seems possible that interaction between HSP101, a member of entirely on cell-free experiments using conditions in which the HSP100 family of oligomeric proteins (Lee et al., 2004), viral TLSs support anomalous labeling of existing proteins bound to both the 5V- and 3V-UTRs could contribute to closed with tritiated amino acid esterified at the –CCA terminus loop formation for TMV RNA. Interestingly, the translation (compare with Barends et al., 2004). regulating properties of 5V-UTR elements are apparently not in Although TMV RNA terminates in a TLS that is capable of all cases enhancing. A 31-nt element in the 5V-UTR of BMV aminoacylation (with histidine) and interaction with eEF1A RNA 2 was shown to reduce translation of the polymerase gene (Mans et al., 1991), its 3V-translational enhancer that acts as a in yeast cells, perhaps serving to limit expression of this protein poly(A) tail substitute (PAS) has been localized to a series of (Noueiry et al., 2000). pseudoknots just upstream of the TLS (Leathers et al., 1993). Although tobamovirus genomes have a 5V-cap, the genome This coincides with sequences essential for of Crucifer-infecting tobamovirus (CrTMV) harbors two CR (Takamatsu et al., 1990) and sequences that could be cross- internal IRESes. The 75-nt sequence called IRES MP,75, linked to eEF1A in an aminoacylation-independent manner upstream of the movement protein gene, and the 148-nt CR (Zeenko et al., 2002). Heat shock chaperone HSP101 is another sequence, IRES CP,148, upstream of and including the first 25 protein that is capable of interacting with the pseudoknots codons of the coat protein ORF, function as cap-independent (Wells et al., 1998), but a role for this protein in 3V-PAS function translation elements (Ivanov et al., 1997). These elements has not been established. It is intriguing to speculate that eEF1A apparently facilitate translation of the viral subgenomic RNAs, CR may participate in the translational enhancement of both TMV which may not be capped. IRESMP,75 was reported to facilitate and TYMV RNAs, although via different RNA interactions. highly efficient translation of a downstream ORF in a It is also intriguing to consider why the TMV TLS is bicistronic reporter mRNA in plant and animal cells (Dorokhov apparently not involved in translational enhancement, despite et al., 2002). The activity of the IRES resides in an A-rich tract sharing similar tRNA-like properties with the TYMV TLS. of purines, and (GAAA)16 had maximum activity (Dorokhov Perhaps TMV RNA is an example of an mRNA that has et al., 2002). Its mechanism of action is unclear, but it reiterates acquired a second 3V-PAS upstream of its usual position at the the recurring theme that plant translational control signals tend 3V-terminus, supplanting the original 3V-terminal PAS element to be more compact than those in animal viral RNAs. (the TLS). Evidently, 3V-PAS elements are relatively free of evolutionary constraints, as evidenced by dissimilarities Viruses with genome-linked proteins between closely related virus genera. For instance, the closest relatives of the tymoviruses are the marafiviruses, whose The genomes of one-fourth of the plant positive strand RNA genomes have a poly(A) tail in place of the TLS (van viral genera, including the largest family of plant viruses, the Regenmortel et al., 2000), while the bromoviruses are closely Potyviridae, have a small protein (VPg) covalently attached to related to AMV yet do not exhibit genome activation by coat the 5V-end instead of an m7G(5V)ppp(5V)N cap structure. protein. Since it seems likely that all positive strand viral Therefore, they must undergo some type of cap-independent RNAs are translated in the closed loop format, the variety translation with novel strategies for closed loop formation. among 3V-UTR properties predicts a variety of 5V–3V bridging Although potyviruses and especially comoviruses resemble interactions. picornaviruses in genome structure and in the presence of a Many viruses with capped mRNAs harbor elements that VPg, their mechanisms of ribosome recruitment for initiation enhance cap-dependent translation, independent of the nature appear to be quite different. The VPg-linked plant viral RNAs of the 3V-UTR. Examples include the 5V-UTRs of the coat lack the long, highly structured 5V-UTRs with embedded AUG protein mRNA of AMV (Jobling and Gehrke, 1987) and the triplets that are typical of the well-characterized picornaviral genomic RNA of Potato virus X (Zelenina et al., 1992). One of IRES elements that confer cap-independent translation (Bel- the best characterized examples is the 68 nt 5V-UTR of TMV, sham and Jackson, 2000). For a more detailed review of plant known as the omega (V) sequence (Gallie et al., 1987a, 1988). virus cap-independent translation, see Pettit Kneller et al. V is a cap-dependent enhancer of translation, increasing (in press). T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 189

The 5V-untranslated regions of Tobacco etch potyvirus detected in a coding region. This IRES, in a highly unusual (TEV) (Carrington and Freed, 1990)andTurnip mosaic location 22 nt downstream of the start codon, directs translation potyvirus (Basso et al., 1994b) have been shown to confer of replication-associated protein 1 (Rap1) from a small ORF cap-independent translation. These 5V-UTRs do function as that overlaps with ORF 1 and is located over 1 kb from the 5V- IRESes when placed downstream of a structure or ORF that end of the genome (Jaag et al., 2003). The sequence blocks ribosome scanning from the 5V-end (Basso et al., 1994b; GGAGAGAGAGG is an essential part of this IRES and, by Niepel and Gallie, 1999), but their action is relatively weak, its purine-rich nature, resembles the CrTMV IRES discussed suggesting a role for the 5V-terminus in ribosome loading. The above. Unexpectedly, no IRES activity has been detected in the 143-nt-long AU-rich 5V-UTR of TEV RNA is predicted to 5V-UTR of Plum pox potyvirus, and cap-independent initiation contain a pseudoknot that has been implicated in translational was preserved despite extensive deletion of the 5V-UTR enhancement (Zeenko and Gallie, 2005), although the (Simon-Buela et al., 1997), suggesting that a variety of presence of alternative structures makes the identification of translation initiation mechanisms may be used in the translation critical features uncertain. A loop within this pseudoknot that of plant viral VPg-linked RNAs. can base-pair directly to 18S rRNA may help to recruit 40S ribosome subunits. Immediately upstream of the pseudoknot Viral genomes with unmodified 5V- and 3V-ends is a CAA-rich sequence reminiscent of the TMV V element that also contributes to cap-independent translation (Zeenko RNAs of viruses in the Tombusviridae and the Luteoviridae and Gallie, 2005). Cap-independent translation supported by families have neither a cap nor a poly(A) tail. The Luteoviridae the TEV IRES is eIF4G-dependent (Gallie, 2001), raising family is quite molecularly divergent, as the RNAs of viruses the possibility that this initiation factor is directly or indirectly in genus Luteovirus (hereafter called luteoviruses) have an recruited to the IRES, stabilizing the closed loop format unmodified 5V-end, while those in genus Polerovirus (also of (Fig. 1E). the Luteoviridae family) have a 5V VPg (above). In general, There appear to have been no studies that compare the replication genes and many gene expression control signals of translatability of potyviral RNA with and without covalently the luteoviruses, but not the poleroviruses, resemble those of bound VPg, although significant translation is clearly possible the Tombusviridae (Miller et al., 2002). In particular, the in the absence of the VPg (Basso et al., 1994a; Niepel and translation control signals of luteoviruses and those of selected Gallie, 1999). The issue is relevant because of several lines of genera in the Tombusviridae are strikingly similar. Perhaps the evidence indicating an involvement of the cap-binding most remarkable shared characteristic is the mediation of cap- translation initiation factor, eIF4E, with potyviral infections. independent translation by sequences in the 3V-UTR. Direct interactions have been detected between eIF4E and The 5V-end of the 3V-UTR of Barley yellow dwarf luteovirus potyviral VPgs (Leonard et al., 2000, 2004; Schaad et al., (BYDV) RNA harbors an approximately 100-nt sequence that 2000), suggesting an additional route for eIF4G recruitment to facilitates highly efficient cap-independent translation initiation the 5V-UTR. Among animal viruses, caliciviral RNA is at the 5V-proximal AUG of the mRNA (Guo et al., 2000; Wang translated more efficiently with VPg intact (Herbert et al., et al., 1997). This BYDV-(like) translation element (BTE) is 1997). While the molecular consequence of an interaction conserved in all luteoviruses (not poleroviruses) and in the between VPg and eIF4E or eIFiso4E is unclear, the Dianthovirus (Mizumoto et al., 2003) and Necrovirus (Meu- interaction is essential for infection (Gao et al., 2004; Kang lewaeter et al., 2004; Shen and Miller, 2004a) genera of the et al., 2005). Thus, mutations in host genes encoding eIF4E Tombusviridae. The BTE is characterized by a 17-nt conserved or eIFiso4E confer resistance to numerous potyviruses (Kang sequence, GGAUCCUGGGAAACAGG, that forms a stem- et al., 2005; Lellis et al., 2002; Nicaise et al., 2003; Ruffel et loop (paired bases are underlined), and by at least one al., 2002; Stein et al., 2005). An analogous interaction, additional stem-loop, whose loop base-pairs to the 5V-UTR between the viral proteinase–VPg fusion protein and eIF- (Guo et al., 2001; Pettit Kneller et al., in press). A tract in this iso4E, has been reported for Tomato ringspot nepovirus element (in italics) also has potential to base-pair near the 3V- (Leonard et al., 2002). end of 18S rRNA (Wang et al., 1997). This may contribute to There has been speculation that the VPg–eIF4E interaction recruitment of the ribosome to the BTE. facilitates cap-independent translation (Gao et al., 2004; Lellis Other viruses of the Tombusviridae harbor cap-independent et al., 2002; Thivierge et al., 2005), although it is possible that translation elements in the 3V-UTR that do not resemble a BTE the VPg–eIF4E interaction inhibits eIF4E-dependent (i.e., cap- (Fabian and White, 2004; Meulewaeter et al., 1998b; Timmer dependent) translation of host mRNA, freeing ribosomes for et al., 1993). Satellite tobacco necrosis virus (STNV) RNA has viral RNA translation. Alternatively, or in addition, the a3V-translation enhancer domain (TED) that functions like a interaction may be relevant to other aspects of the infection, BTE (Meulewaeter et al., 1998a; Meulewaeter et al., 1998b; such as facilitating cell-to-cell movement of viral RNA (Gao et Timmer et al., 1993) but bears no primary or secondary al., 2004). structural similarity to a BTE. The TED binds specifically to Other viruses whose genomes are VPg-linked have been translation initiation factors eIF4E or eIFiso4E, and this less studied. While the 5V-UTRs of genomic and subgenomic binding correlates with cap-independent translation function RNAs of Potato leafroll polerovirus appear to lack translation (Gazo et al., 2004). The BTE of BYDV also interacts with cap- enhancement activity (Juszczuk et al., 2000), an IRES has been binding factors (E. Allen, E. Pettit, W.A. Miller, unpublished 190 T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 results). Thus, TED and BTE elements may recruit the because of interference by translating ribosomes (Gamarnik translation initiation machinery by binding canonical cap- and Andino, 1998). We postulate that this scenario is avoided binding factors, leading to recruitment of the viral RNA to the when the passage of the replicase disrupts both the structure of ribosome (Figs. 1F and G). The ribosome is likely placed in the the BTE and its base-pairing to the 5V-UTR. This would shut vicinity of the 5V-end via long distance base-pairing between off ribosome recruitment and delivery to the 5V-end (Barry and the 3V BTE and a single-stranded region in the 5V-UTR. Such 3V- Miller, 2002). The binding of eEF1A to the TLS at the 3V-end UTR–5V-UTR base-pairing has been demonstrated for the of TYMV RNA is thought to accomplish an analogous BTEs of BYDV (Guo et al., 2001) and TNV (Shen and Miller, regulation, serving as a switch between translation and 2004a) and for a non-BTE cap-independent translation element replication. When eEF1A is bound, translation is facilitated of Tomato bushy stunt virus (Fabian and White, 2004); it has (see above), while dissociation permits access by the replicase been predicted for all Tombusviridae (Fabian and White, 2004) to its CCA initiation box at the 3V-terminus (Matsuda et al., and luteoviruses (Guo et al., 2001). Guo et al. (2001) provided 2004b). evidence that the ribosome must scan from the 5V-end of the The BTE element serves as a 5V-cap mimic and can be mRNA to the first AUG, as for normal cap-dependent functionally replaced by a cap but not by a poly(A) tail (Wang translation. Thus, the BTE demonstrates that a cap-independent et al., 1997). It is positioned well upstream of the 3V-terminus translation element need not be an IRES. and does not functionally replace the poly(A) tail. Indeed, the The 3V location of translation initiation elements may BTE is insufficient for translation in vivo and must be provide a molecular switch to facilitate the shift from augmented by other sequences from the viral 3V-UTR or an translation to replication, competing processes that occur on artificial poly(A) tail (Guo et al., 2000). Recent studies have the same molecule for positive strand RNA viruses. Long- shown that various portions of the 869-nt-long BYDV 3V-UTR distance base-pairing and active cap-independent translation contribute to poly(A) independent translation. A distinctive are postulated to be the default status in the absence of viral stem-loop followed by the sequence RCCC (R = purine) forms protein at the beginning of the infection. Once the viral the 3V-terminus of all luteovirus and Tombusviridae RNAs. In replicase has appeared as a result of this translational activity, it each virus, the RCCC can exist in two conformations, either could begin to transcribe the viral RNA from the 3V-end, single stranded, or base-paired to a site upstream of the moving in the 5V direction on the template. The formation of terminal stem-loop (Koev et al., 2002; Pogany et al., 2003; full-length minus strand product would be unlikely, however, Zhang et al., 2004). The base-paired conformation acts as a

Fig. 2. Expanded expression repertoire resulting from leaky scanning and translational recoding. Panel A depicts in simple terms the coding content of a standard mRNA, which directs protein synthesis between an initiation codon, typically the 5V-most AUG (black diamond), and a termination codon (red hexagon). The encoded protein is indicated by the thick line below the RNA. (B) In many viral RNAs, more than one initiation site can be used. This can occur by leaky scanning when the 5V-most initiation codon is weakly recognized by ribosomes because it is in a weak context, as occurs with pyrimidines (Y) in the À3 and/or +4 positions (as indicated) or if initiation occurs at a non-AUG codon (not shown). When the initiation sites are in different reading frames, two proteins of unrelated sequence are made (as shown); when the initiation sites are in-frame (not shown), the encoded proteins are identical except for the presence of an N-terminal extension on one of the proteins. The product of the downstream ORF is generally less efficiently expressed, indicated by the thinner line. (C) In viral RNAs such as TMV RNA, a suppressible termination codon (cross-hatched hexagon) is embedded in an ORF. In conjunction with the downstream recoding signal CARYYA (R = purine;Y= pyrimidine), a small proportion of ribosomes avoids termination, permitting the synthesis of an elongated version of the upstream protein. (D) Frameshifting (typically À1) occurs in mRNAs, which have a pair of recoding signals: a ‘‘slippery’’ heptanucleotide (XXxNNNZ, where X and N can be any base and Z is any base except G) and a feature such as a pseudoknot that is thought to induce ribosome pausing. The encoded products represent translation of the entire upstream ORF and a longer chimeric protein derived from the different ORFs upstream and downstream of the frameshift point. T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 191 replication silencer, preventing access by the replicase to the 3V- end (Pogany et al., 2003) and perhaps also protects the 3V-end from exonucleases. If this is also the conformation that enhances translation as a poly(A) tail mimic, then the conformational switch of the RCCC selects between translation (base-paired RCCC) and replication (single-stranded RCCC) much like the eEF1A binding to TYMV RNA discussed above.

Strategic consequences of genome compression: leaky scanning, readthrough and frameshifting

Overlapping open reading frames and leaky scanning

Many positive strand RNA plant viral genomes have evolved to expand their genetic repertoire through overlapping ORFs. Tymoviruses have the longest tracts of overlapping ORFs. Some 1.9 kb of TYMV RNA simultaneously encodes the 69-kDa movement protein and 206-kDa replication polyprotein. A further 0.4 kb overlap exists between the coat protein ORF and a readthrough domain of the 206-kDa ORF (Bransom et al., 1995). Overlapping ORFs are found in many other viruses and are particularly evident in the triple gene block arrays of movement protein genes. In most cases, such as with the TYMV 69-kDa and 206-kDa ORFs and the overlapping triple gene block ORFs, both proteins are decoded from a single mRNA by the use of alternative initiation sites. The paradigm for such decoding is the phenomenon of leaky scanning (Kozak, 2002)(Fig. 2B). Initiation typically occurs at the 5V-most AUG triplet, but its efficiency is influenced by the surrounding nucleotides or ‘‘context.’’ For plants, optimum context is (A/G)aaAUGGC for dicots and (A/G)ccAUGGC for monocots (Joshi et al., 1997; Lukaszewicz et al., 2000; Lutcke et al., 1987). Two types of suboptimal initiation sites result in initiation by only a fraction of scanning ribosomes: AUG triplets with pyrimidines at the Fig. 3. Examples of viral genes translated by leaky scanning. Only the ORFs À3 and +4 positions (Fig. 2B) or certain non-AUG triplets, involved in leaky scanning are shown. The genus to which each virus belongs is listed below the virus acronym. Gaps in box outline indicate ORFs that are such as CUG (Shirako, 1998), that are surrounded by optimal not shown to scale. Initiation codon contexts are shown above (first ORF) or or near-optimal context. ‘‘Leaky’’ ribosomes that fail to initiate below (second ORF) the translation start site. Bases at À3 and +4 positions at these sites continue to scan for an appropriate initiation site. relative to the start codon (bold AUG), that fit the optimal context (G at +4, A Such leaky scanning is distinct from shunting, in which at À3) are underlined. In all cases, the second ORF start codon is in a better ribosomes bypass part of the 5V-UTR by interrupting their initiation context than the upstream AUGs. The p20 ORF of SBMV-C contains two AUG codons in weak contexts that do not act as initiation codons (gray linear scanning. Shunting occurs in the expression of Cauli- sequences below p20 ORF) (Sivakumaran and Hacker, 1998). The second start flower mosaic virus proteins (Ryabova and Hohn, 2000). codons of CPMV and PPV are in the same reading frame as the first, yielding an Leaky scanning is a process involving sequential initiation N-terminally truncated protein. The CPMV ORF has an in-frame AUG (gray decisions made as 40S ribosome subunits scan in the 5V to 3V sequence) 12 nt downstream of the second start codon that can act as a start direction (Kozak, 2002). Leaky scanning is thus revealed by codon in artificial contexts, but is unlikely to function under usual conditions (Holness et al., 1989). See text for additional explanation and references. observing that downstream initiation is blocked either by optimization of the upstream initiation site or by the insertion of a new upstream AUG in a strong context. By at least one of it is important to perform experiments that preclude alternative these criteria, the third triple gene block ORF of Barley stripe translation mechanisms such as shunting, internal ribosome mosaic virus (Zhou and Jackson, 1996)(Fig. 3) and Potato entry, or initiation by ribosomes that have completed the virus X (Verchot et al., 1998) is expressed by leaky scanning. translation of an upstream ORF and resumed scanning By similar criteria, the second ORF of Peanut clump virus (reinitiation) (Kozak, 1999). By inserting several different (PCV) RNA2 is also expressed by leaky scanning (Herzog et upstream AUG codons, altering the positional relationship al., 1995), despite the presence of about 600 intervening between the upstream ORF and the downstream AUG, and by nucleotides (lacking any AUG triplets) (Fig. 3). In such cases, using a stable hairpin placed at the 5V-terminus to demonstrate 192 T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 that ribosomes initiate scanning by interacting with the 5V-end, levels of a C-terminally extended version of a protein (Fig. 2C). these issues were addressed in the PCV study (Herzog et al., The best characterized example is the expression of the 126 and 1995). 183 kDa ORFs of TMV. Expression of the catalytic domain of Some further examples of leaky scanning have been the RNA-dependent RNA polymerase (RdRp), which is demonstrated for TYMV RNA (Weiland and Dreher, 1989), located in the C terminal portion of P183, requires readthrough subgenomic RNA1 of Barley yellow dwarf virus (BYDV) of the P126 ORF stop codon (Pelham, 1978). This results in (Dinesh-Kumar and Miller, 1993), the 0.9-kb subgenomic expression of large quantities of P126, which includes helicase mRNA of Cucumber necrosis virus (Johnston and Rochon, and methyl transferase (capping enzyme) domains, and much 1996), Southern bean mosaic virus (cowpea strain) (SBMV-C) smaller amounts of the RdRp (P183), which consists of P126 RNA (Sivakumaran and Hacker, 1998), and Plum pox virus with a 57-kDa C-terminal extension. Translation studies in (PPV) RNA (Simon-Buela et al., 1997)(Fig. 3). Note that the which expression of a reporter gene depended on readthrough observation of leaky scanning associated with BYDV, CNV, revealed that the sequence UAGCARYYA (UAG is the P126 SBMV-C, and PPV RNAs indicates that this expression stop codon, R = purine, Y = pyrimidine) was sufficient for mechanism is not limited to capped mRNAs. wild-type levels of readthrough (Fig. 2C), which was about 5% The two initiation AUGs of TYMV RNA are separated by (Namy et al., 2001; Skuzeski et al., 1991). only four intervening nucleotides. Consistent with normal Unrelated viruses also employ readthrough to express the leaky scanning, mutational inactivation of the upstream AUG catalytic domain of the RdRp. These include most genera of the resulted in increased initiation from the downstream AUG Tombusviridae and various rod-shaped fungus-transmitted (Matsuda et al., 2004a). However, the close proximity of the viruses. The readthrough signals of these viruses are poorly two AUGs represents a special situation in which ribosomes characterized. seem to choose quasi-simultaneously between the alternative The stop codons of the major CP gene of viruses in the start sites. Initiation site selection depends on the AUG Luteoviridae, furoviruses, and Beet necrotic yellow vein virus contexts, but the close spacing potentiates expression from (BNYVV) are leaky, resulting in a C-terminal extension of the the downstream initiation site (D. Matsuda and T. Dreher, CP that is larger than the CP itself. In Luteoviridae, the CP unpublished). As with TYMV RNA, expression from the 5V- alone is sufficient to form infectious virions, but the read- most AUG of BYDV-PAV sgRNA1 increased upon inactiva- through domain (C-terminal extension) is required for aphid tion of the next AUG, 41 nt downstream. It was proposed that a transmission (Brault et al., 1995, 2005). For rod-shaped following ribosome bumps up against a ribosome paused fungally transmitted viruses, such as BNYVV (Tamada et al., during initiation at the downstream AUG, arresting it in the 1996) and Potato mop-top virus (Reavy et al., 1998), the vicinity of the upstream AUG and increasing the likelihood of readthrough domain facilitates fungal transmission. In contrast initiation (Dinesh-Kumar and Miller, 1993). Alternatively, the to the luteoviruses, the readthrough domain is required for downstream ribosome could affect upstream initiation by BNYVV virion assembly (Schmitt et al., 1992). melting part of the rather extensively folded 5V-UTR. The In the Luteoviridae, the cis-acting signals that cause the leaky scanning that occurs on SBMV-C RNA is unusual in ribosome to read through the stop codon include a cytidine-rich bypassing two intervening AUG codons (Sivakumaran and repeat (CCNNNN)8–16 beginning about 20 nt downstream of Hacker, 1998). Several of these examples present properties the stop codon and an essential sequence located over 700 nt that differ from the standard form of leaky scanning (Kozak, downstream (Brown et al., 1996). Thus, the luteovirus read- 1989), suggesting that a variety of idiosyncratic expression through signal is totally different from that of TMV. Read- mechanisms likely awaits discovery as more viruses are through occurs when a non-cognate aminoacyl-tRNA base- investigated in depth. Not only does leaky scanning allow pairs to the stop codon in the ribosomal A site, followed by expression of overlapping ORFs but it controls the relative peptidyl transfer rather than insertion of release factor in the A amounts of protein synthesized from each ORF. For example, site that facilitates termination. It is not known how the diverse alteration of the start codon of the first ORF (P0, suppressor of viral readthrough signals cause a fraction of the ribosomes to silencing) of Beet western yellows polerovirus (Fig. 3)toa be reprogrammed in this way. more efficient or less efficient context prevents virus replica- A smaller set of viruses employs an entirely different tion (Pfeffer et al., 2002). recoding event to achieve a similar end result as readthrough. Instead of reading through a stop codon, ribosomes are induced Translational recoding: readthrough and frameshifting to change reading frames during the elongation phase of translation. Like readthrough, frameshifting generally occurs Recoding is dynamic reprogramming of translation so that for less than 5% of transiting ribosomes. In most plant viruses, the genetic code is temporarily redefined at specific codons, the frameshift causes the ribosome to bypass a stop codon, generally at low frequency (Gesteland et al., 1992; Baranov et providing a C-terminal extension to the protein generated by al., 2002). Here, we discuss examples of stop codon read- canonical translation (Fig. 2D). In most plant viruses known to through and ribosomal frameshifting. The structure of the viral undergo frameshifting, it is the catalytic domain of the RdRp mRNA contains complete information in the form of cis-acting that is expressed by frameshift. The Luteoviridae,the sequences to induce these recoding events. Many viruses Dianthovirus genus of the Tombusviridae, and some sobemo- employ in-frame readthrough of stop codons to express low viruses (Makinen et al., 1995) all employ À1 frameshifting, T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197 193 i.e., the ribosomes back up one base after pausing during Future perspectives translation of the first ORF, thereby shifting into the second ORF. As with readthrough, this appears to be a regulatory Research over the next few years on the topics we have strategy to express the RdRp at low levels. One exception described in this review will bring a better understanding of the appears to be SBMV, in which the frameshift causes the range of regulatory elements present in plant viruses that ribosome to shift out of the RdRp reading frame in order to appear to be quite distinct from those found in animal viruses, translate an ORF needed for virus cell-to-cell movement including the 3V-translational enhancers and IRES elements. A (Sivakumaran et al., 1998). number of other fruitful research directions can be anticipated. Plant viruses share features with the canonical À1 More information is needed on the features that regulate the frameshift signals present in the polymerase genes of the competition between coexisting viral mRNAs and the levels of animal-infecting nidoviruses and retroviruses. This includes the different gene products encoded by a given virus. We have an XXxNNNZ motif at the shifty site, where X is any base, N described the expanded coding possibilities afforded by over- is usually A or U, and Z is any base except G (lower case lapping open reading frames, readthrough, and frameshifting, indicates a consensus with occasional exceptions). The shifty and mentioned the fact that recoding results in low expression site is followed by a highly structured region, usually a levels for one of the proteins. But we do not fully understand pseudoknot, beginning 5–6 nt downstream (Fig. 2D). The the importance of particular expression ratios nor all the factors three-dimensional structure of the small, 28 nt frameshift- that influence those ratios, especially when proteins are made inducing pseudoknot of poleroviruses has been characterized from different mRNAs, including subgenomic RNAs. For at high resolution by X-ray crystallography (Su et al., 1999) instance, we do not understand why subgenomic RNAs and nuclear magnetic resonance (Cornish et al., 2005; encoding coat proteins often translationally out-compete Giedroc et al., 2003) and subjected to saturation mutagenesis genomic RNAs encoding replication proteins (Pyne and Hall, (Kim et al., 2000; Kim et al., 1999). It is a remarkably 1979; Wang et al., 1999). The regulated timing and quantity of compact structure featuring base triplets between the first viral protein accumulation likely are an important ingredient stem of the pseudoknot and an A tract in the second loop. In for a successful infection (Shen and Miller, 2004a, 2004b). contrast, the secondary structures that induce frameshifting on A question that has been insufficiently addressed for plant Red clover necrotic mosaic dianthovirus (RCNMV) (Kim and viruses is the effect of virus infection on host translation. It is Lommel, 1998) and BYDV RNAs consist of a long bulged generally believed that plant viruses, unlike most animal stem-loop, which, in the case of BYDV, must base-pair to a viruses, do not globally shut off host gene expression (Hull, sequence 4 kb downstream (Barry and Miller, 2002). Despite 2002). During animal virus infections, there is active parrying being members of different families, the primary and at the level of the translation machinery in which the host secondary structures of RCNMV and BYDV frameshift attempts to limit the translation of viral proteins while the virus elements are quite similar, as are the amino acid sequences attempts to establish optimal conditions for the selective of the polymerase genes. translation of viral mRNAs (Gale et al., 2000). For instance, Regarding mechanism, it has been proposed that the picornaviruses, whose RNAs lack a 5V-cap, turn that distinction downstream pseudoknot slows or inhibits the advancing to their advantage by undermining cap-dependent translation. It ribosome, perhaps even forcing it back one base, precisely is not known to what extent virus–host interactions of this type when the shifty site codons are in the A and P sites of the occur during infections, but it is important to clarify ribosome (Plant et al., 2003; Yusupova et al., 2001). The this matter. In one set of studies, host gene expression was tRNAs in the A and P sites simultaneously slip back one base transiently reduced at the virus infection front (Wang and on the mRNA and then re-pair. The shifty site sequence usually Maule, 1995), though this appeared not to occur at the level of allows five of the six bases in the anticodons of the tRNAs in translation (Aranda and Maule, 1998). The regulation of the A and P sites to pair to the codons in the À1 reading frame translation is an innate response that is a valuable part of the of the mRNA. antiviral arsenal available to animal cells (Schneider and Mohr, Other less well-characterized frameshift events may be 2003). In plants, understanding the involvement of similar unique to plant viruses. Translation of the Potato virus M responses is only just beginning (Bilgin et al., 2003), but there 12-kDa ORF requires a À1 frameshift to occur when is the possibility that the powerful role of post-transcriptional ribosomes reach the CP stop codon (Gramstat et al., 1994). gene silencing in plants has diminished reliance on antiviral This event requires only four slippery bases that are followed regulation at the translational level. immediately by any stop codon. The stop codon presumably A fertile field for future research should be elucidating the induces pausing, and slippage occurs with a tRNA in only transition from translation to replication during the infection. the P site. The sequences of the closterovirus genomes This is expected to require a clearance of ribosomes from the suggest that a net reading frame shift of +1 must occur to mRNA. Two possible strategies are suggested by studies with express the RdRp (Karasev et al., 1995). If indeed +1 BYDVandTYMVmentionedinthisreview.Butthe frameshifting occurs, this would be of interest because +1 recruitment of viral RNAs to the membranous sites of RNA frameshifting or other events that cause a net +1 frame replication is also part of this transition (Schwartz et al., 2002). change have not been demonstrated in any eukaryotic virus Studies with BMV indicate that the translation of viral RNAs in genome (Baranov et al., 2001). yeast requires a set of host proteins (Lsm1–7p and Pat1p 194 T.W. Dreher, W.A. Miller / Virology 344 (2006) 185–197

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