Journal of General Virology (2013), 94, 1415–1420 DOI 10.1099/vir.0.051755-0

Short A stem–loop structure in the 59 untranslated region Communication of bean pod mottle RNA2 is specifically required for RNA2 accumulation

Junyan Lin,1 Ahmed Khamis Ali,1,3 Ping Chen,1,4 Said Ghabrial,5 John Finer,2 Anne Dorrance,1 Peg Redinbaugh1,6 and Feng Qu1

Correspondence 1Department of Plant Pathology, Ohio Agricultural Research and Development Center, The Ohio Feng Qu State University, 1680 Madison Ave, Wooster, OH 44691, USA [email protected] 2Department of Horticulture and Crop Science, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Ave, Wooster, OH 44691, USA 3Department of Biology, College of Science, University of Mustansiriyah, Bagdad, Iraq 4Key Laboratory of Protection and Utilization of Tropical Crop Germplasm Resources, Hainan University, Haikou, Hainan, People’s Republic of China 5Department of Plant Pathology, University of Kentucky, Lexington, KY 40546, USA 6USDA-ARS, Corn and Research Unit, Wooster, OH 44691, USA

Bean pod mottle virus (BPMV) is a bipartite, positive-sense (+) RNA plant virus of the family . Its RNA1 encodes all proteins needed for replication and is capable of autonomous replication. By contrast, BPMV RNA2 must utilize RNA1-encoded proteins for replication. Here, we sought to identify RNA elements in RNA2 required for its replication. The exchange of 59 untranslated regions (UTRs) between genome segments revealed an RNA2- specific element in its 59 UTR. Further mapping localized a 66 nucleotide region that was predicted to fold into an RNA stem–loop structure, designated SLC. Additional functional analysis Received 21 January 2013 indicated the importance of the middle portion of the stem and an adjacent two-base mismatch. Accepted 5 February 2013 This is the first report of a cis-acting RNA element in RNA2 of a bipartite secovirus.

The replication of viral is a key step in the viral turn carries the corresponding viral to VRCs. The life cycle that depends on both virus-encoded functions latter mechanism is commonly used by with and a multitude of host elements (den Boon & Ahlquist, multipartite genomes, as some of their genome segments 2010; Nagy & Pogany, 2010). For positive-sense (+) RNA encode functions unrelated to genome replication, thus viruses, this step is carried out primarily by RNA- they have to rely on AP and RdRP encoded by other dependent RNA polymerases (RdRPs) translated directly segments for their replication. This situation is best from viral genomic RNAs. In addition, most (+) RNA exemplified by brome (BMV) and red clover viruses also encode a second protein, designated auxiliary mosaic virus (RCNMV). The genetic information of BMV protein (AP), that induces the rearrangement of specific is partitioned in three RNA segments, with RNA1 and 2 organellar membranes to form protective enclosures that encoding 1a and 2a, serving AP and RdRP functions, shield the replication process from the cellular envir- respectively. The smaller RNA3 encodes the cell-to-cell onment (Schwartz et al., 2002). The complete membrane- movement and capsid proteins (MP and CP). Accordingly, based enclosure, consisting of viral RNA, RdRP, AP, as well BMV RNA3 encodes a unique, cis-acting RNA structure as various host proteins recruited to assist in this process, is that specifically interacts with 1a to dramatically enhance often referred to as the viral replication complex (VRC). the replication of RNA3 (Baumstark & Ahlquist, 2001; and references therein). Similarly, RCNMV RNA2, which The question of how viral RNAs access VRCs has been encodes solely the viral MP, is recruited to VRCs through extensively studied for a number of viruses. Many viral the specific interaction between a Y-shaped RNA structure genomic RNAs are also mRNAs for the translation of AP, in its 39 untranslated region (UTR) and the RNA1-encoded hence may be brought to VRCs by APs in a translation- p27, the RCNMV AP (Iwakawa et al., 2011). coupled manner (Yi & Kao, 2008; Wang et al., 2009). Other viral RNAs encode specific secondary structures or stem– (BPMV) is similar to RCNMV in loops (SLs) that are recognized by AP or RdRP, which in that all viral proteins required for genome replication are

051755 Printed in Great Britain 1415 J. Lin and others encoded on RNA1 of its bipartite (+) RNA genome. In fact, used as the RNA2 surrogate, an RNA2 derivative that BPMV RNA1 is known to replicate in single cells in the contained a GFP insert between MP and L-CP (RNA2-GFP absence of RNA2 (Gu & Ghabrial, 2005; J. Lin & F. Qu, or R2G; Fig. 1a), which was shown previously to replicate unpublished). However, unlike RCNMV, each of the two to similar levels as wild-type RNA2 (Zhang et al., 2010). As BPMV RNAs encodes one single polyprotein, from which a negative control, we generated a defective RNA1 cDNA, multiple mature viral proteins are derived through post- referred to as RNA1 m (Fig. 1a), which encodes a non- translational processing by the virus-encoded protease functional RdRP due to the replacement of the highly (Pro). Specifically, the RNA1-encoded polyprotein is the conserved glycine-aspartic acid-aspartic acid (GDD) motif precursor for five proteins: a putative protease co-factor (C- with alanine-alanine-histidine (AAH; Fig. 1a). Finally, we Pro), a putative RNA helicase (HEL) and possibly an AP, a used particle bombardment to deliver these constructs into small protein that covalently binds to the 59 end of the lima bean cotyledons, which were shown to be well suited genomic RNAs (viral protein genome-linked or VPg), Pro, for particle bombardment delivery of plasmid DNA and the viral RdRP (Fig. 1a). Notably, translation of BPMV (Hernandez-Garcia et al., 2010). RNA2 can initiate at two different start codons, leading to Bombardment of lima bean cotyledons with RNA1+R2G, the production of two polyproteins that differ only at their + N-termini (MacFarlane et al., 1991; Fig. 1a). The larger but not with RNA1 m R2G, resulted in rigorous BPMV polyprotein is thought to be processed into three mature replication and cell-to-cell movement as indicated by proteins: an N-terminal 58K protein (p58) with unknown bright green fluorescent infection foci that cover multiple functions, and two CP subunits (L-CP and S-CP. Fig. 1a), cells (Fig. 1b, top row, second and third panels; data whereas the smaller polyprotein produces a smaller N- not shown). Additionally, extracts of the BPMV-positive terminal mature protein that serves as the viral MP (Fig. 1a). cotyledons were highly infectious to soybean plants, leading to severe BPMV symptoms and bright GFP BPMV is a member of the genus in the fluorescence in the systemically infected leaves (Fig. 1b, subfamily of Secoviridae (Sanfac¸on et al., second row, second and third panels; data not shown). The 2009). Members of Secoviridae share a number of unique identity of R2G in both lima bean cotyledons and features, among them icosahedral particle structures, two systemically infected soybean leaves were further confirmed or more different capsid protein subunits, and a very small with strand-specific reverse transcription-PCR (RT-PCR; VPg, which align them closely with the animal-infecting Fig. 1c, d, lanes 2 and 3). In summary, our new approach viruses of Picornavidae (Le Gall et al., 2008). However, permitted the observation of BPMV infection from single- unlike , many secoviruses have bipartite cell levels to whole plants, hence is suited for our study. RNA genomes, the replication of which is not well understood. In particular, it remains to be resolved how We next examined the role of both 59 and 39 UTRs of the replication proteins encoded by one of the genome BPMV RNA2 in RNA2 accumulation using this new segments replicates the other segment (van Bokhoven et al., procedure. To this end, we generated three R2G mutants 1993; Gaire et al., 1999). To begin to understand the in which the 59 UTR, 39 UTR, or both were replaced by replication process of BPMV, we chose to first focus on the their RNA1 counterparts, resulting in constructs R2G– replication requirements of BPMV RNA2, and attempted 1U5, –1U3, and –1U5/1U3, respectively (Fig. 1a). These to identify cis-acting RNA sequences or structures within constructs were then bombarded into lima bean cotyle- RNA2 that are specifically needed for its own accumula- dons together with RNA1. As shown in Fig. 1b, while tion. We report here the identification of an SL structure replacing the 39 UTR of RNA2 with that of RNA1 exerted within the 59 UTR of BPMV RNA2, referred to as SLC, no detectable impact on the infectivity of the virus in both which acts in cis to ensure the accumulation of RNA2 in bombarded lima bean cotyledons and systemically infected cells. We speculate that SLC plays an essential role infected (Fig. 1b, third row, first panel), doing in shepherding RNA2 into VRCs programmed by RNA1- the same with 59 UTRs led to a complete loss of BPMV encoded proteins. infectivity in both types of plants (Fig. 1b, first row, last panel; data not shown). Accordingly, replacing both UTRs Our study began with the development of a reliable also caused the loss of viral infectivity (Fig. 1b, third row, experimental system that allows us to monitor the second panel). These observations suggested that RNA2 59 accumulation of viral RNAs at both the single-cell and UTR contained critical cis-acting element(s) essential for the intact-plant levels. The initial difficulty of developing a RNA2 accumulation. protoplast system amenable to BPMV infections prompted us to adopt an alternative approach. In this new approach, We then made additional modifications within the 59 UTR we placed the cDNAs of BPMV RNA1 and RNA2 between of RNA2. Pairwise comparison revealed that the first 262 the 35S promoter and terminator (P35S and T35S; Fig. 1a) nucleotides (nts) of RNA1 and RNA2 59 UTRs share a very of cauliflower mosaic virus. Consequently, upon delivery of high level of sequence identity (at least 91 %), whereas the the resulting constructs into BPMV host cells, viral sequences after nt position 263 are much more divergent infection can be launched with RNAs transcribed intra- (J. Lin & F. Qu, unpublished; data not shown). We first cellularly, thus bypassing the need for in vitro transcripts. determined whether the few differences within the first Next, to facilitate the tracking of the infection process, we 262 nt could affect the replication of RNA2 by substituting

1416 Journal of General Virology 94 cis-acting RNA element of BPMV RNA2

(a) Fig. 1. RNA2 59 UTR contains key cis-acting elements required for RNA1: RNA2 accumulation. (a) Schematic representation of BPMV P35S C-ProHEL Pro RdRP T35S RNA1, RNA1 m, and RNA2 (R2G) constructs, and R2G mutant 5´UTR VPg 3´UTR 9 9 (1-368) GDD AAH with altered 5 or 3 UTRs. (b) Infectivity of mutant constructs RNA1m: assessed with particle-bombarded lima bean cotyledons and subsequently rub-inoculated soybean plants. The images of GFP (1-368) fluorescent cotyledons were taken 48 h post bombardment, RNA2-GFP 5´UTR (R2G): (1-466) 773 3´UTR whereas those of systemically infected soybean leaves were taken P35S MP GFP L-CP S-CP T35S two week post-inoculation. (c) Verification of active replication in p58 lima bean cotyledons with (-) RNA2-specific RT-PCR. An RT-PCR R2G-1U5: R2G-1U3: product of a lima bean actin mRNA was used as controls. (d) Verification of active replication in systemically infected soybean R2G-1U5(262): leaves with (-) RNA2-specific RT-PCR. An RT-PCR product of a soybean actin mRNA was used as controls. R2G-Δ1-262:

(b) Pos. ctrl.: Neg. ctrl.: RNA1+ by RNA1 and RNA2, as its deletion from R2G led to a Mock RNA1+R2G RNA1m+R2G R2G-1U5 complete loss of BPMV infectivity (Fig. 1b, third row, last Lima bean panel). Together, these results indicated that the RNA2- cotyledons specific cis-acting structure does not reside in the first

Soybean 262 nt of RNA2 59 UTR. systemic leaves We then moved to interrogate the rest of the 59 UTR sequence (nt numbers 263–466) for a potential role in RNA1+ RNA1+ RNA1+ RNA1+ R2G-1U3 R2G-1U5/1U3 R2G-1U5(262) R2G-Δ1-(262) RNA2 replication. We first sought to determine the boundaries of the cis-acting elements by inserting a 36 nt Lima bean cotyledons non-BPMV sequence (termed ‘ha’ as the majority of its sequence was derived from the HA epitope tag cDNA) Soybean systemic between nt positions 466 and 467, and 263 and 264, leaves creating constructs R2G-466 ha and R2G-263 ha, respect- ively (Fig. 2a). When delivered to lima bean cotyledons and subsequently soybean plants to examine their infectivity, RNA1+ (c) Lima bean both of them resulted in productive infections indistin- cotyledons guishable from wild-type R2G (Fig. 2c, top row, last panel; Δ1-(262) and third row, first panel). Thus, the potential RNA2- Pos. ctrl.: Neg. ctrl.: Mock RNA1+R2GRNA1m+R2GR2G-1U5R2G-1U3R2G-1U5/1U3R2G-1U5(262)R2G- specific, cis-acting element(s) must reside within the region (-) RNA2 447bp between nt 263 and 466. Lima bean 390bp We then further delimited the putative cis-acting element actin 12 34567 8 through deletion mutagenesis. To help guide the deletion mutagenesis, we used the MFold algorithm to predict the (d) Soybean RNA1+ RNA secondary structure of this region. As illustrated in Fig. systemic leaves 2b, this region could potentially fold into three SLs, referred

Δ1-(262) to as SLA, SLB and SLC, separated by unstructured sections Pos. ctrl.: Neg. ctrl.: R2G-1U51U3 Mock RNA1+R2GRNA1m+R2GR2G-1U5R2G-1U3 R2G-1U5/2U5R2G- of varying lengths. We thus generated three different deletion mutants of R2G, namely DSLA, DSLB and DSLC, (-) RNA2 447bp each removing one of the SLs together with a few flanking Soybean actin 345bp nts (Fig. 2a). As shown in Fig. 2c, while R2G-DSLA and 12 34567 8 R2G-DSLB caused rigorous BPMV infections essentially identical to wild-type R2G, R2G-DSLC was unable to cause any detectable infection even at the single-cell level (Fig. 2c, third and fourth rows, last three panels; data not shown). this portion of RNA2 59 UTR for its RNA1 counterpart. These results were further confirmed with strand-specific The resulting construct, R2G–1U5(262), was similarly RT-PCR detection of fragments of expected size (Fig. 2d, e). competent as the wild-type R2G (Fig. 1b, third panel in Collectively, they strongly suggest that SLC constitutes a key third and fourth rows; Fig. 1c, d, lane 7). This result cis-acting element for RNA2 propagation in host cells. demonstrated that the first 262 nt of RNA1 and RNA2 59 UTRs are interchangeable and thus do not contain cis- We next conducted a preliminary evaluation of the acting elements unique for RNA2. Nevertheless, this sequence and structural requirements of SLC. We started section of 59 UTR contains essential cis-elements shared with the middle section of the stem because it contains the http://vir.sgmjournals.org 1417 J. Lin and others

(a) (c) Pos. ctrl.: Neg. ctrl.: RNA1+ RNA2-GFP 5´UTR 3´UTR Mock RNA1+R2G RNA1m+R2G R2G-466ha P35S p58/Mp GFP L-CP S-CP T35S (R2G): Lima bean 1 263 466 cotyledons

ha R2G-466ha: Soybean systemic 263 466 467 leaves ha R2G-263ha: RNA1+ RNA1+ RNA1+ 466 RNA1+ 263 264 R2G-263ha R2G-DSLA R2G-DSLB R2G-DSLC R2G-DSLA: 263 307 466 Lima bean cotyledons R2G-DSLB: 263 308 399 466 Soybean R2G-DSLC: systemic leaves 263 400 460 466

(d) Lima bean RNA1+ (b) cotyledons SLB SLC DSLA DSLB DSLC Pos. ctrl.: Neg. ctrl.: Mock RNA1+R2GRNA1m+R2GR2G-466haR2G-263haR2G- R2G- R2G- 747bp 710bp (-) RNA2 667bp 610bp Lima bean 390bp actin 12 34567 8

(e) Soybean RNA1+ systemic SLA leaves

DSLA DSLB DSLC Pos. ctrl.: Neg. ctrl.: Mock RNA1+R2GRNA1m+R2GR2G-466haR2G-263haR2G- R2G- R2G- 747bp (-) RNA2 710bp 667bp 610bp Soybean actin 345bp 12 34567 8

Fig. 2. Identification of SLC as a cis-acting element essential for RNA2 accumulation. (a) Schematic representation of R2G mutants containing various forms of insertions/deletions within the second half of 59 UTR of RNA2 (nt position 263–466). (b) RNA secondary structure of the nt 263–466 region predicted using the MFold algorithm. The three putative stem–loop structures are referred to as SLA, SLB, and SLC, respectively. (c) Infectivity of mutant constructs assessed with particle- bombarded lima bean cotyledons and subsequently rub-inoculated soybean plants. Mutants tested are indicated above the respective panels. Images were recorded in a manner similar to those in Fig. 1b. (d) and (e) Verification of active replication in lima bean cotyledons and systemically infected soybean leaves with (-) RNA2-specific RT-PCR.

longest double-stranded stretch of SLC, with seven undis- We then examined the importance of the bottom part of rupted base pairs (Fig. 3a). We first designed disruptions of the SLC stem consisting of one C–U mismatch and six base the base pairs by changing four nts on the left arm of the pairs (Fig. 3a). We disrupted this portion of structure by stem to their complements on the right (UUCA to gggu), inserting four nts (aucc) after nt number 461, hence creating mutRR (Fig. 3a). A reciprocal mutLL mutant was creating a BamHI site in the corresponding cDNA. The similarly created by replacing UGGG on the right with acuu resulting mutant, referred to as R2G-460BamHI, comple- (Fig. 3a). In agreement with a critical importance of tely abolished the BPMV infectivity (Fig. 3b, third row, last maintaining the stem, both mutRR and mutLL completely panel). It should be noted that MFold predicted that the lost the ability to infect lima bean cotyledons (Fig. 3b, top upper two-thirds of SLC would remain essentially undis- row, last panel, and third row, first panel). However, when turbed in this mutant. In addition, the six base pair stem at the two mutations were combined to create mutRL, in the bottom of SLC also remained intact despite some which the stem structure but not its original sequence was changes in nt identity (Fig. 3a). If this predicted structural restored, wild-type infectivity was observed (Fig. 3b, third consequence is correct, then either the two base (C–U) and fourth rows, middle panels; data not shown). These mismatch needs to be faithfully preserved, or the identity results indicated that the integrity of the middle section of of certain nts within this section of SLC is critically the stem is critical for the function of SLC. important. In summary, our results support a critical role

1418 Journal of General Virology 94 cis-acting RNA element of BPMV RNA2

for SLC as a unique cis-acting RNA structure required for (a) RNA2 accumulation in the host cells of BPMV. 460BamHI SLC Our study reveals the first RNA2-specific cis-acting element mutRR in a Secoviridae member. While similar elements have been identified in BMV and RCNMV, two well-studied multi- mutRL partite (+) RNA viruses that adopt different coding and replication strategies than BPMV (Baumstark & Ahlquist, mutLL 2001; Iwakawa et al., 2011), such elements have not been identified in other Secoviridae members. Although cowpea mosaic virus (CPMV), another Secoviridae member with a similar genome organization as BPMV, has been subjected to UTR exchanges in a manner similar to our current study, no RNA2-specific cis-acting element was identified, 9 9 (b) Pos. ctrl.: Neg. ctrl.: RNA1+ as both 5 and 3 UTRs of CPMV RNA2 could be replaced Mock RNA1+R2G RNA1m+R2G R2G-mutLL by their RNA1 counterparts without affecting RNA2 replication (van Bokhoven et al., 1993; Rohll et al., Lima bean cotyledons 1993). We speculate that CPMV RNA2 could possess a cis-acting element within its polyprotein-coding sequence.

Soybean Alternatively, CPMV RNA2 could be brought to VRCs systemic leaves using a mechanism that does not rely on a unique cis- acting RNA element. RNA1+ RNA1+ RNA1+ R2G-mutRR R2G-mutRL R2G-460BamHI Importantly, the conservation of SLC structure was also

Lima bean supported by phylogenetic analysis. As shown in Fig. 3c, cotyledons among the nine BPMV isolates for which RNA2 sequences

Soybean are available through GenBank, six of them contained the systemic exact same sequence as the isolate we examined (IA-Di1). leaves The other three isolates (two KY, one OH), while containing a number of base pair covariations, maintained the same SL structure (Fig. 3c). While it remains to be (c) determined how SLC facilitates the accumulation of BPMV RNA2, the fact that SLC mutants were unable to accumulate even in single cells implies that SLC might help deliver RNA2 to RNA1-programmed VRCs. However, other possibilities, such as enhancing the translation or stability of RNA2, cannot be completely ruled out. Our next goals are to test if SLC interacts with RNA1-encoded proteins, and to elucidate the intra-cellular pathway through which RNA2 associates with VRCs.

Acknowledgements We greatly appreciate the generosity of Drs Steve Whitham and John Hill at Iowa State University in providing us with the RNA2-GFP construct. We thank members of the Qu and Finer laboratories for stimulating discussions and technical assistances, Dr Lucy Stewart and other members of the USDA, ARS Corn and Soybean Research Unit for sharing equipment. This study was supported in part by an Fig. 3. Functionality of SLC depends on the integrity of the central OARDC Graduate Seed award to J. L. Soybean virus-related research portion of the stem. (a) Schematic representations of mutRR, in the Qu lab has been supported in part by grants from the North mutLL, mutRL, and 460BamHI. The altered nts in each mutant are Central Soybean Research Program and Ohio Soybean Council. We in lower-case letters. (b) Infectivity of mutant constructs assessed gratefully acknowledge Dr Andy White for critical reading of the manuscript and numerous suggestions. with particle-bombarded lima bean cotyledons and subsequently rub-inoculated soybean plants. (c) Conservation of SLC structure in RNA2 of various BPMV isolates. The SLC element was initially References identified from an Iowa (IA-Di1) isolate, and shared with 100 % identity by five additional isolates. Limited nt variations were found Baumstark, T. & Ahlquist, P. (2001). The brome mosaic virus RNA3 in two Kentucky (KY) isolates and one Ohio (OH) isolate, none of intergenic replication enhancer folds to mimic a tRNA TyC-stem which caused significant changes in the overall structure. loop and is modified in vivo. RNA 7, 1652–1670. http://vir.sgmjournals.org 1419 J. Lin and others den Boon, J. A. & Ahlquist, P. (2010). Organelle-like membrane bean pod mottle virus middle component RNA. 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