The Bovine Leukemia Virus Encapsidation Signal Is Composed of RNA Secondary Structures

The encapsidation signal of bovine leukemia virus (BLV) was previously shown by deletion analysis to be discontinuous and to extend into the 5′ end of the gag gene (L. Mansky et al., J. Virol. 69:3282–3289, 1995). The global minimum-energy optimal folding for the entire BLV RNA, including the previously mapped primary and secondary encapsidation signal regions, was analyzed. Two stable stem-loop structures (located just downstream of the gag start codon) were predicted within the primary signal region, and one stable stem-loop structure (in the gag gene) was predicted in the secondary signal region. Based on these predicted structures, we introduced a series of mutations into the primary and secondary encapsidation signals in order to explore the sequence and structural information contained within these regions. The replication efficiency and levels of cytoplasmic and virion RNA were analyzed for these mutants. Mutations that disrupted either or both of the predicted stem-loop structures of the primary signal reduced the replication efficiency by factors of 7 and 40, respectively; similar reductions in RNA encapsidation efficiency were observed. The mutant with both stem-loop structures disrupted had a phenotype similar to that of a mutant containing a deletion of the entire primary signal region. Mutations that disrupted the predicted stem-loop structure of the secondary signal led to similar reductions (factors of 4 to 6) in both the replication and RNA encapsidation efficiencies. The introduction of compensatory mutations into mutants from both the primary and secondary signal regions, which restored the predicted stem-loop structures, led to levels of replication and RNA encapsidation comparable to those of virus containing the wild-type encapsidation signal. Replacement of the BLV RNA region containing the primary and secondary encapsidation signals with a similar region from human T-cell leukemia virus (HTLV) type 1 or type 2 led to virus replication at three-quarters or one-fifth of the level of the parental virus, respectively. The results from both the compensatory mutants and BLV-HTLV chimeras indicate that the encapsidation sequences are recognized largely by their secondary or tertiary structures.     

Structural requirements of the higher order RNA kissing element in the enteroviral 3'UTR.

The origin of replication ( oriR ) involved in the initiation of (-) strand enterovirus RNA synthesis is a quasi-globular multi-domain RNA structure which is maintained by a tertiary kissing interaction. The kissing interaction is formed by base pairing of complementary sequences within the predominant hairpin-loop structures of the enteroviral 3' untranslated region. In this report, we have fully characterised the kissing interaction. Site-directed mutations which affected the different base pairs involved in the kissing interaction were generated in an infectious coxsackie B3 virus cDNA clone. The kissing interaction appeared to consist of 6 bp. Distortion of the interaction by mispairing of each of the base pairs involved in this higher order RNA structure resulted in either temperature sensitive or lethal phenotypes. The nucleotide constitution of the base which gaps the major groove of the kissing domain was not relevant for virus growth. The reciprocal exchange of the complete sequence involved in the kissing resulted in a mutant virus with wild type virus growth characteristics arguing that the base pair constitution is of less importance for the initiation of (-) strand RNA synthesis than the existence of the tertiary structure itself.     

Recurring features of local tertiary structural elements in RNA molecules exemplified by hepatitis D virus RNA

Elements of local tertiary structure in RNA molecules are important in understanding structure-function relationships. The loop E motif, first identified in several eukaryotic RNAs at functional sites which share an exceptional propensity for UV crosslinking between specific bases, was subsequently shown to have a characteristic tertiary structure. Common sequences and secondary structures have allowed other examples of the E-loop motif to be recognized in a number of RNAs at sites of protein binding or other biological function. We would like to know if more elements of local tertiary structure, in addition to the E-loop, can be identified by such common features. The highly structured circular RNA genome of the hepatitis D virus (HDV) provides an ideal test molecule because it has extensive internal structure, a UV-crosslinkable tertiary element, and specific sites for functional interactions with proteins including host PKR. We have now found a UV-crosslinkable element of local tertiary structure in antigenomic HDV RNA which, although differing from the E-loop, has a very similar pattern of sequence and secondary structure to the UV-crosslinkable element found in the genomic strand. Despite the fact that the two structures map close to one another, the sequences comprising them are not the templates for each other. Instead, the template regions for each element are additional sites for potential higher order structure on their respective complementary strands. This wealth of recurring sequences interspersed with base-paired stems provides a context to examine other RNA species for such features and their correlations with biological function.     

Efficient algorithms for probing the RNA mutation landscape

The diversity and importance of the role played by RNAs in the regulation and development of the cell are now well-known and well-documented. This broad range of functions is achieved through specific structures that have been (presumably) optimized through evolution. State-of-the-art methods, such as McCaskill's algorithm, use a statistical mechanics framework based on the computation of the partition function over the canonical ensemble of all possible secondary structures on a given sequence. Although secondary structure predictions from thermodynamics-based algorithms are not as accurate as methods employing comparative genomics, the former methods are the only available tools to investigate novel RNAs, such as the many RNAs of unknown function recently reported by the ENCODE consortium. In this paper, we generalize the McCaskill partition function algorithm to sum over the grand canonical ensemble of all secondary structures of all mutants of the given sequence. Specifically, our new program, RNAmutants, simultaneously computes for each integer k the minimum free energy structure MFE(k) and the partition function Z(k) over all secondary structures of all k-point mutants, even allowing the user to specify certain positions required not to mutate and certain positions required to base-pair or remain unpaired. This technically important extension allows us to study the resilience of an RNA molecule to pointwise mutations. By computing the mutation profile of a sequence, a novel graphical representation of the mutational tendency of nucleotide positions, we analyze the deleterious nature of mutating specific nucleotide positions or groups of positions. We have successfully applied RNAmutants to investigate deleterious mutations (mutations that radically modify the secondary structure) in the Hepatitis C virus cis-acting replication element and to evaluate the evolutionary pressure applied on different regions of the HIV trans-activation response element. In particular, we show qualitative agreement between published Hepatitis C and HIV experimental mutagenesis studies and our analysis of deleterious mutations using RNAmutants. Our work also predicts other deleterious mutations, which could be verified experimentally. Finally, we provide evidence that the 3' UTR of the GB RNA virus C has been optimized to preserve evolutionarily conserved stem regions from a deleterious effect of pointwise mutations. We hope that there will be long-term potential applications of RNAmutants in de novo RNA design and drug design against RNA viruses. This work also suggests potential applications for large-scale exploration of the RNA sequence-structure network. Binary distributions are available at http://RNAmutants.csail.mit.edu/.     

A long-range pseudoknot is required for activity of the Neurospora VS ribozyme.

Four small RNA self-cleaving domains, the hammerhead, hairpin, hepatitis delta virus and Neurospora VS ribozymes, have been identified previously in naturally occurring RNAs. The secondary structures of these ribozymes are reasonably well understood, but little is known about long-range interactions that form the catalytically active tertiary conformations. Our previous work, which identified several secondary structure elements of the VS ribozyme, also showed that many additional bases were protected by magnesium-dependent interactions, implying that several tertiary contacts remained to be identified. Here we have used site-directed mutagenesis and chemical modification to characterize the first long-range interaction identified in VS RNA. This interaction contains a 3 bp pseudoknot helix that is required for tertiary folding and self-cleavage activity of the VS ribozyme.     

A base-paired structure in the avian sarcoma virus 5' leader is required for efficient encapsidation of RNA.

Selective encapsidation of avian sarcoma-leukosis virus genomic RNA within virions requires recognition of a cis-acting signal (termed psi) located in the 5' leader of the RNA between the primer binding site and the splice donor site. Computer analyses indicate the potential for numerous secondary structure interactions within this region, including alternative conformations with similar free energy levels. We have constructed mutations designed to disrupt and restore potential secondary structure interactions within psi to investigate the role of these structures in RNA packaging. To test for the ability of psi mutants to package a heterologous reporter gene into virions, chimeric constructs bearing avian sarcoma virus 5' sequences fused to lacZ were transiently cotransfected with a nonpackageable helper construct into chicken embryo fibroblasts. lacZ virions produced from cotransfected cells were used to infect new cultures of chicken embryo fibroblasts, and then an in situ assay for individual cells expressing lacZ was done. Results obtained with this assay were confirmed in direct analyses of isolated virion RNA by RNase protection assays. Two mutations, predicted to disrupt a potential stem structure forming between elements located at nucleotides 160 to 167 and 227 to 234, severely inhibited packaging when either element was mutated. A construct in which these mutations were combined to restore potential base pairing between the two elements displayed a partially restored packaging phenotype. These results strongly suggest that the structure, referred to as the O3 stem, is required for efficient encapsidation of avian sarcoma virus RNA. Site-directed mutagenesis of additional sequence elements located in the O3 loop reduced packaging as measured by the indirect assay, suggesting that these sequences may also be components of the encapsidation signal. The possible implications of the O3 stem structure with regard to translation of avian sarcoma-leukosis virus short upstream open reading frames are discussed.     

Lead-catalyzed cleavage of ribonuclease P RNA as a probe for integrity of tertiary structure.

Pb(2+)-catalyzed cleavage of RNA has been shown previously to be a useful probe for tertiary structure. In the present study, Pb2+ cleavage patterns were identified for ribonuclease P RNAs from three phylogenetically disparate organisms, Escherichia coli, Chromatium vinosum, Bacillus subtilis, and for E. coli RNase P RNAs that had been altered by deletions. Each of the native RNAs undergoes cleavage at several sites in the core structure that is common to all bacterial RNase P RNAs. All the cleavages occur in non-paired regions of the secondary structure models of the RNAs, in regions likely to be involved in tertiary interactions. Two cleavage sites occur at homologous positions in all the native RNAs, regardless of sequence variation, suggesting common tertiary structural features. The Pb2+ cleavage sites in four deletion mutants of E. coli RNase P RNA differed from the native pattern, indicating alterations in the tertiary structures of the mutant RNAs. This conclusion is consistent with previously characterized properties of the mutant RNAs. The Pb2+ cleavage assay is thus a useful probe to reveal alteration of tertiary structure in RNase P RNA.     

Identification of potential conserved RNA secondary structure throughout influenza A coding regions

Influenza A is a negative sense RNA virus of significant public health concern. While much is understood about the life cycle of the virus, knowledge of RNA secondary structure in influenza A virus is sparse. Predictions of RNA secondary structure can focus experimental efforts. The present study analyzes coding regions of the eight viral genome segments in both the (+) and (-) sense RNA for conserved secondary structure. The predictions are based on identifying regions of unusual thermodynamic stabilities and are correlated with studies of suppression of synonymous codon usage (SSCU). The results indicate that secondary structure is favored in the (+) sense influenza RNA. Twenty regions with putative conserved RNA structure have been identified, including two previously described structured regions. Of these predictions, eight have high thermodynamic stability and SSCU, with five of these corresponding to current annotations (e.g., splice sites), while the remaining 12 are predicted by the thermodynamics alone. Secondary structures with high conservation of base-pairing are proposed within the five regions having known function. A combination of thermodynamics, amino acid and nucleotide sequence comparisons along with SSCU was essential for revealing potential secondary structures.     

Genetic interactions between an essential 3' cis-acting RNA pseudoknot, replicase gene products, and the extreme 3' end of the mouse coronavirus genome

The upstream end of the 3' untranslated region (UTR) of the mouse hepatitis virus genome contains two essential and overlapping RNA secondary structures, a bulged stem-loop and a pseudoknot, which have been proposed to be elements of a molecular switch that is critical for viral RNA synthesis. It has previously been shown that a particular six-base insertion in loop 1 of the pseudoknot is extremely deleterious to the virus. We have now isolated multiple independent second-site revertants of the loop 1 insertion mutant, and we used reverse-genetics methods to confirm the identities of suppressor mutations that could compensate for the original insertion. The suppressors were localized to two separate regions of the genome. Members of one class of suppressor were mapped to the portions of gene 1 that encode nsp8 and nsp9, thereby providing the first evidence for specific interactions between coronavirus replicase gene products and a cis-acting genomic RNA element. The second class of suppressor was mapped to the extreme 3' end of the genome, a result which pointed to the existence of a direct base-pairing interaction between loop 1 of the pseudoknot and the genomic terminus. The latter finding was strongly supported by phylogenetic evidence and by the construction of a deletion mutant that reduced the 3' UTR to its minimal essential elements. Taken together, the interactions revealed by the two classes of suppressors suggest a model for the initiation of coronavirus negative-strand RNA synthesis.     

Hairpin Loop Structure in the 3′ Arm of the Influenza A Virus Virion RNA Promoter Is Required for Endonuclease Activity

Previous studies have shown that the 5' arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3' arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3' arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3' hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3' arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.     

Secondary structure of the human T-cell leukemia virus type 1 rex-responsive element is essential for rex regulation of RNA processing and transport of unspliced RNAs.

Rex protein of human T-cell leukemia virus type 1 (HTLV-1) induces cytoplasmic expression of unspliced gag/pol mRNA and singly spliced env mRNA and thus is essential for replication of the virus. This regulation requires a cis-acting rex-responsive element (RXE), located in the 3' region of the viral RNA. By external deletion, we have identified RXE composed of 205 nucleotides. The secondary structure of RXE was confirmed by studies on its susceptibility to nuclease digestions to consist of four stem-loops and a long stretch of stem structure. Substitution and deletion mutations revealed that two regions of the stem-loops and their secondary structures are essential for rex regulation. Similar secondary structures were found in the corresponding regions of HTLV-2, bovine leukemia virus and human immunodeficiency virus. Furthermore, a sequence of 11 nucleotides in the RXE was found to be conserved in the secondary structures of HTLV-1, HTLV-2, and bovine leukemia virus. These observations suggest that the secondary structure as well as the conserved sequence may be important in expression of unspliced RNA even with diverged sequences as observed in these viruses.     

Effect of single-base substitutions in the central domain of virus-associated RNA I on its function.

Adenoviruses use virus-associated RNA I (VAI RNA) to counteract the cellular antiviral response mediated by the interferon-induced, double-stranded-RNA-activated protein kinase PKR. VAI RNA is a highly structured small RNA which consists of two long duplex regions connected at the center by a complex, short stem-loop. This short stem-loop and the adjacent base-paired regions, referred to as the central domain, bind to PKR and inactivate it. Currently it is not known whether binding of VAI RNA to PKR is dependent solely on the secondary (and tertiary) structure of the central domain or whether nucleotide sequences in the central domain are also critical for this interaction. To address this question, 54 VAI mutants with single-base substitution mutations in the central domain of the RNA were constructed, and their capacities to inhibit the autophosphoryation of PKR in vitro were determined. It was found that although about half of the mutants inhibited PKR activity as efficiently as the wild type, a significant number of mutants lost the inhibitory activity substantially, without a perceptible change in their secondary structures. These results indicate that, in addition to secondary structure, at least some nucleotides in the central domain may be critical for the efficient function of VAI RNA.     

Sequence requirements of the hammerhead RNA self-cleavage reaction.

A previously well-characterized hammerhead catalytic RNA consisting of a 24-nucleotide substrate and a 19-nucleotide ribozyme was used to perform an extensive mutagenesis study. The cleavage rates of 21 different substrate mutations and 24 different ribozyme mutations were determined. Only one of the three phylogenetically conserved base pairs but all nine of the conserved single-stranded residues in the central core are needed for self cleavage. In most cases the mutations did not alter the ability of the hammerhead to assemble into a bimolecular complex. In the few cases where mutant hammerheads did not assemble, it appeared to be the result of the mutation stabilizing an alternate substrate or ribozyme secondary structure. All combinations of mutant substrate and mutant ribozyme were less active than the corresponding single mutations, suggesting that the hammerhead contains few, if any, replaceable tertiary interactions as are found in tRNA. The refined consensus hammerhead resulting from this work was used to identify potential hammerheads present in a variety of Escherichia coli gene sequences.     

Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA.

Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wild-type RNA forms a 5' stem-loop that enhances electrophoretic mobility. All mutations that disrupt the stem of this hairpin decrease mobility of the RNA. In contrast, mutations that change the sequence of the stem without disrupting it (e.g. change G.U to A.U) do not affect mobility. Nearly all mutations in single-stranded regions of the structure also have no effect on mobility. Confirmation of the proposed 5' stem-loop was obtained by constructing and analyzing compensatory double mutants. Combinations of mutations that restore a base-pair of the stem also restore mobility. The genetic phenotypes of sar mutants confirm that the proposed secondary structure is correct and is essential for optimal activity of the antisense RNA in vivo.     

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.     

The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures.

We analyzed the leader region of human immunodeficiency virus type 1 (HIV-1) RNA to decipher the nature of the cis-acting E/psi element required for encapsidation of viral RNA into virus particles. Our data indicate that, for RNA encapsidation, there are at least two functional subregions in the leader region. One subregion is located at a position immediately proximal to the major splice donor, and the second is located between the splice donor and the beginning of the gag gene. This suggests that at least two discrete cis-acting elements are recognition signals for encapsidation. To determine whether specific putative RNA secondary structures serve as the signal(s) for encapsidation, we constructed primary base substitution mutations that would be expected to destabilize these potential structures and second-site compensatory mutations that would restore secondary structure. Analysis of these mutants allowed the identification of two discrete hairpins that facilitate RNA encapsidation in vivo. Thus, the HIV-1 E/psi region is a multipartite element composed of specific and functional RNA secondary structures. Compensation of the primary mutations by the second-site mutations could not be attained in trans. This indicates that interstrand base pairing between these two stem regions within the hairpins does not appear to be the basis for HIV-1 RNA dimer formation. Comparison of the hypothetical RNA secondary structures from 10 replication-competent HIV-1 strains suggests that a subset of the hydrogen-bonded base pairs within the stems of the hairpins is likely to be required for function in cis.     

The hepatitis C virus internal ribosome entry site adopts an ion-dependent tertiary fold.

Hepatitis C virus (HCV) contains an internal ribosome entry site (IRES) located in the 5' untranslated region of the genomic RNA that drives cap-independent initiation of translation of the viral message. The approximate secondary structure and minimum functional length of the HCV IRES are known, and extensive mutagenesis has established that nearly all secondary structural domains are critical for activity. However, the presence of an IRES RNA tertiary fold and its functional relevance have not been established. Using chemical and enzymatic probes of the HCV IRES RNA in solution, we show that the IRES adopts a unique three-dimensional structure at physiological salt concentrations in the absence of additional cofactors or the translation apparatus. Folding of the IRES involves cooperative uptake of magnesium and is driven primarily by charge neutralization. This tertiary structure contains at least two independently folded regions which closely correspond to putative binding sites for the 40 S ribosomal subunit and initiation factor 3 (eIF3). Point mutations that inhibit IRES folding also inhibit its function, suggesting that the IRES tertiary structure is essential for translation initiation activity. Chemical and enzymatic probing data and small-angle X-ray scattering (SAXS) experiments in solution show that upon folding, the IRES forms an extended structure in which functionally important loops are exposed. These results suggest that the 40 S ribosomal subunit and eIF3 bind an HCV IRES that is prefolded to spatially organize recognition domains.     

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)