trans splicing of polycistronic Caenorhabditis elegans pre-mRNAs: analysis of the SL2 RNA

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop prevents trans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.     

The nucleotides on the stem-loop RNA structure in the junction region of the hepatitis E virus genome are critical for virus replication

The roles of conserved nucleotides on the stem-loop (SL) structure in the intergenic region of the hepatitis E virus (HEV) genome in virus replication were determined by using Huh7 cells transfected with HEV SL mutant replicons containing reporter genes. One or two nucleotide mutations of the AGA motif on the loop significantly reduced HEV replication, and three or more nucleotide mutations on the loop abolished HEV replication. Mutations on the stem and of the subgenome start sequence also significantly inhibited HEV replication. The results indicated that both the sequence and the SL structure in the junction region play important roles in HEV replication.     

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.     

The SL1 stem-loop structure at the 5'-end of potato virus X RNA is required for efficient binding to host proteins and for viral infectivity

The 5'-region of Potato virus X (PVX) RNA, which contains an AC-rich, single-stranded region and stem-loop structure 1 (SL1), affects RNA replication and assembly. Using Systemic Evolution of Ligands by EXponential enrichment (SELEX) and the electrophoretic mobility shift assay, we demonstrate that SL1 interacts specifically with tobacco protoplast protein extracts (S100). The 36 nucleotides that correspond to the top region of SL1, which comprises stem C, loop C, stem D, and the tetra loop (TL), were randomized and bound to the S100. Remarkably, the wild-type (wt) sequence was selected in the second round, and the number of wt sequences increased as selection proceeded. All of the selected clones from the fifth round contained the wt sequence. Secondary structure predictions (mFOLD) of the recovered sequences revealed relatively stable stem-loop structures that resembled SL1, although the nucleotide sequences therein were different. Moreover, many of the clones selected in the fourth round conserved the TL and C-C mismatch, which suggests the importance of these elements in host protein binding. The SELEX clone that closely resembled the wt SL1 structure with the TL and C-C mismatch was able to replicate and cause systemic symptoms in plants, while most of the other winners replicated poorly only on inoculated leaves. The RNA replication level on protoplasts was also similarly affected. Taken together, these results indicate that the SL1 of PVX interacts with host protein(s) that play important roles related to virus 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.     

Structure-function analysis of the 3' stem-loop of hepatitis C virus genomic RNA and its role in viral RNA replication

Previous studies indicate that the 3' terminal 46 nt of the RNA genome of hepatitis C virus (HCV) are highly conserved among different viral strains and essential for RNA replication. Here, we describe a mutational analysis of the 3' terminal hairpin (stem-loop I) that is putatively formed by this sequence and demonstrate its role in replication of the viral RNA. We show that single base substitutions within the 6-nt loop at positions adjacent to the stem abrogate replication of a subgenomic RNA, whereas substitutions in the three apical nucleotides were well tolerated without loss of replication competence. Single point mutations were also well tolerated within the middle section of the duplex, but not at the penultimate nucleotide positions near either end of the stem. However, complementary substitutions at the -19 and -28 positions (from the 3' end) restored replication competence, providing strong evidence for the existence of the structure and its involvement in RNA replication. This was confirmed by rescue of replicating RNAs from mutants containing complementary 10-nt block substitutions at the base of the stem. Each of these RNAs contained an additional U at the 3' terminus. Further experiments indicated a strong preference for U at the 3' terminal position (followed in order by C, A, and G), and a G at the -2 position. These features of stem-loop I are likely to facilitate recognition of the 3' end of the viral RNA by the viral RNA replicase.     

Structural lability in stem-loop 1 drives a 5' UTR-3' UTR interaction in coronavirus replication

The leader RNA of the 5′ untranslated region (UTR) of coronaviral genomes contains two stem-loop structures denoted SL1 and SL2. Herein, we show that SL1 is functionally and structurally bipartite. While the upper region of SL1 is required to be paired, we observe strong genetic selection against viruses that contain a deletion of A35, an extrahelical nucleotide that destabilizes SL1, in favor of genomes that contain a diverse panel of destabilizing second-site mutations, due to introduction of a noncanonical base pair near A35. Viruses containing destabilizing SL1-ΔA35 mutations also contain one of two specific mutations in the 3′ UTR. Thermal denaturation and imino proton solvent exchange experiments reveal that the lower half of SL1 is unstable and that second-site SL1-ΔA35 substitutions are characterized by one or more features of the wild-type SL1. We propose a “dynamic SL1” model, in which the base of SL1 has an optimized lability required to mediate a physical interaction between the 5′ UTR and the 3′ UTR that stimulates subgenomic RNA synthesis. Although not conserved at the nucleotide sequence level, these general structural characteristics of SL1 appear to be conserved in other coronaviral genomes.     

The RNA binding site of bacteriophage MS2 coat protein.

The coat protein of the RNA bacteriophage MS2 binds a specific stem-loop structure in viral RNA to accomplish encapsidation of the genome and translational repression of replicase synthesis. In order to identify the structural components of coat protein required for its RNA binding function, a series of repressor-defective mutants has been isolated. To ensure that the repressor defects were due to substitution of binding site residues, the mutant coat proteins were screened for retention of the ability to form virus-like particles. Since virus assembly presumably requires native structure, this approach eliminated mutants whose repressor defects were secondary consequences of protein folding or stability defects. Each of the variant coat proteins was purified and its ability to bind operator RNA in vitro was measured. DNA sequence analysis identified the nucleotide and amino acid substitutions responsible for reduced RNA binding affinity. Localization of the substituted sites in the three-dimensional structure of coat protein reveals that amino acid residues on three adjacent strands of the coat protein beta-sheet are required for translational repression and RNA binding. The sidechains of the affected residues form a contiguous patch on the interior surface of the viral coat.     

Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5' splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5' splice donor site, are required for efficient RNA packaging and dimerization.     

trans regulation of cap-independent translation by a viral subgenomic RNA

Many positive-strand RNA viruses generate 3'-coterminal subgenomic mRNAs to allow translation of 5'-distal open reading frames. It is unclear how viral genomic and subgenomic mRNAs compete with each other for the cellular translation machinery. Translation of the uncapped Barley yellow dwarf virus genomic RNA (gRNA) and subgenomic RNA1 (sgRNA1) is driven by the powerful cap-independent translation element (BTE) in their 3' untranslated regions (UTRs). The BTE forms a kissing stem-loop interaction with the 5' UTR to mediate translation initiation at the 5' end. Here, using reporter mRNAs that mimic gRNA and sgRNA1, we show that the abundant sgRNA2 inhibits translation of gRNA, but not sgRNA1, in vitro and in vivo. This trans inhibition requires the functional BTE in the 5' UTR of sgRNA2, but no translation of sgRNA2 itself is detectable. The efficiency of translation of the viral mRNAs in the presence of sgRNA2 is determined by proximity to the mRNA 5' end of the stem-loop that kisses the 3' BTE. Thus, the gRNA and sgRNA1 have "tuned" their expression efficiencies via the site in the 5' UTR to which the 3' BTE base pairs. We conclude that sgRNA2 is a riboregulator that switches off translation of replication genes from gRNA while permitting translation of structural genes from sgRNA1. These results reveal (i) a new level of control of subgenomic-RNA gene expression, (ii) a new role for a viral subgenomic RNA, and (iii) a new mechanism for RNA-mediated regulation of translation.     

3' RNA elements in hepatitis C virus replication: kissing partners and long poly(U)

The hepatitis C virus (HCV) genomic RNA possesses conserved structural elements that are essential for its replication. The 3′ nontranslated region (NTR) contains several of these elements: a variable region, the poly(U/UC) tract, and a highly conserved 3′ X tail, consisting of stem-loop 1 (SL1), SL2, and SL3. Studies of drug-selected, cell culture-adapted subgenomic replicons have indicated that an RNA element within the NS5B coding region, 5BSL3.2, forms a functional kissing-loop tertiary structure with part of the 3′ NTR, 3′ SL2. Recent advances now allow the efficient propagation of unadapted HCV genomes in the context of a complete infectious life cycle (HCV cell culture [HCVcc]). Using this system, we determine that the kissing-loop interaction between 5BSL3.2 and 3′ SL2 is required for replication in the genotype 2a HCVcc context. Remarkably, the overall integrity of the 5BSL3 cruciform is not an absolute requirement for the kissing-loop interaction, suggesting a model in which trans-acting factor(s) that stabilize this interaction may interact initially with the 3′ X tail rather than 5BSL3. The length and composition of the poly(U/UC) tract were also critical determinants of HCVcc replication, with a length of 33 consecutive U residues required for maximal RNA amplification. Interrupting the U homopolymer with C residues was deleterious, implicating a trans-acting factor with a preference for U over mixed pyrimidine nucleotides. Finally, we show that both the poly(U) and kissing-loop RNA elements can function outside of their normal genome contexts. This suggests that the poly(U/UC) tract does not function simply as an unstructured spacer to position the kissing-loop elements.     

Mutation of mapped TIA-1/TIAR binding sites in the 3' terminal stem-loop of West Nile virus minus-strand RNA in an infectious clone negatively affects genomic RNA amplification

Previous data showed that the cellular proteins TIA-1 and TIAR bound specifically to the West Nile virus 3' minus-strand stem-loop [WNV3'(-)SL] RNA (37) and colocalized with flavivirus replication complexes in WNV- and dengue virus-infected cells (21). In the present study, the sites on the WNV3'(-)SL RNA required for efficient in vitro T-cell intracellular antigen-related (TIAR) and T-cell intracellular antigen-1 (TIA-1) protein binding were mapped to short AU sequences (UAAUU) located in two internal loops of the WNV3'(-)SL RNA structure. Infectious clone RNAs with all or most of the binding site nucleotides in one of the 3' (-)SL loops deleted or substituted did not produce detectable virus after transfection or subsequent passage. With one exception, deletion/mutation of a single terminal nucleotide in one of the binding sequences had little effect on the efficiency of protein binding or virus production, but mutation of a nucleotide in the middle of a binding sequence reduced both the in vitro protein binding efficiency and virus production. Plaque size, intracellular genomic RNA levels, and virus production progressively decreased with decreasing in vitro TIAR/TIA-1 binding activity, but the translation efficiency of the various mutant RNAs was similar to that of the parental RNA. Several of the mutant RNAs that inefficiently interacted with TIAR/TIA-1 in vitro rapidly reverted in vivo, indicating that they could replicate at a low level and suggesting that an interaction between TIAR/TIA-1 and the viral 3'(-)SL RNA is not required for initial low-level symmetric RNA replication but instead facilitates the subsequent asymmetric amplification of genome RNA from the minus-strand template.     

Identification of the in vitro HIV-2/SIV RNA dimerization site reveals striking differences with HIV-1

Although their genomes cannot be aligned at the nucleotide level, the HIV-1/SIVcpz and the HIV-2/SIVsm viruses are closely related lentiviruses that contain homologous functional and structural RNA elements in their 5'-untranslated regions. In both groups, the domains containing the trans-activating region, the 5'-copy of the polyadenylation signal, and the primer binding site (PBS) are followed by a short stem-loop (SL1) containing a six-nucleotide self-complementary sequence in the loop, flanked by unpaired purines. In HIV-1, SL1 is involved in the dimerization of the viral RNA, in vitro and in vivo. Here, we tested whether SL1 has the same function in HIV-2 and SIVsm RNA. Surprisingly, we found that SL1 is neither required nor involved in the dimerization of HIV-2 and SIV RNA. We identified the NarI sequence located in the PBS as the main site of HIV-2 RNA dimerization. cis and trans complementation of point mutations indicated that this self-complementary sequence forms symmetrical intermolecular interactions in the RNA dimer and suggested that HIV-2 and SIV RNA dimerization proceeds through a kissing loop mechanism, as previously shown for HIV-1. Furthermore, annealing of tRNA(3)(Lys) to the PBS strongly inhibited in vitro RNA dimerization, indicating that, in vivo, the intermolecular interaction involving the NarI sequence must be dissociated to allow annealing of the primer tRNA.     

Position dependence of functional hairpins important for human immunodeficiency virus type 1 RNA encapsidation in vivo.

At least two hairpins in the 5' untranslated leader region, stem-loops 1 and 3 (SL1 and SL3), contribute to human immunodeficiency virus type 1 RNA encapsidation in vivo. We used a competitive assay, which measures the relative encapsidation efficiency of mutant viral RNA in the presence of competing wild-type RNA, to compare the contributions of SL1, SL3, and two adjacent secondary structures, SL2 and SL4, to encapsidation. SL2 is not required for RNA encapsidation, while SL1, SL3, and SL4 all contribute approximately equally to encapsidation. To determine whether these hairpins function in a position-dependent manner, we interchanged the positions of two of these stem-loop structures. This resulted in substantial diminution of encapsidation, indicating that the secondary structures that comprise E, the encapsidation signal, function only in their correct contexts. Mutation of nucleotides flanking SL1 and SL3 had little effect on encapsidation. We also showed that SL1, while present on both genomic and subgenomic viral RNAs, nonetheless contributes to selective encapsidation of genomic RNA. Taken together, these data are consistent with the formation of a higher-order RNA structure, partially composed of SL1, SL3, and SL4, that functions to effect concurrent encapsidation of full-length RNA and exclusion of subgenomic RNA. Finally, it has been reported that E is required for efficient translation of Gag mRNA in vivo. However, we have found that a variety of mutants, including a mutant lacking the entire region encompassing SL1, SL2, and SL3, still produce RNAs that are efficiently translated. These data indicate that E is unlikely to contribute to efficient Gag mRNA translation in vivo.     

Avipoxviruses: infection biology and their use as vaccine vectors

Background

It has been well documented that the 5' untranslated region (5' UTR) of many positive-stranded RNA viruses contain key cis-acting regulatory sequences, as well as high-order structural elements. Little is known for such regulatory elements controlling porcine arterivirus replication. We investigated the roles of a conserved stem-loop 2 (SL2) that resides in the 5'UTR of the genome of a type II porcine reproductive and respiratory syndrome virus (PRRSV).

Results

We provided genetic evidences demonstrating that 1) the SL2 in type II PRRSV 5' UTR, N-SL2, could be structurally and functionally substituted by its counterpart in type I PRRSV, E-SL2; 2) the functionality of N-SL2 was dependent upon the G-C rich stem structure, while the ternary-loop size was irrelevant to RNA synthesis; 3) serial deletions showed that the stem integrity of N-SL2 was crucial for subgenomic mRNA synthesis; and 4) when extensive base-pairs in the stem region was deleted, an alternative N-SL2-like structure with different sequence was utilized for virus replication.

Conclusion

Taken together, we concluded that the phylogenetically conserved SL2 in the 5' UTR was crucial for PRRSV virus replication, subgenomic mRNA synthesis in particular.