Downloaded from rnajournal.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press A second functional RNA domain in the 5؅ UTR of the Tomato bushy stunt virus genome: Intra- and interdomain interactions mediate viral RNA replication DEBASHISH RAY, BAODONG WU, and K. ANDREW WHITE Department of Biology, York University, Toronto, Ontario, Canada M3J 1P3 ABSTRACT The 5؅ untranslated regions (UTRs) of (+)-strand RNA viruses play a variety of roles in the reproductive cycles of these infectious agents. Tomato bushy stunt virus (TBSV) belongs to this class of RNA virus and is the prototype member of the genus Tombusvirus. Previous studies have demonstrated that a T-shaped domain (TSD) forms in the 5؅ half of the TBSV 5؅ UTR and that it plays a central role in viral RNA replication. Here we have extended our structure–function analysis to the 3؅ half of the (5؅ UTR. Investigation of this region in the context of a model viral replicon (i.e., a TBSV-derived defective interfering [DI] RNA revealed that this segment contains numerous functionally relevant structural features. In vitro solution structure probing along with comparative and computer-aided RNA secondary structure analyses predicted the presence of a simple stem loop (SL5) followed by a more complex downstream domain (DSD). Both structures were found to be essential for efficient DI RNA accumulation when tested in a plant protoplast system. For SL5, maintenance of the base of its stem was the principal feature required for robust in vivo accumulation. In the DSD, both helical and unpaired regions containing conserved sequences were necessary for efficient DI RNA accumulation. Additionally, optimal DI RNA accumulation required a TSD–DSD interaction mediated by a pseudoknot. Modifications that reduced accumulation did not appreciably affect DI RNA stability in vivo, indicating that the DSD and SL5 act to facilitate viral RNA replication. Keywords: RNA replication; RNA structure; Tombusvirus; (+)-strand RNA virus; plant virus; Tombusviridae; TBSV; DI RNA INTRODUCTION 1994; Bink et al. 2002; Sasaki and Taniguchi 2003). Given the importance and range of functions for viral 5Ј UTRs, it The genomes of (+)-strand RNA viruses are involved in is not surprising that modifications in this region can mark- many processes during viral infections. For many viruses, edly influence viral pathogenicity (Sarnow2003). RNA elements involved in one or more process have been Many aspects of viral reproduction have been studied in identified within the 5Ј untranslated regions (UTR) of their members of the family Tombusviridae (Russo et al. 1994). genomes. For instance, several diverse plant and animal Tomato bushy stunt virus (TBSV), the prototype member of (+)-strand RNA viruses contain RNA structures in this re- this family, possesses a 4.8-kb (+)-strand RNA genome that gion that facilitate viral protein translation (Gallie 2001; encodes five proteins (Fig. 1A; Hearne et al. 1990). These Guo et al. 2001; Vagner et al. 2001), genome replication proteins are translated via a 5Ј-cap- and 3Ј-poly(A)-inde- (Andino et al. 1990; Miller et al. 1998; Chen et al. 2001; pendent mechanism (Wu and White 1999). Productive ge- Mason et al. 2002; Luo et al. 2003) or subgenomic (sg) nome replication requires viral protein p33 and its transla- mRNA transcription (van Marle et al. 1999). In both fila- tional readthrough product p92 (the RNA-dependent RNA mentous and icosahedral viruses, RNA encapsidation sig- polymerase [RdRp]; Oster et al. 1998). In addition to these nals that bind capsid protein and promote virus particle viral proteins (and unidentified host factors), this process assembly have been identified in their 5Ј UTRs (Sit et al. uses RNA signals within the viral template to promote and regulate replication—a process that proceeds through a Reprint requests to: K. AndrewWhite, Department of Biology, York (−)-strand RNA intermediate (Buck 1996). University, 4700 Keele Street, Toronto, Ontario, Canada M3J 1P3; e-mail: For TBSV, viral RNA replication has been studied exten- [email protected]; fax: (416) 736-5698. Article and publication are at http://www.rnajournal.org/cgi/doi/ sively using defective interfering (DI) RNAs (White and 10.1261/rna.5630203. Morris 1994a,b; Chang et al. 1995; Wu and White 1998; Ray 1232 RNA (2003), 9:1232–1245. Published by Cold Spring Harbor Laboratory Press. Copyright © 2003 RNA Society. Downloaded from rnajournal.cshlp.org on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press A second RNA domain in the TBSV 5؅ UTR when the 5Ј UTR is present, mutations within this region act in a dominant-negative manner (Wu et al. 2001). In this study, we used a TBSV DI RNA to define a struc- ture–function model for the previously uncharacterized 3Ј portion of the 5Ј UTR. This region was found to contain two major structural elements, a simple stem–loop (SL), called SL5, and a more elaborate structure, termed the downstream domain (DSD). Formation of both SL5 and helical regions within the DSD were very important for DI RNA accumulation. Additionally, conserved unpaired se- quences within the DSD contributed substantially to DI RNA amplification, and optimal accumulation required formation of a pseudoknot between the TSD and DSD. The results confirm an important role for structures in the 3Ј half of the 5Ј UTR in viral RNA replication. RESULTS FIGURE 1. (A) The TBSV RNA genome. The genome is represented by a thick black line with coding regions depicted as boxes with ap- RNA secondary structure of the 3؅ half of the TBSV proximate molecular masses (in kDa) of the encoded proteins. Two ؅ subgenomic mRNAs produced during infection are shown as arrows 5 UTR above the genome. (B) A prototypical TBSV DI RNA (DI-72SXP). Previously, we investigated the structure of the 5Ј half of the Shaded boxes represent TBSV genomic segments present in DI RNA, Ј whereas black lines represent genomic segments that are absent. TBSV 5 UTR (coordinates 1–78) and provided evidence (C) Expanded linear representation of the TBSV 5Ј UTR. The T- that it folds into a functionally relevant TSD (Fig. 1D; Wu shaped domain (TSD; black) was defined previously (Wu et al. 2001). et al. 2001). In the present study, we have extended our Elements defined in this study are stem–loop 5 (SL5; light gray) and structural analysis to the 3Ј half of the 5Ј UTR (coordinates the downstream domain (DSD; dark gray). Coordinates correspond- ing to the boundaries of each of the three elements are provided. 79–169). Solution structure probing of this region, within (D) RNA secondary structure model for the TBSV TSD (Wu et al. the context of a prototypical DI RNA, predicts that it adopts 2001). RNA stem (S) and loop (L) structures are labeled and coordi- two distinct helical regions, both of which are supported by nates are given at the beginning and end of the sequence. MFOLD analysis (Fig. 2). At the extreme 5Ј end of this segment, a relatively large hairpin, termed SL5, is predicted. This hairpin is separated from a more complex downstream and White 1999, 2003; Nagy and Pogany 2000; Panavas et helical region by a single-stranded (ss) intervening sequence al. 2002a,b; Panavas and Nagy 2003). DI RNAs are genome- (is), defined as is5/6. The second helical region consists of a derived deletion mutants that are noncoding but maintain lower helix (S6) separated by a 12-nt bulge (B2) from an important RNA replication elements that allowthem to be upper helix (S7). S7 is interrupted by a single A-bulge and amplified when p33 and p92 are provided in trans (White is capped by a UNCG-type super-stable tetraloop (L7). The 1996). Thus, when coinoculated with the wild-type (wt) 3Ј-proximal sequence, which ends with the start codon for TBSV genome, DI RNAs are replicated very efficiently and p33/92, has been termed sequence 8 (s8) and, collectively, provide convenient model templates to study cis-acting se- is5/6, S6, B1, B2, SL7, and s8 define the downstream do- quences involved specifically in replication. main (DSD; Fig. 2). Solution structure analysis of the se- A prototypical TBSV DI RNA is comprised of four re- quence encompassing SL5 and the DSD revealed reactivities gions (RI through RIV) derived from noncontiguous seg- of single-strand (ss)-specific chemicals (CMCT and DEPC) ments within the viral genome (Fig. 1B; White 1996). RI and a ribonuclease (RNase T1) with predicted unpaired corresponds to the TBSV 5Ј UTR and includes the start residues. These include strong modifications of L5, is5/6, codon for p33/p92. In vitro studies have shown that the L7, and B2 (Fig. 2). Additionally, weaker modifications were core promoter for plus-strand synthesis is contained in the also observed for the bulged adenylate comprising B1 and complement of this region and corresponds to the 3Ј-ter- also at the 5Ј end of s8. The reactivities seen within S5, S6, minal 11 nt in the (−)-strand (Panavas et al. 2002a). Other and the lower half of S7 are likely the result of transient in vivo studies have shown that the 5Ј-proximal portion of disruption of these helices at less stable locales. Interest- the 5Ј UTR forms a T-shaped domain (TSD) in the (+)- ingly, no significant modification of the majority of s8 was strand and that this structure is crucial for DI RNA repli- observed, indicating that this region may interact with other cation (Fig. 1C,D; Wu et al. 2001). Interestingly, DI RNAs RNA sequences. In contrast, the extended unpaired seg- lacking the entire 5Ј UTR are viable and accumulate to ments in L5, is5/6, and B2, as well as the single A-bulge in ∼10% that of wild type (Wu and White 1998).