Conformational organization of the 3' untranslated region in the tomato bushy stunt virus genome

The 3' untranslated regions (UTRs) of positive-strand RNA viruses often form complex structures that facilitate various viral processes. We have examined the RNA conformation of the 352 nucleotide (nt) long 3' UTR of the Tomato bushy stunt virus (TBSV) genome with the goal of defining both local and global structures that are important for virus viability. Gel mobility analyses of a 3'-terminal 81 nt segment of the 3' UTR revealed that it is able to form a compact RNA domain (or closed conformation) that is stabilized by a previously proposed tertiary interaction. RNA-RNA gel shift assays were used to provide the first physical evidence for the formation of this tertiary interaction and revealed that it represents the dominant or "default" structure in the TBSV genome. Further analysis showed that the tertiary interaction involves five base pairs, each of which contributes differently to overall complex stability. Just upstream from the 3'-terminal domain, a long-distance RNA-RNA interaction involving 3' UTR sequences was found to be required for efficient viral RNA accumulation in vivo and to also contribute to the formation of the 3'-terminal domain in vitro. Collectively, these results provide a comprehensive overview of the conformational and functional organization of the 3' UTR of the TBSV genome.     

A second functional RNA domain in the 5' UTR of the Tomato bushy stunt virus genome: intra- and interdomain interactions mediate viral RNA replication

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.     

Structural properties of a multifunctional T-shaped RNA domain that mediate efficient tomato bushy stunt virus RNA replication

In positive-strand RNA viruses, 5' untranslated regions (5' UTRs) mediate many essential viral processes, including genome replication. Previously, we proposed that the 5'-terminal portion of the genomic leader sequence of Tomato bushy stunt virus (TBSV) forms an RNA structure containing a 3-helix junction, termed the T-shaped domain (TSD). In the present study, we have carried out structure-function analysis of the proposed TSD and have confirmed an important role for this domain in mediating efficient viral RNA amplification. Using a model TBSV defective interfering RNA replicon and a protoplast system, we demonstrated that various TSD subelements contribute to the efficiency of viral RNA replication. In particular, the stabilities of all three stems (S1, S2, and S4) forming the 3-helix junction are important, while stem-loop 3-a terminal extension of S2-is largely dispensable. Additionally, some of the sequences forming the 3-helix junction are required in an identity-dependent manner. Thus, both secondary structure and nucleotide identity are important for TSD-mediated viral RNA replication. Importantly, these results are fully consistent with the dual functions we defined previously for the sequences corresponding to loops 3 and 4, respectively, in facilitating 5' cap- and 3' poly(A) tail-independent translation of the genome by forming a loop-loop interaction with the 3'-proximal translational enhancer and in mediating viral RNA replication through formation of a pseudoknot with the adjacent downstream RNA domain. Also, since comparable TSDs and associated interactions are predicted in the 5' UTRs of all sequenced Aureusvirus genomes, members of at least one other genus in the family Tombusviridae appear to utilize this type of multifunctional RNA domain.     

On the rigidity of RNA in tomato bushy stunt virus

The motional state of RNA in tomato bushy stunt virus, both in the crystalline state and in solution, has been investigated using ³¹P nuclear magnetic resonance methods. It has been found that the RNA is highly immobile in the native virus and it is suggested that the lack of a high-resolution X-ray diffraction pattern for either the RNA or the N-terminal regions of the protein coat molecules (Harrison et al., 1978) is due to static disorder in the crystals. Dynamic disorder has been detected in the virus after treatment with EDTA, which causes a structural change and an increase in particle size.     

An internally located RNA hairpin enhances replication of Tomato bushy stunt virus RNAs

Defective interfering (DI) RNAs of Tomato bushy stunt virus (TBSV), a plus-sense RNA virus, comprise four conserved noncontiguous regions (I through IV) derived from the viral genome. Region III, a 70-nucleotide-long sequence corresponding to a genomic segment located 378 nucleotides upstream of the 3' terminus of the genome, has been found to enhance DI RNA accumulation by approximately 10-fold in an orientation-independent manner (D. Ray and K. A. White, Virology 256:162-171, 1999). In this study, a more detailed structure-function analysis of region III was conducted. RNA secondary-structure analyses indicated that region III contains stem-loop structures in both plus and minus strands. Through deletion analyses of a DI RNA, a primary determinant of region III activity was mapped to the 5'-proximal 35-nucleotide segment. Compensatory-type mutational analyses showed that a stem-loop structure in the minus strand of this subregion was required for enhanced DI RNA replication. The same stem-loop structure was also found to function in a position-independent manner in a DI RNA (albeit at reduced levels) and to be important for efficient accumulation within the context of the TBSV genome. Taken together, these observations suggest that the 5'-proximal segment of region III is a modular RNA replication element that functions primarily through the formation of an RNA hairpin structure in the minus strand.     

Poly(C)-binding protein 2 interacts with sequences required for viral replication in the hepatitis C virus (HCV) 5' untranslated region and directs HCV RNA replication through circularizing the viral genome

Sequences in the 5' untranslated region (5'UTR) of hepatitis C virus (HCV) RNA is important for modulating both translation and RNA replication. The translation of the HCV genome depends on an internal ribosome entry site (IRES) located within the 341-nucleotide 5'UTR, while RNA replication requires a smaller region. A question arises whether the replication and translation functions require different regions of the 5'UTR and different sets of RNA-binding proteins. Here, we showed that the 5'-most 157 nucleotides of HCV RNA is the minimum 5'UTR for RNA replication, and it partially overlaps with the IRES. Stem-loops 1 and 2 of the 5'UTR are essential for RNA replication, whereas stem-loop 1 is not required for translation. We also found that poly(C)-binding protein 2 (PCBP2) bound to the replication region of the 5'UTR and associated with detergent-resistant membrane fractions, which are the sites of the HCV replication complex. The knockdown of PCBP2 by short hairpin RNA decreased the amounts of HCV RNA and nonstructural proteins. Antibody-mediated blocking of PCBP2 reduced HCV RNA replication in vitro, indicating that PCBP2 is directly involved in HCV RNA replication. Furthermore, PCBP2 knockdown reduced IRES-dependent translation preferentially from a dual reporter plasmid, suggesting that PCBP2 also regulated IRES activity. These findings indicate that PCBP2 participates in both HCV RNA replication and translation. Moreover, PCBP2 interacts with HCV 5'- and 3'UTR RNA fragments to form an RNA-protein complex and induces the circularization of HCV RNA, as revealed by electron microscopy. This study thus demonstrates the mechanism of the participation of PCBP2 in HCV translation and replication and provides physical evidence for HCV RNA circularization through 5'- and 3'UTR interaction.     

A stem-loop motif formed by the immediate 5' terminus of the bovine viral diarrhea virus genome modulates translation as well as replication of the viral RNA

Bovine viral diarrhea virus (BVDV), a Pestivirus member of the Flaviviridae family, has a positive-stranded RNA genome which consists of a single open reading frame (ORF) and untranslated regions (UTRs) at the 5' and 3' ends. The 5' UTR harbors extensive RNA structure motifs; most of them were shown to contribute to an internal ribosomal entry site (IRES), which mediates cap-independent translation of the ORF. The extreme 5'-terminal region of the BVDV genome had so far been believed not to be required for IRES function. By structure probing techniques, we initially verified the existence of a computer-predicted stem-loop motif at the 5' end of the viral genome (hairpin Ia) as well as at the 3' end of the complementary negative-strand replication intermediate [termed hairpin Ia (-)]. While the stem of this structure is mainly constituted of nucleotides that are conserved among pestiviruses, the loop region is predominantly composed of variable residues. Taking a reverse genetics approach to a subgenomic BVDV replicon RNA (DI9c) which could be equally employed in a translation as well as replication assay system based on BHK-21 cells, we obtained the following results. (i) Proper folding of the Ia stem was found to be crucial for efficient translation. Thus, in the context of an authentic replication-competent viral RNA, the 5'-terminal motif operates apparently as an integral functional part of the ribosome entry. (ii) An intact loop structure and a stretch of nucleotide residues that constitute a portion of the stem of the Ia or the Ia (-) motif, respectively, were defined to represent important determinants of the RNA replication pathway. (iii) Formation of the stem structure of the Ia (-) motif was determined to be not critical for RNA replication. In summary, our findings affirmed that the 5'-terminal region of the BVDV genome encodes a bifunctional secondary structure motif which may enable the viral RNA to switch from the translation to the replicative cycle and vice versa.     

The 3' untranslated region of picornavirus RNA: features required for efficient genome replication.

The role of the 3' untranslated region (3'UTR) in the replication of enteroviruses has been studied with a series of mutants derived from either poliovirus type 3 (PV3) or a PV3 replicon containing the reporter gene chloramphenicol acetyltransferase. Replication was observed when the PV3 3'UTR was replaced with that of either coxsackie B4 virus, human rhinovirus 14 (HRV14), bovine enterovirus, or hepatitis A virus, despite the lack of sequence and secondary structure homology of the 3'UTRs of these viruses. The levels of replication observed for recombinants containing the 3'UTRs of hepatitis A virus and bovine enterovirus were lower than those for PV3 and the other recombinants. Extensive site-directed mutagenesis of the single stem-loop structure formed by the HRV14 3'UTR indicated the importance of (i) the loop sequence, (ii) the stability of the stem, and (iii) the location of the stem immediately upstream of the poly(A) tail. The role of a 4-bp motif at the base of the HRV14 stem, highly conserved among rhinoviruses, was examined by site-directed mutagenesis of individual base pairs. This analysis did not pinpoint a particular base pair as crucial for function. The requirement for immediate adjacent positioning of the open reading frame and the 3'UTR was examined by insertion of a 1.1-kb heterologous sequence. A replicon containing this insert replicated to about 30% of the level observed for the wild type. However, the corresponding virus consistently deleted most of the inserted fragment, suggesting that its presence was incompatible with a full replication cycle.     

Role of the 5'-proximal stem-loop structure of the 5' untranslated region in replication and translation of hepatitis C virus RNA

Sequences of the untranslated regions at the 5' and 3' ends (5'UTR and 3'UTR) of the hepatitis C virus (HCV) RNA genome are highly conserved and contain cis-acting RNA elements for HCV RNA replication. The HCV 5'UTR consists of two distinct RNA elements, a short 5'-proximal stem-loop RNA element (nucleotides 1 to 43) and a longer element of internal ribosome entry site. To determine the sequence and structural requirements of the 5'-proximal stem-loop RNA element in HCV RNA replication and translation, a mutagenesis analysis was preformed by nucleotide deletions and substitutions. Effects of mutations in the 5'-proximal stem-loop RNA element on HCV RNA replication were determined by using a cell-based HCV replicon replication system. Deletion of the first 20 nucleotides from the 5' end resulted in elimination of cell colony formation. Likewise, disruption of the 5'-proximal stem-loop by nucleotide substitutions abolished the ability of HCV RNA to induce cell colony formation. However, restoration of the 5'-proximal stem-loop by compensatory mutations with different nucleotides rescued the ability of the subgenomic HCV RNA to replicate in Huh7 cells. In addition, deletion and nucleotide substitutions of the 5'-proximal stem-loop structure, including the restored stem-loop by compensatory mutations, all resulted in reduction of translation by two- to fivefold, suggesting that the 5'-proximal stem-loop RNA element also modulates HCV RNA translation. These findings demonstrate that the 5'-proximal stem-loop of the HCV RNA is a cis-acting RNA element that regulates HCV RNA replication and translation.     

Natural infection of sugar beet with tomato bushy stunt virus

A virus transmissible toChenopodium quinoa was isolated from leaves of sugar beet showing large chlorotic ring spots and line pattern. The virus was serologically unrelated to tobacco necrosis virus and tomato black ring virus or to its beet ringspot strain either. A positive result was obtained with antiserum against tomato bushy stunt virus. Reactions of herbaceous indicators and properties of the virus in crude sap were in accordance with the serological diagnosis. A survey of natural hosts of tomato bushy stunt virus demonstrated recently by the authors is given.     

Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.     

Studies on the occurrence of tomato bushy stunt virus in English rivers

Tomato bushy stunt virus (TBSV) of unknown source was isolated from water of the River Thames, near Oxford. The isolate designated TBSV-T was mechanically transmissible to several tomato (Lycopersicon esculentum) cvs and to other species including Petunia hybrida, pepper (Capsicum annuum). eggplant (Solanum melongena), Nicotiana clevelandii, Chenopodium amaranticolor and C. quinoa in which it caused systemic symptoms. It caused no infection of globe artichoke (Cynara scolymus) or Pelargonium domesticum. The virus was not adsorbed to soil and could be isolated from leachate of soil in which systemically-infected tomato or C. quinoa plants were grown. Tomato plants became infected when grown in soil watered with virus suspensions. TBSV-T was infective after 10 min at 80°C but not at 90°C and when diluted to 10^-5 but not to 10^-6. Purified virus preparations contained C. 30 nm isometric particles. In gel-diffusion serological tests, TBSV-T reacted with homologous anti-serum and with antiserum to petunia asteroid mosaic virus but not to pelargonium leaf curl virus. Seed-borne infection (50–65%) of TBSV was demonstrated in plants grown from seed of symptomlessly-infected tomato fruit. TBSV was isolated from symptomlessly-infected tomato fruit imported from Morocco during October-April 1981. One of the isolates (TBSV-M) was indistinguishable from TBSV-T in host range, symptomatology and serological reactions. TBSV was also found in tomato plants growing extraneously in primary settlement beds at sewage works; such plants having been derived from undigested seeds in sewage. Because of its ‘alimentary-resistance’ in man, it is possible that one ecological route whereby TBSV enters rivers is by man's consumption of TBSV-infected tomatoes and eventual sewage dispersal into rivers.     

Authentic replication and recombination of Tomato bushy stunt virus RNA in a cell-free extract from yeast

To study the replication of Tomato bushy stunt virus (TBSV), a small tombusvirus of plants, we have developed a cell-free system based on a Saccharomyces cerevisiae extract. The cell-free system was capable of performing a complete replication cycle on added plus-stranded TBSV replicon RNA (repRNA) that led to the production of approximately 30-fold-more plus-stranded progeny RNAs than the minus-stranded replication intermediate. The cell-free system also replicated the full-length TBSV genomic RNA, which resulted in production of subgenomic RNAs as well. The cell-free system showed high template specificity, since a mutated repRNA, minus-stranded repRNA, or a heterologous viral RNA could not be used as templates by the tombusvirus replicase. Similar to the in vivo situation, replication of the TBSV replicon RNA took place in a membraneous fraction, in which the viral replicase-RNA complex was RNase and protease resistant but sensitive to detergents. In addition to faithfully replicating the TBSV replicon RNA, the cell-free system was also capable of generating TBSV RNA recombinants with high efficiency. Altogether, tombusvirus replicase in the cell-free system showed features remarkably similar to those of the in vivo replicase, including carrying out a complete cycle of replication, high template specificity, and the ability to recombine efficiently.     

Rubella virus capsid protein modulates viral genome replication and virus infectivity

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.