Mutations in a conserved enteroviral RNA oligonucleotide sequence affect positive strand viral RNA synthesis.

We showed earlier that a transition mutation U234C, located within the completely conserved 5 nucleotide (nt) tract 5'-CGUUA (nt232-236) in the 5' non-translated region (NTR) of the coxsackievirus B3 (CVB3) genome, attenuated CVB3 cardiovirulence in mice. To further explore the role of the sequence, we induced two single and one double transversion mutations in the conserved 5mer in a cardiovirulent CVB3 genome. The mutated sites partially or totally reverted to parental wild-type when progeny viruses were passaged at 37 degrees C, but remained stable when transfection and subsequent passages were performed at 33.5 degrees C. Viral replication in cell culture was attenuated at 37 degrees C or 39.5 degrees C relative to replication at 33.5 degrees C. While Western blot analysis demonstrated the level of protein translation consistent with virus replication, the ratios of positive to negative strand viral RNA at 37 degrees C in murine cells demonstrated a 2-5 fold diminution from those measured at 33.5 degrees C. Mutant CVB3 strains failed to replicate productively when inoculated into mice. The biological data are consistent with an hypothesis that proposes a lesion with primary effects at the level of positive strand viral RNA synthesis that results in attenuation of viral replication at physiologic temperature.

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.

Asystoles during infancy recorded by home memory monitors. Benign events?

OBJECTIVES: To assess the frequency and clinical significance of asystole (sinus arrest > or = 2.0 seconds) and the incidence of bradycardia in infants prescribed home cardiorespiratory monitors and to test the hypothesis that asystoles are more likely to occur in preterm infants. DESIGN: Prospective, consecutive sample of monitor printouts. METHODS: All 291 printouts from the memory monitors of 161 patients received during a 2-month period were analyzed. SETTING: University hospital providing primary and referral care. MAIN OUTCOME MEASURES: Asystoles and bradycardias; clinical course of patients with asystoles. RESULTS: Eight patients (5.0%) had 32 episodes of asystole lasting 2.0 to 4.3 seconds (group 1). Fifty-three patients (32.9%) had true bradycardia alarms but no asystoles (group 2). One hundred patients (62.1%) had neither asystoles nor bradycardias (group 3). Preterm infants constituted 88% of group 1 and 81% of group 2 but only 58% of group 3. Infants were more likely to be full-term in group 3 than in the other 2 groups (chi 2, P = .02). Birth weights were lower in group 1 than in group 3 (P < .05, 1-tailed t test). There were 479 true bradycardias; 72.2% lasted 10 seconds or less, 26.3% were longer than 10 seconds but no more than 20 seconds, and 1.5% were longer than 20 seconds. None of the 8 patients with asystoles required resuscitation for their asystoles; all survived and were free of any life-threatening events after their monitors were discontinued and up until their first birthday. CONCLUSIONS: Asystoles occur more commonly in preterm infants; those pauses in the 2.0- to 4.0-second range seem to be benign. Studies of long-term recordings are needed to redefine asystole in both normal preterm and fullterm infants. These data would help further refine current guidelines for pacemaker implantation during infancy.

The cardiovirulent phenotype of coxsackievirus B3 is determined at a single site in the genomic 5' nontranslated region.

We report the construction of chimeric coxsackievirus B3 (CVB3) strains in which sequences of an infectious cDNA copy of a noncardiovirulent CVB3 genome were replaced by the homologous sequences from a cardiovirulent CVB3 genome to identify which of 10 predicted genetic sites determine cardiovirulence. Cardiovirulent phenotype expression was consistently linked to nucleotide 234 (U in cardiovirulent CVB3 and C in avirulent CVB3) in the 5' nontranslated region. Reconstructions of the parental noncardiovirulent CVB3 genome from chimeras restored the noncardiovirulent phenotype when tested in mice. Inoculation of severe combined immunodeficient (scid) mice with the noncardiovirulent CVB3 strain resulted in massive cardiomyocyte necrosis in all animals. Sequence analysis of viral genomes isolated from twelve scid mouse hearts showed that only nucleotide position 234 was different (a C-->U transition) from that in the input parental noncardiovirulent CVB3 genome. Higher-order RNA structures predicted by two different algorithms did not demonstrate an obvious local effect caused by the C-->U change at nucleotide 234. Initial studies of parental and chimeric CVB3 replication in primary cultures of fetal murine heart fibroblasts and in adult murine cardiac myocytes demonstrated that viral RNA transcriptional efficiency is approximately 10-fold lower for noncardiovirulent CVB3 than for cardiovirulent CVB3. CVB3 did not shut off protein synthesis in murine cardiac fibroblasts, nor were levels of viral protein synthesis significantly different as a function of viral phenotype. Taken together, these data support a significant role for determination of the CVB3 cardiovirulence phenotype by nucleotide 234 in the 5' nontranslated region, possibly via a transcriptional mechanism.

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 model for mouse beta Maj globin mRNA derived from enzymatic digestion data, comparative sequence and computer analysis.

A model for the secondary structure of mouse beta Maj globin messenger RNA is presented based on enzymatic digestion data, comparative sequence and computer analysis. Using 5'-32P-end-labeled beta globin mRNA as a substrate, single-stranded regions were determined with S1 and T1 nucleases and double-stranded regions with V1 ribonuclease from cobra venom. The structure data obtained for ca. 75% of the molecule was introduced into a computer algorithm which predicts secondary structures of minimum free energy consistent with the enzymatic data. Two prominent base paired regions independently derived by phylogenetic analysis were also present in the computer generated structure lending support for the model. An interesting feature of the model is the presence of long-range base pairing interactions which permit the beta globin mRNA to fold back on itself, thereby bringing the 5'- and 3'-noncoding regions within close proximity. This feature is consistent with data from other laboratories suggesting an interaction of the 5'- and 3'-domains in the mammalian globin mRNAs.

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.

Increased neurovirulence associated with a single nucleotide change in a noncoding region of the Sabin type 3 poliovaccine genome.

Most of the small number of cases of poliomyelitis which occur in countries where Sabin's attenuated poliovirus vaccines are used are temporally associated with administration of vaccine and involve polioviruses of types 2 and 3 (ref. 1). Recent studies have provided convincing evidence that the Sabin type 2 and 3 viruses themselves may revert to a neurovirulent phenotype on passage in man. We report here that a point mutation in the 5' noncoding region of the genome of the poliovirus type 3 vaccine consistently reverts to wild type in strains isolated from cases of vaccine-associated poliomyelitis. Virus with this change is rapidly selected on passage through the human gastrointestinal tract. The change is associated with a demonstrable increase in the neurovirulence of the virus.

Generating non-overlapping displays of nucleic acid secondary structure.

A new algorithm is presented which permits the display of nucleic acid secondary structure by computer. This algorithm circumvents the problem of overlapping portions of the molecule which is inherent in some other drawing programs. The results from this algorithm may also be used as input to the drawing algorithm previously reported in this journal [1] to untangle most of a drawing. The algorithm also represents the molecule in a form which makes visual comparisons for similarity quite easy since it guarantees that comparable features will reside in the same relative position in the drawings when the drawings are normalized.

Holoprosencephaly in infants of diabetic mothers.

We report seven infants of diabetic mothers, affected with holoprosencephaly malformation sequence. An additional 15 cases assembled from personal communications and the literature indicate that holoprosencephaly, like neural tube, cardiac, and caudal defects, is specifically increased in children of diabetic mothers. Incidence figures from newborn surveys demonstrate a risk for holoprosencephaly in infants of diabetic mothers comparable to the 1% risk for caudal regression malformation sequence. The embryologic timing of cranial, cardiac, and caudal defects emphasizes the need for pregnancy planning and diabetes control.