Long-range RNA-RNA interactions circularize the dengue virus genome

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.     

Genetic evidence of a long-range RNA-RNA interaction between the genomic 5' untranslated region and the nonstructural protein 1 coding region in murine and bovine coronaviruses

Higher-order RNA structures in the 5' untranslated regions (UTRs) of the mouse hepatitis coronavirus (MHV) and bovine coronavirus (BCoV), separate species in the betacoronavirus genus, appear to be largely conserved despite an ～36% nucleotide sequence divergence. In a previous study, each of three 5'-end-proximal cis-acting stem-loop domains in the BCoV genome, I/II, III, and IV, yielded near-wild-type (wt) MHV phenotypes when used by reverse genetics to replace its counterpart in the MHV genome. Replacement with the BCoV 32-nucleotide (nt) inter-stem-loop fourth domain between stem-loops III and IV, however, required blind cell passaging for virus recovery. Here, we describe suppressor mutations within the transplanted BCoV 32-nt domain that along with appearance of potential base pairings identify an RNA-RNA interaction between this domain and a 32-nt region ～200 nt downstream within the nonstructural protein 1 (Nsp1)-coding region. Mfold and phylogenetic covariation patterns among similarly grouped betacoronaviruses support this interaction, as does cotransplantation of the BCoV 5' UTR and its downstream base-pairing domain. Interestingly, cotransplantation of the BCoV 5' UTR and BCoV Nsp1 coding region directly yielded an MHV wt-like phenotype, which demonstrates a cognate interaction between these two BCoV regions, which in the MHV genome act in a fully interspecies-compliant manner. Surprisingly, the 30-nt inter-stem-loop domain in the MHV genome can be deleted and viral progeny, although debilitated, are still produced. These results together identify a previously undescribed long-range RNA-RNA interaction between the 5' UTR and Nsp1 coding region in MHV-like and BCoV-like betacoronaviruses that is cis acting for viral fitness but is not absolutely required for viral replication in cell culture.     

5'-3' RNA-RNA interaction facilitates cap- and poly(A) tail-independent translation of tomato bushy stunt virus mrna: a potential common mechanism for tombusviridae

Tomato bushy stunt virus (TBSV) is the prototypical member of the genus Tombusvirus in the family Tombusviridae. The (+)-strand RNA genome of TBSV lacks both a 5' cap and a 3' poly(A) tail and instead contains a 3'-terminal RNA sequence that acts as a cap-independent translational enhancer (3' CITE). In this study, we have determined the RNA secondary structure of the translation-specific central segment of the 3' CITE, termed region 3.5 (R3.5). MFOLD structural modeling combined with solution structure mapping and comparative sequence analysis indicate that R3.5 adopts a branched structure that contains three major helices. Deletion and substitution studies revealed that two of these extended stem-loop (SL) structures are essential for 3' CITE activity in vivo. In particular, the terminal loop of one of these SLs, SL-B, was found to be critical for translation. Compensatory mutational analysis showed that SL-B functions by base pairing with another SL, SL3, in the 5' untranslated region of the TBSV genome. Thus, efficient translation of TBSV mRNA in vivo requires a 5'-3' RNA-RNA interaction that effectively circularizes the message. Similar types of interactions are also predicted to occur in TBSV subgenomic mRNAs between their 5' untranslated regions and the 3' CITE, and both genomic and subgenomic 5'-3' interactions are well conserved in all members of the genus Tombusvirus. In addition, a survey of other genera in Tombusviridae revealed the potential for similar 5'-3' RNA-RNA-based interactions in their viral mRNAs, suggesting that this mechanism extends throughout this large virus family.     

The 5'-terminal region of the Aichi virus genome encodes cis-acting replication elements required for positive- and negative-strand RNA synthesis

Aichi virus is a member of the family Picornaviridae. It has already been shown that three stem-loop structures (SL-A, SL-B, and SL-C, from the 5' end) formed at the 5' end of the genome are critical elements for viral RNA replication. In this study, we further characterized the 5'-terminal cis-acting replication elements. We found that an additional structural element, a pseudoknot structure, is formed through base-pairing interaction between the loop segment of SL-B (nucleotides [nt] 57 to 60) and a sequence downstream of SL-C (nt 112 to 115) and showed that the formation of this pseudoknot is critical for viral RNA replication. Mapping of the 5'-terminal sequence of the Aichi virus genome required for RNA replication using a series of Aichi virus-encephalomyocarditis virus chimera replicons indicated that the 5'-end 115 nucleotides including the pseudoknot structure are the minimum requirement for RNA replication. Using the cell-free translation-replication system, we examined the abilities of viral RNAs with a lethal mutation in the 5'-terminal structural elements to synthesize negative- and positive-strand RNAs. The results showed that the formation of three stem-loops and the pseudoknot structure at the 5' end of the genome is required for negative-strand RNA synthesis. In addition, specific nucleotide sequences in the stem of SL-A or its complementary sequences at the 3' end of the negative-strand were shown to be critical for the initiation of positive-strand RNA synthesis but not for that of negative-strand synthesis. Thus, the 5' end of the Aichi virus genome encodes elements important for not only negative-strand synthesis but also positive-strand synthesis.     

Analysis of a 3'-translation enhancer in a tombusvirus: a dynamic model for RNA-RNA interactions of mRNA termini

Tomato bushy stunt virus is a (+)-strand RNA virus that is neither 5'-capped nor 3'-polyadenylated. Translation of viral proteins is instead mediated by an RNA element, the 3'-cap-independent translational enhancer (3'CITE), which is located in its 3' untranslated region (UTR). The 3'CITE is proposed to recruit the translational machinery to the viral message, while a long-distance RNA-RNA interaction between the 3'CITE and 5' UTR is thought to deliver the 43S ribosomal subunit to the 5' end of the viral mRNA. Here we provide the first evidence that the 5' UTR and 3'CITE interact physically. Mutational analysis showed that formation of this RNA-RNA interaction in vitro correlates well with efficient translation in vivo, thus supporting its functional relevance. Other analyses of the 3'CITE confirmed an overall Y-shaped RNA secondary structure and demonstrated the importance of numerous minor structural features for efficient translation of viral mRNAs. Functional studies on the role of the 5' UTR revealed that despite the absence of a cap structure, 43S subunits load at the very 5' end and scan in a 3' direction. These results indicate that the 5'-3' RNA-RNA interaction is likely disrupted by scanning ribosomal subunits and suggest a dynamic model for the interaction of mRNA termini during active translation.     

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.     

Ribosome binding to a 5' translational enhancer is altered in the presence of the 3' untranslated region in cap-independent translation of turnip crinkle virus

Plus-strand RNA viruses without 5' caps require noncanonical mechanisms for ribosome recruitment. A translational enhancer in the 3' untranslated region (UTR) of Turnip crinkle virus (TCV) contains an internal T-shaped structure (TSS) that binds to 60S ribosomal subunits. We now report that the 63-nucleotide (nt) 5' UTR of TCV contains a 19-nt pyrimidine-rich element near the initiation codon that supports translation of an internal open reading frame (ORF) independent of upstream 5' UTR sequences. Addition of 80S ribosomes to the 5' UTR reduced the flexibility of the polypyrimidine residues and generated a toeprint consistent with binding to this region. Binding of salt-washed 40S ribosomal subunits was reduced 6-fold when the pyrimidine-rich sequence was mutated. 40S subunit binding generated the same toeprint as 80S ribosomes but also additional ones near the 5' end. Generation of out-of-frame AUGs upstream of the polypyrimidine region reduced translation, which suggests that 5'-terminal entry of 40S subunits is followed by scanning and that the polypyrimidine region is needed for an alternative function that requires ribosome binding. No evidence for RNA-RNA interactions between 5' and 3' sequences was found, suggesting that TCV utilizes an alternative means for circularizing its genome. Combining 5' and 3' UTR fragments in vitro had no discernible effect on the structures of the RNAs. In contrast, when 80S ribosomes were added to both fragments, structural changes were found in the 5' UTR polypyrimidine tract that were not evident when ribosomes interacted with the individual fragments. This suggests that ribosomes can promote an interaction between the 5' and 3' UTRs of TCV.     

Specific interaction of hepatitis C virus protease/helicase NS3 with the 3'-terminal sequences of viral positive- and negative-strand RNA

The hepatitis C virus (HCV)-encoded protease/helicase NS3 is likely to be involved in viral RNA replication. We have expressed and purified recombinant NS3 (protease and helicase domains) and Delta pNS3 (helicase domain only) and examined their abilities to interact with the 3'-terminal sequence of both positive and negative strands of HCV RNA. These regions of RNA were chosen because initiation of RNA synthesis is likely to occur at or near the 3' untranslated region (UTR). The results presented here demonstrate that NS3 (and Delta pNS3) interacts efficiently and specifically with the 3'-terminal sequences of both positive- and negative-strand RNA but not with the corresponding complementary 5'-terminal RNA sequences. The interaction of NS3 with the 3'-terminal negative strand [called 3'(-) UTR(127)] was specific in that only homologous (and not heterologous) RNA competed efficiently in the binding reaction. A predicted stem-loop structure present at the 3' terminus (nucleotides 5 to 20 from the 3' end) of the negative-strand RNA appears to be important for NS3 binding to the negative-strand UTR. Deletion of the stem-loop structure almost totally impaired NS3 (and Delta pNS3) binding. Additional mutagenesis showed that three G-C pairs within the stem were critical for helicase-RNA interaction. The data presented here also suggested that both a double-stranded structure and the 3'-proximal guanosine residues in the stem were important determinants of protein binding. In contrast to the relatively stringent requirement for 3'(-) UTR binding, specific interaction of NS3 (or Delta pNS3) with the 3'-terminal sequences of the positive-strand RNA [3'(+) UTR] appears to require the entire 3'(+) UTR of HCV. Deletion of either the 98-nucleotide 3'-terminal conserved region or the 5' half sequence containing the variable region and the poly(U) and/or poly(UC) stretch significantly impaired RNA-protein interaction. The implication of NS3 binding to the 3'-terminal sequences of viral positive- and negative-strand RNA in viral replication is discussed.     

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.     

Mutagenic analysis of the 3' cis-acting elements of the rubella virus genome

Thermodynamically predicted secondary structure analysis of the 3'-terminal 305 nucleotides (nt) of the rubella virus (RUB) genome, a region conserved in all RUB defective interfering RNAs, revealed four stem-loop (SL) structures; SL1 and SL2 are both located in the E1 coding region, while SL3 and SL4 are within the 59-nt 3' untranslated region (UTR) preceding the poly(A) tract. SL2 is a structure shown to interact with human calreticulin (CAL), an autoantigen potentially involved in RUB RNA replication and pathogenesis. RNase mapping indicated that SL2 and SL3 are in equilibrium between two conformations, in the second of which the previously proposed CAL binding site in SL2, a U-U bulge, is not formed. Site-directed mutagenesis of the 3' UTR with a RUB infectious clone, Robo302, revealed that most of the 3' UTR is required for viral viability except for the 3'-terminal 5 nt and the poly(A) tract, although poly(A) was rapidly regenerated during subsequent replication. Maintenance of the overall SL3 structure, the 11-nt single-stranded sequence between SL3 and SL4, and the sequences forming SL4 were all important for viral viability. Studies on the interaction between host factors and the 3' UTR showed the formation of three RNA-protein complexes by gel mobility shift assay, and UV-induced cross-linking detected six host protein species, with molecular masses of 120, 80, 66, 55, 48, and 36 kDa, interacting with the 3' UTR. Site-directed mutagenesis of SL2 by nucleotide substitutions showed that maintenance of SL2 stem rather than the U-U bulge was critical in CAL binding since mutants having the U-U bulge base paired had a similar binding activity for CAL as the native structure whereas mutants having the SL2 stem destabilized had much lower binding activity. However, all of these mutations gave rise to viable viruses when introduced into Robo302, indicating that binding of CAL to SL2 is independent of viral viability.     

An optimal cis-replication stem-loop IV in the 5' untranslated region of the mouse coronavirus genome extends 16 nucleotides into open reading frame 1

The 288-nucleotide (nt) 3' untranslated region (UTR) in the genome of the bovine coronavirus (BCoV) and 339-nt 3' UTR in the severe acute respiratory syndrome (SARS) coronavirus (SCoV) can each replace the 301-nt 3' UTR in the mouse hepatitis coronavirus (MHV) for virus replication, thus demonstrating common 3' cis-replication signals. Here, we show that replacing the 209-nt MHV 5' UTR with the ～63%-sequence-identical 210-nt BCoV 5' UTR by reverse genetics does not yield viable virus, suggesting 5' end signals are more stringent or possibly are not strictly 5' UTR confined. To identify potential smaller, 5'-common signals, each of three stem-loop (SL) signaling domains and one inter-stem-loop domain from the BCoV 5' UTR was tested by replacing its counterpart in the MHV genome. The SLI/II domain (nucleotides 1 to 84) and SLIII domain (nucleotides 85 to 141) each immediately enabled near-wild-type (wt) MHV-like progeny, thus behaving similarly to comparable 5'-proximal regions of the SCoV 5' UTR as shown by others. The inter-stem-loop domain (nt 142 to 173 between SLs III and IV) enabled small plaques only after genetic adaptation. The SLIV domain (nt 174 to 210) required a 16-nt extension into BCoV open reading frame 1 (ORF1) for apparent stabilization of a longer BCoV SLIV (nt 174 to 226) and optimal virus replication. Surprisingly, pleiomorphic SLIV structures, including a terminal loop deletion, were found among debilitated progeny from intra-SLIV chimeras. The results show the inter-stem-loop domain to be a potential novel species-specific cis-replication element and that cis-acting SLIV in the viral genome extends into ORF1 in a manner that stabilizes its lower stem and is thus not 5' UTR confined.     

A balance between circular and linear forms of the dengue virus genome is crucial for viral replication

The plasticity of viral plus strand RNA genomes is fundamental for the multiple functions of these molecules. Local and long-range RNA-RNA interactions provide the scaffold for interacting proteins of the translation, replication, and encapsidation machinery. Using dengue virus as a model, we investigated the relevance of the interplay between two alternative conformations of the viral genome during replication. Flaviviruses require long-range RNA-RNA interactions and genome cyclization for RNA synthesis. Here, we define a sequence present in the viral 3'UTR that overlaps two mutually exclusive structures. This sequence can form an extended duplex by long-range 5'-3' interactions in the circular conformation of the RNA or fold locally into a small hairpin (sHP) in the linear form of the genome. A mutational analysis of the sHP structure revealed an absolute requirement of this element for viral viability, suggesting the need of a linear conformation of the genome. Viral RNA replication showed high vulnerability to changes that alter the balance between circular and linear forms of the RNA. Mutations that shift the equilibrium toward the circular or the linear conformation of the genome spontaneously revert to sequences with different mutations that tend to restore the relative stability of the two competing structures. We propose a model in which the viral genome exists in at least two alternative conformations and the balance between these two states is critical for infectivity.     

Structure and function of the 3' terminal six nucleotides of the west nile virus genome in viral replication

Using a self-replicating reporting replicon of West Nile (WN) virus, we performed a mutagenesis analysis to define the structure and function of the 3'-terminal 6 nucleotides (nt) (5'-GGAUCU(OH)-3') of the WN virus genome in viral replication. We show that mutations of nucleotide sequence or base pair structure of any of the 3'-terminal 6 nt do not significantly affect viral translation, but exert discrete effects on RNA replication. (i). The flavivirus-conserved terminal 3' U is optimal for WN virus replication. Replacement of the wild-type 3' U with a purine A or G resulted in a substantial reduction in RNA replication, with a complete reversion to the wild-type sequence. In contrast, replacement with a pyrimidine C resulted in a replication level similar to that of the 3' A or G mutants, with only partial reversion. (ii). The flavivirus-conserved 3' penultimate C and two upstream nucleotides (positions 78 and 79), which potentially base pair with the 3'-terminal CU(OH), are absolutely essential for viral replication. (iii). The base pair structures, but not the nucleotide sequences at the 3rd (U) and the 4th (A) positions, are critical for RNA replication. (iv). The nucleotide sequences of the 5th (G) position and its base pair nucleotide (C) are essential for viral replication. (v). Neither the sequence nor the base pair structure of the 6th nucleotide (G) is critical for WN virus replication. These results provide strong functional evidence for the existence of the 3' flavivirus-conserved RNA structure, which may function as contact sites for specific assembly of the replication complex or for efficient initiation of minus-sense RNA synthesis.     

cis-acting sequences required for coronavirus infectious bronchitis virus defective-RNA replication and packaging

The parts of the RNA genome of infectious bronchitis virus (IBV) required for replication and packaging of the RNA were investigated using deletion mutagenesis of a defective RNA (D-RNA) CD-61 (6.1 kb) containing a chloramphenicol acetyltransferase reporter gene. A D-RNA with the first 544, but not as few as 338, nucleotides (nt) of the 5' terminus was replicated; the 5' untranslated region (UTR) comprises 528 nt. Region I of the 3' UTR, adjacent to the nucleocapsid protein gene, comprised 212 nt and could be removed without impairment of replication or packaging of D-RNAs. A D-RNA with the final 338 nt, including the 293 nt in the highly conserved region II of the 3' UTR, was replicated. Thus, the 5'-terminal 544 nt and 3'-terminal 338 nt contained the necessary signals for RNA replication. Phylogenetic analysis of 19 strains of IBV and 3 strains of turkey coronavirus predicted a conserved stem-loop structure at the 5' end of region II of the 3' UTR. Removal of the predicted stem-loop structure abolished replication of the D-RNAs. D-RNAs in which replicase gene 1b-derived sequences had been removed or replaced with all the downstream genes were replicated well but were rescued poorly, suggesting inefficient packaging. However, no specific part of the 1b gene was required for efficient packaging.     

Specific interaction between the classical swine fever virus NS5B protein and the viral genome.

The NS5B protein of the classical swine fever virus (CSFV) is the RNA-dependent RNA polymerase of the virus and is able to catalyze the viral genome replication. The 3' untranslated region is most likely involved in regulation of the Pestivirus genome replication. However, little is known about the interaction between the CSFV NS5B protein and the viral genome. We used different RNA templates derived from the plus-strand viral genome, or the minus-strand viral genome and the CSFV NS5B protein obtained from the Escherichia coli expression system to address this problem. We first showed that the viral NS5B protein formed a complex with the plus-strand genome through the genomic 3' UTR and that the NS5B protein was also able to bind the minus-strand 3' UTR. Moreover, it was found that viral NS5B protein bound the minus-strand 3' UTR more efficiently than the plus-strand 3' UTR. Further, we observed that the plus-strand 3' UTR with deletion of CCCGG or 21 continuous nucleotides at its 3' terminal had no binding activity and also lost the activity for initiation of minus-strand RNA synthesis, which similarly occurred in the minus-strand 3' UTR with CATATGCTC or the 21 nucleotide fragment deleted from the 3' terminal. Therefore, it is indicated that the 3' CCCGG sequence of the plus-strand 3' UTR, and the 3' CATATGCTC fragment of the minus-strand are essential to in vitro synthesis of the minus-strand RNA and the plus-strand RNA, respectively. The same conclusion is also appropriate for the 3' 21 nucleotide terminal site of both the 3' UTRs.     

Accumulation of a 3'-terminal genome fragment in Japanese encephalitis virus-infected mammalian and mosquito cells

Japanese encephalitis virus (JEV) contains a single positive-strand RNA genome nearly 11 kb in length and is not formally thought to generate subgenomic RNA molecules during replication. Here, we report the abundant accumulation of a 3'-terminal 521- to 523-nucleotide (nt) genome fragment, representing a major portion of the 585-nt 3' untranslated region, in both mammalian (BHK-21) and mosquito (C6/36) cells infected with any of nine strains of JEV. In BHK-21 cells, the viral genome was detected as early as 24 h postinfection, the small RNA was detected as early as 28 h postinfection, and the small RNA was 0.25 to 1.5 times as abundant as the genome on a molar basis between 28 and 48 h postinfection. In C6/36 cells, the genome and small RNA were present 5 days postinfection and the small RNA was 1.25 to 5.14 times as abundant as the genome. The 3'-terminal 523-nt small RNA contains a 5'-proximal stable hairpin (nt 6 to 56) that may play a role in its formation and the conserved flavivirus 3'-cyclization motif (nt 413 to 420) and the 3'-terminal long stable hairpin structure (nt 440 to 523) that have postulated roles in genome replication. Abundant accumulation of the small RNA during viral replication in both mammalian and mosquito cells suggests that it may play a biological role, perhaps as a regulator of RNA synthesis.     

A defective interfering RNA that contains a mosaic of a plant virus genome

A symptom-modulating RNA associated with tomato bushy stunt virus (TBSV) was investigated with respect to physical and biological properties. Linear RNA of approximately 396 nucleotides was packaged in viral coat protein and was dependent on TBSV for replication. Coinoculation of the small RNA with TBSV resulted in the attenuation of TBSV-induced symptoms and depression of virus synthesis in whole plants. Nucleotide sequence analysis revealed that the symptom-modulating RNA was derived from 5', 3', and internal segments of the TBSV genome. The identification of this symptom-modulating RNA as a co-linear deletion mutant of the helper virus genome establishes it as the first definitive defective interfering RNA (DI RNA) to be identified in association with a plant virus.     

Secondary structure of the 3' terminus of hepatitis C virus minus-strand RNA

The 3'-terminal ends of both the positive and negative strands of the hepatitis C virus (HCV) RNA, the latter being the replicative intermediate, are most likely the initiation sites for replication by the viral RNA-dependent RNA polymerase, NS5B. The structural features of the very conserved 3' plus [(+)] strand untranslated region [3' (+) UTR] are well established (K. J. Blight and C. M. Rice, J. Virol. 71:7345-7352, 1997). However, little information is available concerning the 3' end of the minus [(-)] strand RNA. In the present work, we used chemical and enzymatic probing to investigate the conformation of that region, which is complementary to the 5' (+) UTR and the first 74 nucleotides of the HCV polyprotein coding sequence. By combining our experimental data with computer predictions, we have derived a secondary-structure model of this region. In our model, the last 220 nucleotides, where initiation of the (+) strand RNA synthesis presumably takes place, fold into five stable stem-loops, forming domain I. Domain I is linked to an overall less stable structure, named domain II, containing the sequences complementary to the pseudoknot of the internal ribosomal entry site in the 5' (+) UTR. Our results show that, even though the (-) strand 3'-terminal region has the antisense sequence of the 5' (+) UTR, it does not fold into its mirror image. Interestingly, comparison of the replication initiation sites on both strands reveals common structural features that may play key functions in the replication process.     

The rhinovirus type 14 genome contains an internally located RNA structure that is required for viral replication.

Cis-acting RNA signals are required for replication of positive-strand viruses such as the picornaviruses. Although these generally have been mapped to the 5' and/or 3' termini of the viral genome, RNAs derived from human rhinovirus type 14 are unable to replicate unless they contain an internal cis-acting replication element (cre) located within the genome segment encoding the capsid proteins. Here, we show that the essential cre sequence is 83-96 nt in length and located between nt 2318-2413 of the genome. Using dicistronic RNAs in which translation of the P1 and P2-P3 segments of the polyprotein were functionally dissociated, we further demonstrate that translation of the cre sequence is not required for RNA replication. Thus, although it is located within a protein-coding segment of the genome, the cre functions as an RNA entity. Computer folds suggested that cre sequences could form a stable structure in either positive- or minus-strand RNA. However, an analysis of mutant RNAs containing multiple covariant and non-covariant nucleotide substitutions within these putative structures demonstrated that only the predicted positive-strand structure is essential for efficient RNA replication. The absence of detectable minus-strand synthesis from RNAs that lack the cre suggests that the cre is required for initiation of minus-strand RNA synthesis. Since a lethal 3' noncoding region mutation could be partially rescued by a compensating mutation within the cre, the cre appears to participate in a long-range RNA-RNA interaction required for this process. These data provide novel insight into the mechanisms of replication of a positive-strand RNA virus, as they define the involvement of an internally located RNA structure in the recognition of viral RNA by the viral replicase complex. Since internally located RNA replication signals have been shown to exist in several other positive-strand RNA virus families, these observations are potentially relevant to a wide array of related viruses.