An RNA domain within the 5' untranslated region of the tomato bushy stunt virus genome modulates viral RNA replication

The terminal half of the 5' untranslated region (UTR) in the (+)-strand RNA genome of tomato bushy stunt virus was analyzed for possible roles in viral RNA replication. Computer-aided thermodynamic analysis of secondary structure, phylogenetic comparisons for base-pair covariation, and chemical and enzymatic solution structure probing were used to analyze the 78 nucleotide long 5'-terminal sequence. The results indicate that this sequence adopts a branched secondary structure containing a three-helix junction core. The T-shaped domain (TSD) formed by this terminal sequence is closed by a prominent ten base-pair long helix, termed stem 1 (S1). Deletion of either the 5' or 3' segment forming S1 (coordinates 1-10 or 69-78, respectively) in a model subviral RNA replicon, i.e. a prototypical defective interfering (DI) RNA, reduced in vivo accumulation levels of this molecule approximately 20-fold. Compensatory-type mutational analysis of S1 within this replicon revealed a strong correlation between formation of the predicted S1 structure and efficient DI RNA accumulation. RNA decay studies in vivo did not reveal any notable changes in the physical stabilities of DI RNAs containing disrupted S1s, thus implicating RNA replication as the affected process. Further investigation revealed that destabilization of S1 in the (+)-strand was significantly more detrimental to DI RNA accumulation than (-)-strand destabilization, therefore S1-mediated activity likely functions primarily via the (+)-strand. The essential role of S1 in DI RNA accumulation prompted us to examine the 5'-proximal secondary structure of a previously identified mutant DI RNA, RNA B, that lacks the 5' UTR but is still capable of low levels of replication. Mutational analysis of a predicted S1-like element present within a cryptic 5'-terminal TSD confirmed the importance of the former in RNA B accumulation. Collectively, these data support a fundamental role for the TSD, and in particular its S1 subelement, in tombusvirus RNA replication.     

Sequence and structural elements at the 3' terminus of bovine viral diarrhea virus genomic RNA: functional role during RNA replication

Bovine viral diarrhea virus (BVDV), a member of the genus Pestivirus in the family Flaviviridae, has a positive-stranded RNA genome consisting of a single open reading frame and untranslated regions (UTRs) at the 5' and 3' ends. Computer modeling suggested the 3' UTR comprised single-stranded regions as well as stem-loop structures-features that were suspected of being essentially implicated in the viral RNA replication pathway. Employing a subgenomic BVDV RNA (DI9c) that was shown to function as an autonomous RNA replicon (S.-E. Behrens, C. W. Grassmann, H. J. Thiel, G. Meyers, and N. Tautz, J. Virol. 72:2364-2372, 1998) the goal of this study was to determine the RNA secondary structure of the 3' UTR by experimental means and to investigate the significance of defined RNA motifs for the RNA replication pathway. Enzymatic and chemical structure probing revealed mainly the conserved terminal part (termed 3'C) of the DI9c 3' UTR containing distinctive RNA motifs, i.e., a stable stem-loop, SL I, near the RNA 3' terminus and a considerably less stable stem-loop, SL II, that forms the 5' portion of 3'C. SL I and SL II are separated by a long single-stranded intervening sequence, denoted SS. The 3'-terminal four C residues of the viral RNA were confirmed to be single stranded as well. Other intramolecular interactions, e.g., with upstream DI9c RNA sequences, were not detected under the experimental conditions used. Mutagenesis of the DI9c RNA demonstrated that the SL I and SS motifs do indeed play essential roles during RNA replication. Abolition of RNA stems, which ought to maintain the overall folding of SL I, as well as substitution of certain single-stranded nucleotides located in the SS region or SL I loop region, gave rise to DI9c derivatives unable to replicate. Conversely, SL I stems comprising compensatory base exchanges turned out to support replication, but mostly to a lower degree than the original structure. Surprisingly, replacement of a number of residues, although they were previously defined as constituents of a highly conserved stretch of sequence of the SS motif, had little effect on the replication ability of DI9c. In summary, these results indicate that RNA structure as well as sequence elements harbored within the 3'C region of the BVDV 3' UTR create a common cis-acting element of the replication process. The data further point at possible interaction sites of host and/or viral proteins and thus provide valuable information for future experiments intended to identify and characterize these factors.     

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.     

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.     

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.     

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.     

Sequences at the 3' untranslated region of bamboo mosaic potexvirus RNA interact with the viral RNA-dependent RNA polymerase

The 3' untranslated region (UTR) of bamboo mosaic potexvirus (BaMV) genomic RNA was found to fold into a series of stem-loop structures including a pseudoknot structure. These structures were demonstrated to be important for viral RNA replication and were believed to be recognized by the replicase (C.-P. Cheng and C.-H. Tsai, J. Mol. Biol. 288:555-565, 1999). Electrophoretic mobility shift and competition assays have now been used to demonstrate that the Escherichia coli-expressed RNA-dependent RNA polymerase domain (Delta 893) derived from BaMV open reading frame 1 could specifically bind to the 3' UTR of BaMV RNA. No competition was observed when bovine liver tRNAs or poly(I)(C) double-stranded homopolymers were used as competitors, and the cucumber mosaic virus 3' UTR was a less efficient competitor. Competition analysis with different regions of the BaMV 3' UTR showed that Delta 893 binds to at least two independent RNA binding sites, stem-loop D and the poly(A) tail. Footprinting analysis revealed that Delta 893 could protect the sequences at loop D containing the potexviral conserved hexamer motif and part of the stem of domain D from chemical cleavage.     

The combined effect of environmental and host factors on the emergence of viral RNA recombinants

Viruses are masters of evolution due to high frequency mutations and genetic recombination. In spite of the significance of viral RNA recombination that promotes the emergence of drug-resistant virus strains, the role of host and environmental factors in RNA recombination is poorly understood. Here we report that the host Met22p/Hal2p bisphosphate-3'-nucleotidase regulates the frequency of viral RNA recombination and the efficiency of viral replication. Based on Tomato bushy stunt virus (TBSV) and yeast as a model host, we demonstrate that deletion of MET22 in yeast or knockdown of AHL, SAL1 and FRY1 nucleotidases/phosphatases in plants leads to increased TBSV recombination and replication. Using a cell-free TBSV recombination/replication assay, we show that the substrate of the above nucleotidases, namely 3'-phosphoadenosine-5'-phosphate pAp, inhibits the activity of the Xrn1p 5'-3' ribonuclease, a known suppressor of TBSV recombination. Inhibition of the activity of the nucleotidases by LiCl and NaCl also leads to increased TBSV recombination, demonstrating that environmental factors could also affect viral RNA recombination. Thus, host factors in combination with environmental factors likely affect virus evolution and adaptation.     

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 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.     

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.     

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 single-stranded loop in the 5' untranslated region of cucumber mosaic virus RNA 4 contributes to competitive translational activity

The 5' untranslated region (UTR) of cucumber mosaic virus (CMV) RNA 4 confers a highly competitive translational advantage on a heterologous luciferase open reading frame. Here we investigated whether secondary structure in the 5' UTR contributes to this translational advantage. Stabilization of the 5' UTR RNA secondary structure inhibited competitive translational activity. Alteration of a potential single-stranded loop to a stem by substitution mutations greatly inhibited the competitive translational activity. Tobacco plants infected with wild type virus showed a 2.5-fold higher accumulation of maximal coat protein than did plants infected with a loop-mutant virus. Amplification of viral RNA in these plants could not explain the difference in accumulation of coat protein. Phylogenetic comparison showed that potential single-stranded loops of 12-23 nucleotides in length exist widely in subgroups of CMV.     

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.     

Tomato bushy stunt virus co-opts the RNA-binding function of a host metabolic enzyme for viral genomic RNA synthesis

Tomato bushy stunt virus (TBSV), a plus-stranded [(+)] RNA plant virus, incorporates the host metabolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the viral replicase complex. Here, we show that, during TBSV replication in yeast, the yeast GAPDH Tdh2p moves from the cytosol to the peroxisomal membrane surface, the site of viral RNA synthesis. In yeast cells lacking Tdh2p, decreasing the levels of its functionally redundant homolog Tdh3p inhibited TBSV replication and resulted in equivalent levels of (+) and minus-stranded [(-)] viral RNA, in contrast to the hallmark excess of (+)RNA. Tdh2p specifically bound an AU pentamer sequence in the (-)RNA, suggesting that GAPDH promotes asymmetric RNA synthesis by selectively retaining the (-)RNA template in the replicase complex. Downregulation of GAPDH in a natural plant host decreased TBSV genomic RNA accumulation. Thus, TBSV co-opts the RNA-binding function of a metabolic protein, helping convert the host cell into a viral factory.     

RNA Polymerase Activity and Specific RNA Structure Are Required for Efficient HCV Replication in Cultured Cells

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3′X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3′ untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3′X region. We next analyzed the 3′ structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3′UTR were required for J6CF replication in cultured cells.     

Role of the 5' TAR stem--loop and the U5-AUG duplex in dimerization of HIV-1 genomic RNA

The HIV-1 genome consists of two identical RNAs that are linked together through noncovalent interactions involving nucleotides from the 5' untranslated region (5' UTR) of each RNA strand. The 5' UTR is the most conserved part of the HIV-1 RNA genome, and its 335 nucleotide residues form regulatory motifs that mediate multiple essential steps in the viral replication cycle. Here, studying the effect of selected mutations both singly and together with mutations disabling SL1 (SL1 is a 5' UTR stem-loop containing a palindrome called the dimerization initiation site), we have done a rather systematic survey of the 5' UTR requirements for full genomic RNA dimerization in grown-up (i.e., predominantly >/=10 h old) HIV-1 viruses produced by transfected human and simian cells. We have identified a role for the 5' transactivation response element (5' TAR) and a contribution of a long-distance base pairing between a sequence located at the beginning of the U5 region and nucleotides surrounding the AUG Gag initiation codon. The resulting intra- or intermolecular duplex is called the U5-AUG duplex. The other regions of the 5' UTR have been shown to play no systematic role in genomic RNA dimerization, except for a sequence located around the 3' end of a large stem-loop enclosing the primer binding site, and the well-documented SL1. Our data are consistent with a direct role for the 5' TAR in genomic RNA dimerization (possibly via a palindrome encompassing the apical loop of the 5' TAR).     

A potential mechanism for selective control of cap-independent translation by a viral RNA sequence in cis and in trans.

Highly efficient cap-independent translation initiation at the 5'-proximal AUG is facilitated by the 3' translation enhancer sequence (3'TE) located near the 3' end of barley yellow dwarf virus (BYDV) genomic RNA. The role of the 3'TE in regulating viral translation was examined. The 3'TE is required for translation and thus replication of the genomic RNA that lacks a 5' cap (Allen et al., 1999, Virology253:139-144). Here we show that the 3'TE also mediates translation of uncapped viral subgenomic mRNAs (sgRNA1 and sgRNA2). A 109-nt viral sequence is sufficient for 3'TE activity in vitro, but additional viral sequence is necessary for cap-independent translation in vivo. The 5' extremity of the sequence required in the 3' untranslated region (UTR) for cap-independent translation in vivo coincides with the 5' end of sgRNA2. Thus, sgRNA2 has the 3'TE in its 5' UTR. Competition studies using physiological ratios of viral RNAs showed that, in trans, the 109-nt 3'TE alone, or in the context of 869-nt sgRNA2, inhibited translation of genomic RNA much more than it inhibited translation of sgRNA1. The divergent 5' UTRs of genomic RNA and sgRNA1 contribute to this differential susceptibility to inhibition. We propose that sgRNA2 serves as a novel regulatory RNA to carry out the switch from early to late gene expression. Thus, this new mechanism for temporal control of translation control involves a sequence that stimulates translation in cis and acts in trans to selectively inhibit translation of viral mRNA.     

Selective repression of translation by the brome mosaic virus 1a RNA replication protein

Differential expression of viral replication proteins is essential for successful infection. We report here that overexpression of the brome mosaic virus (BMV) 1a protein can repress viral RNA replication in a dosage-dependent manner. Using RNA replication-incompetent reporter constructs, repression of translation from BMV RNA1 and RNA2 was observed, suggesting that the effect on translation of the BMV RNA replication proteins is responsible for the decrease in RNA levels. Furthermore, repression of translation by 1a required the B box in the 5'-untranslated region (5' UTR); BMV RNA3 that lacks a B box in its 5' UTR is not subject to 1a-mediated translational inhibition. Mutations in either the methyltransferase or the helicase-like domains of 1a reduced the repression of replication and translation. These results suggest that in addition to its known functions in BMV RNA synthesis, 1a also regulates viral gene expression.