Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities.

The prediction of the complete matrix of base pairing probabilities was applied to the 3'' noncoding region (NCR) of flavivirus genomes. This approach identifies not only well-defined secondary structure elements, but also regions of high structural flexibility. Flaviviruses, many of which are important human pathogens, have a common genomic organization, but exhibit a significant degree of RNA sequence diversity in the functionally important 3''-NCR. We demonstrate the presence of secondary structures shared by all flaviviruses, as well as structural features that are characteristic for groups of viruses within the genus reflecting the established classification scheme. The significance of most of the predicted structures is corroborated by compensatory mutations. The availability of infectious clones for several flaviviruses will allow the assessment of these structural elements in processes of the viral life cycle, such as replication and assembly.     

Five pseudoknots are present at the 204 nucleotides long 3'' noncoding region of tobacco mosaic virus RNA.

The 104 nucleotides long 3' terminal region of TMV RNA was shown previously to contain two pseudoknotted structures (Rietveld et al. (1984), EMBO J. 3, 2613-2619). We here present evidence for the occurrence, within the 204 nucleotides long 3' noncoding region, of another highly structured domain located immediately adjacent to the tRNA-like structure of 95 nucleotides (Joshi et al. (1985) Nucleic Acids Res. 13, 347-354). A model for the three-dimensional folding of this region, containing three more pseudoknots, is proposed on the basis of chemical modification and enzymatic digestion. The existence of these three consecutive pseudoknots was supported by sequence comparisons with the RNA from the related tobamoviruses TMV-L, CcTMV and CGMMV. Coaxial stacking of the six double helical segments involved gives rise to the formation of a 25 basepair long quasi-continuous double helix. The results show that the three-dimensional folding of the 3' non-translated region of tobamoviral RNAs is largely maintained by the formation of five pseudoknots. The organisation of this region in the RNA of the tobamovirus CcTMV suggests that recombinational events among aminoacylatable plant viral RNAs have to be considered.     

Replication-competent picornaviruses with complete genomic RNA 3' noncoding region deletions.

The genomic RNA 3' noncoding region is believed to be a major cis-acting molecular genetic determinant for regulating picornavirus negative-strand RNA synthesis by promoting replication complex recognition. We report the replication of two picornavirus RNAs harboring complete deletions of the genomic RNA 3' noncoding regions. Our results suggest that while specific 3'-terminal RNA sequences and/or secondary structures may have evolved to promote or regulate negative-strand RNA synthesis, the basic mechanism of replication initiation is not strictly template specific and may rely primarily upon the proximity of newly translated viral replication proteins to the 3' terminus of template RNAs within tight membranous replication complexes.     

Sequence of the Kluyveromyces lactis beta-galactosidase: comparison with prokaryotic enzymes and secondary structure analysis.

The LAC4 gene encoding the beta-galactosidase (beta Gal) of the yeast, Kluyveromyces lactis, was cloned on a 7.2-kb fragment by complementation of a lacZ-deficient Escherichia coli strain. The nucleotide sequence of the structural gene, with 42 bp and 583 bp of the 5'- and 3'-flanking sequences, respectively, was determined. The deduced amino acid (aa) sequence of the K. lactis beta Gal predicts a 1025-aa polypeptide with a calculated M(r) of 117618 and reveals extended sequence homologies with all the published prokaryotic beta Gal sequences. This suggests that the eukaryotic beta Gal is closely related, evolutionarily and structurally, to the prokaryotic beta Gal's. In addition, sequence similarities were observed between the highly conserved N-terminal two-thirds of the beta Gal and the entire length of the beta-glucuronidase (beta Glu) polypeptides, which suggests that beta Glu is clearly related, structurally and evolutionarily, to the N-terminal two-thirds of the beta Gal. The structural analysis of the beta Gal alignment, performed by mean secondary structure prediction, revealed that most of the invariant residues are located in turn or loop structures. The location of the invariant residues is discussed with respect to their accessibility and their possible involvement in the catalytic process.     

The 5' and 3' downstream AUG region elements are required for mosquito-borne flavivirus RNA replication

Flaviviruses require complementarity between the 5' and 3' ends of the genome for RNA replication. For mosquito-borne flaviviruses, the cyclization sequences (CS) and upstream of AUG region (UAR) elements at the genomic termini are necessary for viral RNA replication, and a third motif, the downstream of AUG region (DAR), was recently designated for dengue virus. The 3' DAR sequence is also part of a hairpin (HP-3'SL), and both complementarity between 5' and 3' DAR motifs and formation of the HP-3'SL in the absence of the 5' end are conserved among mosquito-borne flaviviruses. Using West Nile virus as a model, we demonstrate that 5'-3' DAR complementarity and HP-3'SL formation are essential for viral RNA replication.     

Phylogenetic analysis of the apolipoprotein B mRNA-editing region. Evidence for a secondary structure between the mooring sequence and the 3' efficiency element

Apolipoprotein (apo) B mRNA editing is the deamination of C(6666) to uridine, which changes the codon at position 2153 from a genomically encoded glutamine (CAA) to an in-frame stop codon (UAA). The apoB mRNA-editing enzyme complex recognizes the editing region of the apoB pre-mRNA with exquisite precision. Four sequence elements spanning 139 nucleotides (nt) on the apoB mRNA have been identified that specify this precision. In cooperation with the indispensable mooring sequence and spacer element, a 5' efficiency element and a 3' efficiency element enhance editing in vitro. A phylogenetic comparison of 32 species showed minor differences in the apoB mRNA sequence, and the apoB mRNA from 31 species was robustly edited in vitro. However, guinea pig mRNA was poorly edited. Compared with the consensus sequences of these 31 species, guinea pig apoB mRNA has three variations in the 3' efficiency element, and the conversion of these to the consensus sequence increased editing to the levels in the other species. From this information, a model for the secondary structure was formulated in which the mooring sequence and the 3' efficiency element form a double-stranded stem. Thirty-one mammalian apoB mRNA sequences are predicted to form this stem positioning C(6666) two nucleotides upstream of the stem. However, the guinea pig apoB mRNA has a mutation in the 3' efficiency element (C(6743) to U) that predicts an extension of the stem and hence the lower editing efficiency. A test of this model demonstrated that a single substitution at 6743 (U to C) in the guinea pig apoB mRNA, that should reduce the stem, enhanced editing, and mutations in the 3' efficiency element that extended the stem for three base pairs dramatically reduced editing. Furthermore, the addition of a 20-nucleotide 3' efficiency element RNA, to a 58-nucleotide guinea pig apoB mRNA lacking the 3' efficiency element more than doubled the in vitro editing activity. Based on these results, a model is proposed in which the mooring sequence and the 3' efficiency element form a double-stranded stem, thus suggesting a mechanism of how the 3' efficiency element enhances editing.     

Cell-dependent role for the poliovirus 3' noncoding region in positive-strand RNA synthesis

We previously reported the isolation of a mutant poliovirus lacking the entire genomic RNA 3' noncoding region. Infection of HeLa cell monolayers with this deletion mutant revealed only a minor defect in the levels of viral RNA replication. To further analyze the consequences of the genomic 3' noncoding region deletion, we examined viral RNA replication in a neuroblastoma cell line, SK-N-SH cells. The minor genomic RNA replication defect in HeLa cells was significantly exacerbated in the SK-N-SH cells, resulting in a decreased capacity for mutant virus growth. Analysis of the nature of the RNA replication deficiency revealed that deleting the poliovirus genomic 3' noncoding region resulted in a positive-strand RNA synthesis defect. The RNA replication deficiency in SK-N-SH cells was not due to a major defect in viral translation or viral protein processing. Neurovirulence of the mutant virus was determined in a transgenic mouse line expressing the human poliovirus receptor. Greater than 1,000 times more mutant virus was required to paralyze 50% of inoculated mice, compared to that with wild-type virus. These data suggest that, together with a cellular factor(s) that is limiting in neuronal cells, the poliovirus 3' noncoding region is involved in positive-strand synthesis during genome replication.     

Secondary structure at the 3' terminal region of RNA coliphages: comparison with tRNA

Secondary structure models for the 3' non-coding region of the four groups of coliphage RNA are proposed based on comparative sequence analysis and on previously published data on the sensitivity of nucleotides in MS2 RNA to chemical modification and enzymes. We report the following observations. (1) In contrast to the coding regions, the structure at the 3' terminus is characterized by stable regular helices. We note the occurrence of the loop sequences 5'-GUUCGC and 5'-CGAAAG, that are reported to confer exceptional stability to stem structures. These features are probably present to promote the segregation of mother and daughter strands during replication. (2) Comparison of homologous helices indicates that only those base pair substitutions are allowed that maintain the thermodynamic stability. (3) We have compared the structure of phage RNA with tRNA. Overall similarity is low, but one common element may exist. It is a quasi-continuous helix of 12 basepairs that could be the equivalent of the 12 basepair long coaxially stacked helix, formed by the T psi C arm and the aminoacyl acceptor arm in tRNA. As in tRNA, this structure element starts after the fourth nucleotide from the 3' end. (4) Phage RNA contains a large variable region of about 35 nucleotides bulging out from the quasi-continuous helix. We speculate that the variable loop in present-day tRNA could be the remnant of the variable region found in phage RNA. The variable region contains overlapping binding sites for the replicase enzyme and the maturation protein. This common binding site may serve as a switch from replication to packaging.     

Predicted secondary structure of the foraminiferal SSU 3' major domain reveals a molecular synapomorphy for granuloreticulosean protists

The small subunit ribosomal RNA genes of foraminiferal protists are the largest and most divergent of any eukaryote. We demonstrate that this foraminiferal sequence alteration represents a substantial modification to the small subunit ribosomal RNA structure, including a large (up to 350 nt) novel helix in a very well-conserved portion of the head domain. This modification dates from the beginning of the foraminiferal radiation and, within modern orders, is partially conserved at the sequence level, suggesting that it is a functional part of the ribosome. The pattern of conservation makes it particularly useful for determining lower-taxon relationships in morphologically ambiguous allogromiid foraminifera.     

Sequence analysis of genes and genomes

A major step towards understanding of the genetic basis of an organism is the complete sequence determination of all genes in its genome. The development of powerful techniques for DNA sequencing has enabled sequencing of large amounts of gene fragments and even complete genomes. Important new techniques for physical mapping, DNA sequencing and sequence analysis have been developed. To increase the throughput, automated procedures for sample preparation and new software for sequence analysis have been applied. This review describes the development of new sequencing methods and the optimisation of sequencing strategies for whole genome and cDNA analysis, as well as discusses issues regarding sequence analysis and annotation.     

5' and 3' untranslated regions of pestivirus genome: primary and secondary structure analyses.

Within the conserved 5' untranslated region (UTR) of the pestivirus genome three highly variable regions were identified. Preceding the polyprotein start codon, multiple cryptic AUG codons and several small open reading frames are characteristic for all the five pestiviruses. Inspection of the context of AUGs revealed that the polyprotein initiation AUG of pestivirus has a weak context for efficient translation initiation. The most favorable context was found in two of the cryptic AUGs. Two oligopyrimidine-rich tracts upstream to the conserved either cryptic or authentic AUG in the 5'-UTR of pestivirus were identified and 83.3% of their nucleotide sequences are complementary to the consensus sequence at the 3' terminus of eucaryotic 18S rRNA. A secondary structure model for the 5'-UTR of pestivirus was predicted. Nucleotide sequence comparison among five pestiviruses led to the identification of a variable region and a conserved region in the 3'-UTR. A deletion of 41 nucleotides was found within the variable region in Osloss. A secondary structure model for the 3'-UTR was also predicted. The structural similarity of the 5'-UTR between pestiviruses and picornaviruses and hepatitis C viruses was demonstrated and the possible implications of features of the 5' and 3'-UTR of pestiviruses are discussed.     

Characterization of an essential RNA secondary structure in the 3' untranslated region of the murine coronavirus genome

We have previously identified a functionally essential bulged stem-loop in the 3' untranslated region of the positive-stranded RNA genome of mouse hepatitis virus. This 68-nucleotide structure is composed of six stem segments interrupted by five bulges, and its structure, but not its primary sequence, is entirely conserved in the related bovine coronavirus. The functional importance of individual stem segments of this stem-loop was characterized by genetic analysis using targeted RNA recombination. We also examined the effects of stem segment mutations on the replication of mouse hepatitis virus defective interfering RNAs. These studies were complemented by enzymatic and chemical probing of the stem-loop. Taken together, our results confirmed most of the previously proposed structure, but they revealed that the terminal loop and an internal loop are larger than originally thought. Three of the stem segments were found to be essential for viral replication. Further, our results suggest that the stem segment at the base of the stem-loop is an alternative base-pairing structure for part of a downstream, and partially overlapping, RNA pseudoknot that has recently been shown to be necessary for bovine coronavirus replication.