Secondary structure computer prediction of the poliovirus 5' non-coding region is improved by a genetic algorithm.

Comparison of the secondary structure of the 5' non-coding region of poliovirus 3 RNA derived from the genetic algorithm with the model of Skinner et al. (J. Mol. Biol., 207, 379-392, 1989) demonstrates many of the confirmed structural elements. The genetic algorithm (Shapiro and Navetta, J. Supercomput., 8, 195-201, 1994) generates a population of all possible stems, then mixes, combines, and recombines these stems in multiple iterations on a massively parallel computer, ultimately selecting a most fit structure based on its energy. The secondary structure of the region containing the determinants of neurovirulence was better predicted using the genetic algorithm, whereas the dynamic programming algorithm (Zuker, Science, 244, 48-52, 1989) required phylogenetic comparative sequence analysis to arrive at the correct conclusion. In addition, artificial mutations were introduced throughout this region of the genome and although rearrangements in structure may occur, many structures persisted, suggesting that the given structures thus selected may have evolved to withstand isolated mutations. The genetic algorithm-derived structure for the 5' non-coding region compares favorably with the biological data and functions previously described, and contains all of the 'persistent' structures, suggesting also that the persistence factor may be an aid to validating structures.

A computational procedure for assessing the significance of RNA secondary structure.

In our recent series of papers, we have used the structures of statistical significance from Monte Carlo simulations to improve the predictions of secondary structure of RNA and to analyze the possible role of locally significant structures in the life cycle of human immunodeficiency virus. Because of intensive computational requirements for Monte Carlo simulation, it becomes impractical even using a supercomputer to assess the significance of a structure with a window size greater than 200 along an RNA sequence of 1000 bases or more. In this paper, we have developed a new procedure that drastically reduces the time needed to assess the significance of structures. In fact, the efficiency of this new method allows us to assess structures on the VAX as well as the CRAY.

A program for predicting significant RNA secondary structures.

We describe a program for the analysis of RNA secondary structure. There are two new features in this program. (i) To get vector speeds on a vector pipeline machine (such as Cray X-MP/24) we have vectorized the secondary structure dynamic algorithm. (ii) The statistical significance of a locally 'optimal' secondary structure is assessed by a Monte Carlo method. The results can be depicted graphically including profiles of the stability of local secondary structures and the distribution of the potentially significant secondary structures in the RNA molecules. Interesting regions where both the potentially significant secondary structures and 'open' structures (single-stranded coils) occur can be identified by the plots mentioned above. Furthermore, the speed of the vectorized code allows repeated Monte Carlo simulations with different overlapping window sizes. Thus, the optimal size of the significant secondary structure occurring in the interesting region can be assessed by repeating the Monte Carlo simulation. The power of the program is demonstrated in the analysis of local secondary structures of human T-cell lymphotrophic virus type III (HIV).

Studies of frequently recurring substructures in human alpha-like globin mRNA precursors.

In general, the results obtained from secondary structure prediction algorithms are often inconsistent with those obtained experimentally. The reason for this disagreement is that the experimentally determined structures have higher free energies (as judged by the currently used "energy rules") than the predicted ones. To overcome this limitation we have developed a new approach which incorporates the frequencies of occurrence of substructures in the growing mRNA chain. This has been accomplished by simulating the folding process of pre-mRNAs. Using this approach we have significantly improved current helical structural prediction for 142 analyzed tRNAs and 16 S rRNA. We have next applied this method to the human alpha-like globins. Comparison of the structures obtained by running the currently used algorithms with those computed by the new method indicates that the final most stable secondary structure contains some infrequently occurring substructures. In addition, some of the frequently recurring substructures are not included in the final structure. Comparison of the simulated folding processes of the human alpha-like globin pre-mRNAs reveals some conserved helices and hairpin loop structures in those frequently recurring substructures. Among these several compensating base changes (transitions and transversions) have been identified.

Potential secondary and tertiary structure in the genomic RNA of foot and mouth disease virus.

The nucleotide sequence of the 5' untranslated region of foot and mouth disease virus (FMDV), serotype A10 has been determined. This completes the first total genomic sequence for any one serotype of FMDV. Analysis of the sequence to the 3' side of the poly (C) tract reveals the presence of a 24 nucleotide repeated motif which has homologies with a sequence located upstream of the transcriptional initiation site from several mammalian fibrinogen genes. The function of this element in FMDV is unclear. However, computer analysis of this region predicts the presence of a high degree of secondary and tertiary structure in which these repeats form an important part. The implications of these predictions are discussed.

Secondary structure of poliovirus RNA: correlation of computer-predicted with electron microscopically observed structure.

A secondary structure map of poliovirus 1, strain Mahoney, RNA was determined by psoralen crosslinking the (+) strand and visualizing the structures in the electron microscope. Hairpins and looped hairpins were observed, and the size and distribution were measured. To orient map features the 3' end of the RNA was linked to polybromodeoxyuridine [poly(BUdR)]SV40 and histograms were constructed from these measurements. Secondary structure maps of the RNA were likewise constructed from the results of computer prediction programs for secondary structure. The programs used were those of M. Zuker (RNA2 and FOLD) which calculate a minimal global energy for a given sequence. Many single hairpins predicted by both RNA2 and FOLD showed a correlation with the histograms of hairpin structures of RNA crosslinked with psoralen. A secondary structure map was also constructed for the entire 7433 bases using the option in FOLD which allows multi-branch loops by folding uniformly stepped overlapping segments. Any structure that occurred at or greater than a given frequency was selected and mapped with respect to genome position. A correlation in structured regions was seen between psoralen-derived and computer-predicted maps of secondary structure. Furthermore, a region of large loops from base position 681 to 3899 was noted that corresponded to frequently observed large loop(s) in electron micrographs of psoralen preparations of RNA. Agreement between the two methods of determining secondary structure strengthens the credibility of the computer-aided methods used for predicting secondary structure and allows us to suggest an overall secondary structure map for poliovirus RNA.

Functional analysis of reverse transcription by a frameshift pol mutant of murine leukemia virus.

Endogenous reverse transcription by wild-type murine leukemia virus (MuLV) was compared to that catalyzed by clone 23, a pol mutant containing a reverse transcriptase protein which lacks the carboxyl-terminal third of the molecule (J. G. Levin, S. C. Hu, A. Rein, L. I. Messer, and B. I. Gerwin (1984), J. Virol. 51, 470-478). Competition immunoassays revealed that mutant virions contain normal amounts of polymerase protein, indicating that the lack of carboxyl-terminal sequences does not alter normal processing of enzyme precursors. Although the mutant enzyme was previously shown to have the ability to copy and degrade RNA:DNA hybrids, the present study demonstrates that it is defective in functions required to generate full-length copies of viral DNA. Analysis of products of endogenous reverse transcription showed that minus-strand strong-stop DNA is formed and that mutant virions synthesize a series of minus-strand DNA intermediates up to 2.2 kb in length. Comparison of mutant and wild-type MuLV reaction products indicated that the 2.2-kb termination site of the mutant corresponds to a normal pausing region for the wild-type enzyme. Computer analysis of sequences and structure within pausing regions suggested the involvement of C-rich consensus sequences plus multibranch loop structures in the general phenomenon of enzyme-pausing during reverse transcription.