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PubMed1978

Management of hypercholesterolemia: evaluation of practical clinical approaches in healthy young adults.

A work site-located clinic screened 6,000 employees (91 percent participation) and identified 146 hypercholesterolemic subjects (100 percent initial participation, 12 percent subsequent dropout rate). The subjects, aged 20 to 50 years, were randomly classified into four groups: Group A, treatment in a lipid intervention clinic with diet for 6 weeks, then diet plus clofibrate for the subsequent 18 weeks; Group B, diet treatment from a clinic nutritionist with the cooperation of the subject's private physician; Group C, referral for treatment by a private physician; and Group D, no intervention. Initial mean cholesterol was 294 mg/100 ml. At 24 weeks, all intervention groups had decreases in serum cholesterol (Group A, 12 percent; Group B, 15 percent; Group C, 17 percent; P less than 0.001). The control group (D) had a small decrease in cholesterol (4 percent). Decreases in cholesterol were correlated with weight loss and decrease in fasting serum triglycerides but not with the use of clofibrate. Serum cholesterol can be reduced in healthy young adults by several practical methods.

Adult↗

PubMed2006

Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling.

The Epstein-Barr virus (EBV) noncoding RNAs, EBV-encoded RNA 1 (EBER1) and EBER2, are the most abundant viral transcripts in all types of latently infected human B cells, but their function remains unknown. We carried out heterokaryon assays using cells that endogenously produce EBERs to address their trafficking, as well as that of the La protein, because EBERs are quantitatively bound by La in vivo. Both in this assay and in oocyte microinjection assays, EBERs are confined to the nucleus, suggesting that their contribution to viral latency is purely nuclear. EBER1 does not bind exportin 5; therefore, it is unlikely to act by interfering with microRNA biogenesis. In contrast, La, which is a nuclear phosphoprotein, undergoes nucleocytoplasmic shuttling independent of the nuclear export protein Crm1. To ensure that small RNA shuttling can be detected in cells that are negative for EBER shuttling, we demonstrate the shuttling of U1 small nuclear RNA.

Active Transport, Cell Nucleus↗

PubMed2006

RNASEN regulates cell proliferation and affects survival in esophageal cancer patients.

PURPOSE: MicroRNAs (miRNA) are small noncoding RNAs thought to be involved in physiologic and developmental processes by negatively regulating the expression of target genes. Little is known about the role of miRNAs in normal and cancer cells. It is possible that deregulation of miRNA may contribute to the oncogenesis of some cancers. We studied the expression level of the miRNA processing enzyme (DICER1, DGCR8, and RNASEN) in esophageal squamous cell carcinoma (ESCC). EXPERIMENTAL DESIGN: The expression levels of DICER1, DGCR8, and RNASEN mRNA in 73 ESCC tissues were compared with that in corresponding normal esophageal epithelium by Taqman real-time reverse-transcription PCR. We also examined RNASEN protein expression in 27 cell lines. The role of RNASEN in cell proliferation in ESCC cells was assessed by small interfering RNA. Paraffin sections of ESCC patients were immunohistochemically investigated. RESULTS: We found that RNASEN expression levels were enhanced in a fraction of esophageal cancers. Multivariate Cox regression analysis showed that the prognostic effect of RNASEN (P = 0.0036) seems to be independent of disease stage (P = 0.0060). Knockdown of RNASEN in esophageal cancer cell lines resulted in a 46% to 85% reduction in cell number. In an immunohistochemical study, the intensity of RNASEN expression was often increased in the tumor compared with that in normal epithelium. CONCLUSIONS: The relationship between the RNASEN expression and the prognosis of the ESCC patients warrants a further study on the role of miRNA and tumor progression.

Adult↗

PubMed1997

Sequence analysis of hepatitis C virus variants producing discrepant results with two different genotyping assays.

Methods for identifying the genotype of hepatitis C virus (HCV) in clinical specimens are frequently based upon the direct characterisation of viral RNA sequences by polymerase chain reaction (PCR) amplification, or by serologically based methods, in which the infecting genotype is inferred from the pattern of antibody reactivity to type-specific peptides or recombinant proteins used as antigens in an Enzyme Linked Immunosorbent Assay (ELISA). Although genotyping by direct, PCR-based methods show generally highly concordant results with the genotype inferred from serological typing assays (> 95% agreement), there exist a small number of samples that produce discrepant results. To investigate the underlying reasons for the discrepancies, we obtained eleven samples from haemophiliacs and four samples from patients with chronic hepatitis C that produced discordant results between a PCR based assay (InnoLipa I and II) and a serotyping assay (Murex HC02). Nucleotide sequences in the 5'noncoding region (5'NCR), core, and NS4 region were used to identify the genotype of the circulating virus and to identify amino acid changes in NS4 that might alter antigenicity. In 14 samples, sequence analysis of all three regions was concordant with the results of the InnoLipa assay. There were few if any amino acid substitutions in NS4 that might have accounted for the discrepant serotyping results, which were found predominantly in samples from individuals with a history of multiple exposure to HCV. It remains unclear whether the detection of antibody in such discrepant samples corresponds to previous expression of a different genotype than detected by PCR, or whether the virus population in plasma is more restricted in genotype diversity than the population in the liver or at other sites of viral replication.

Amino Acid Sequence↗

PubMed2003

A panoramic view of yeast noncoding RNA processing.

Predictive analysis using publicly available yeast functional genomics and proteomics data suggests that many more proteins may be involved in biogenesis of ribonucleoproteins than are currently known. Using a microarray that monitors abundance and processing of noncoding RNAs, we analyzed 468 yeast strains carrying mutations in protein-coding genes, most of which have not previously been associated with RNA or RNP synthesis. Many strains mutated in uncharacterized genes displayed aberrant noncoding RNA profiles. Ten factors involved in noncoding RNA biogenesis were verified by further experimentation, including a protein required for 20S pre-rRNA processing (Tsr2p), a protein associated with the nuclear exosome (Lrp1p), and a factor required for box C/D snoRNA accumulation (Bcd1p). These data present a global view of yeast noncoding RNA processing and confirm that many currently uncharacterized yeast proteins are involved in biogenesis of noncoding RNA.

Cells, Cultured↗

PubMed2000

Severe phenotypes associated with inactive ring X chromosomes.

Mental retardation and congenital malformations in individuals with small ring X chromosomes are often due to the functional disomy that results from failure of these chromosomes to undergo X inactivation. Such chromosomes either lack the XIST locus or do not express it. We have carried out genetic analysis of the ring X chromosomes from two girls with a 45,X/46,X,r(X) karyotype, mental retardation, and a constellation of abnormalities characteristic of the severe phenotype due to X disomy. In each case the ring X chromosome included an intact XIST locus which was expressed; the breakpoints were distal to DXS128, and therefore outside the XIC region; transcription analysis of alleles at the androgen receptor locus confirmed that these were inactive chromosomes. The characteristics of the XIST RNA were similar to the wild-type. Additional studies in cultured fibroblasts showed a second ring in a small percentage of the cells. The association of severe phenotype with an inactive X chromosome most likely reflects the presence of a second ring X chromosome which was active at least in some tissues during embryogenesis, but is no longer prominent in the tissues we analyzed.

Dosage Compensation, Genetic↗

PubMed1991

Transcripts of simian virus 41 (SV41) matrix gene are exclusively dicistronic with the fusion gene which is also transcribed as a monocistron.

The complete nucleotide sequences of the matrix (M) and fusion (F) genes of simian virus 41 (SV41) were determined. Deduced amino acid sequences confirmed the close relationship of SV41 with human parainfluenza type 2 virus (PIV2). Analyses of noncoding regions between the F and the hemagglutinin-neuraminidase (HN) genes suggested the absence of the small hydrophobic gene, which is present between the F and the HN genes of simian virus 5 and mumps virus. It was striking that there was no apparent consensus gene end sequence between the M and the F genes and that the M gene was transcribed exclusively as a dicistron with the F gene. The number of monocistronic transcripts of the F gene was approximately half that of the dicistronic transcripts. However, the F protein of SV41 seemed to be efficiently translated, since viral multiplication and fusion from within were as efficient as in PIV2. These results suggest that the lack of a consensus gene end sequence resulted in the readthrough of viral RNA polymerases between the M and the F genes and that the initiation of F gene transcription could occur by newly entered polymerases independently of the polymerases that started the upstream M gene transcription.

Amino Acid Sequence↗

PubMed1991

Host-derived 5' ends and overlapping complementary 3' ends of the two mRNAs transcribed from the ambisense S segment of Uukuniemi virus.

Two mRNAs, coding for the N and NSS proteins, are transcribed from the small (S) Uukuniemi virus RNA segment by an ambisense strategy (J. F. Simons, U. Hellman, and R. F. Pettersson, J. Virol. 64:247-255, 1990). In this report, we describe the analysis of the 5' and 3' ends of the two mRNAs. Primer extension as well as cloning and sequencing of individual mRNAs showed that the 5' ends of both mRNAs contained nonviral sequences ranging from 7 to 25 residues in length (mean, 12 residues), indicating a cap-snatching mechanism similar to the one originally described for priming of influenza virus mRNA synthesis. In 35% of the cases, the first virion-specified nucleotide (an A residue) was substituted with a G residue. Between the translation termination codons of N and NSS, there is a 74-residue-long noncoding intergenic region (Simons et al., J. Virol. 64:247-255, 1990). Nuclease protection assays using both RNA and DNA hybridization probes showed that the 3' ends of the N and NSS mRNAs overlap each other by about 100 nucleotides. The 3' end of the NSS mRNA extends into the coding sequence of the N mRNA, whereas the N mRNA is terminated just prior to the stop codon of NSS. To our knowledge, this is the first example of overlapping complementary mRNAs in viruses with an ambisense coding strategy. No obvious transcription termination sequence was identified. However, because of a short palindromic sequence in the intergenic region, the 3' ends of both mRNAs (and consequently also the template RNAs) can be folded into an A/U-rich hairpin structure. It remains to be determined whether this structure plays any role in transcription termination.

Amino Acid Sequence↗

PubMed1998

Alterations of per RNA in noncoding regions affect periodicity of circadian behavioral rhythms.

Circadian rhythms in Drosophila depend on a molecular feedback loop that includes products of the period (per) and timeless (tim) genes. RNA and protein products of both genes cycle with a circadian period and the proteins feedback to inhibit expression of their own mRNAs. While cyclic expression of PER protein appears to be necessary for rhythmic behavior, the function of per RNA cycling is somewhat controversial. Rhythmic transcription accounts, in part, for cycling of per RNA, but it is clear now that posttranscriptional mechanisms also contribute to the cyclic expression of both per RNA and protein. As posttranscriptional mechanisms, such as mRNA stability and translation, are frequently mediated by 3' untranslated regions (UTR) of genes, the authors examined the role of this region of per in the regulation of circadian rhythms. Removal of most of per's 3' UTR had a small effect on the function of a per transgene. However, replacement of per's 3'UTR with corresponding sequences of the tubulin gene led to the rescue of behavioral rhythms in per01 flies with periods that were 3 h shorter than those generated by a wild-type per transgene. The hybrid RNA cycles, but the protein produced by it accumulates earlier in a day-night cycle than the PER protein produced by a control per transgene carrying its own 3'UTR, perhaps because the tubulin sequences counteract the effect of destabilizing elements in the per RNA at earlier points in the circadian cycle. These data indicate that the appropriate regulation of per RNA expression, effected by transcriptional as well as posttranscriptional mechanisms, is critical for the determination of circadian period.

Animals↗

PubMed2006

Escherichia coli Hfq binds A18 and DsrA domain II with similar 2:1 Hfq6/RNA stoichiometry using different surface sites.

Hfq is a RNA-binding protein in Escherichia coli that plays an essential role in post-transcriptional regulation of mRNAs by facilitating pairing of noncoding RNAs (ncRNAs) to mRNA target sites. Recent work has provided evidence that E. coli Hfq has two distinct RNA-binding surfaces. In this study, a comparative sequence-structure analysis of hfq genes in bacterial genomes was employed to identify conserved residues that may be involved in binding RNA. A covariance of residue properties at neighboring positions 12 and 39 and conserved surface residues with high propensities at binding sites of RNA-binding proteins suggested several sites for Hfq-RNA interactions. On the basis of these predictions, eight mutant Hfq proteins were produced and their interactions were examined with the 38 nucleotide (nt) domain II of DsrA ncRNA (DsrA(DII)) and A(18) by a gel-mobility shift assay, fluorescence anisotropy, and fluorescence quenching. Mutations on the proximal surface of Hfq had a small affect on Hfq binding to A(18) (<or=2-fold), while the mutations Y25A and K31A on the distal surface decreased affinity to A(18) by 100-fold in solution. Mutations F39A and R16A on the proximal surface reduced affinity to DsrA(DII) by 6-8-fold, while other mutations on the distal or proximal surfaces affected affinity to DsrA(DII) by <or=2-fold using the gel-mobility shift assay. The F39A/L12F double mutation partially regained the affinity for DsrA(DII) lost by the F39A mutation. The latter observation is consistent with the implied importance of an aromatic residue at position 12 or 39 suggested by the sequence covariance. Titration experiments indicate a 2:1 Hfq(6)/RNA stoichiometry for the strong binding complexes of Hfq with either A(18) or DsrA(DII) and suggests that RNA-induced dimer formation of Hfq(6) is a common feature of Hfq-RNA interactions.

Amino Acid Sequence↗

PubMed2005

MicroRNA-binding viral protein interferes with Arabidopsis development.

MicroRNAs (miRNAs) are small (approximately 21 nt), noncoding RNAs that negatively regulate target mRNAs at the posttranscriptional level that are involved in development. In plants, virus-induced disease symptoms often result in developmental abnormalities resembling perturbation of miRNA-mediated function. Here, we report that expression in transgenic plants of a geminivirus-encoded AC4 protein from African cassava mosaic virus Cameroon Strain (ACMV), a suppressor of posttranscriptional gene silencing, was correlated with decreased accumulation of host miRNAs and increased development abnormalities in Arabidopsis. Down-regulation of miRNA correlated with an up-regulation of target mRNA level. In vitro binding assays revealed the ability of AC4 of ACMV (A-AC4) but not East African cassava mosaic Cameroon virus AC2 to bind single-stranded forms of miRNAs and short interfering RNAs but not double-stranded RNA forms. Normally, a labile intermediate during the miRNA biogenesis/RNA-induced silencing complex assembly, miRNA*, was below the level of detection, indicating that AC4 might interfere at a point downstream of the miRNA duplex unwinding process. The association of AC4 with miRNA was demonstrated by the association of A-AC4-GFP fusion protein, extracted from Arabidopsis protoplasts, with 2'-O-methyloligonucleotide complementary to miR159 (miR159*) and by the presence of miRNA with the A-AC4-GFP fusion protein after immunoprecipitation with antibody against GFP. In both assays, A-AC4 protein and miRNA complexes were copurified. These results provide direct evidence that AC4 is a unique virus-encoded posttranscriptional gene-silencing suppressor protein that binds to and presumably inactivates mature miRNAs and thus blocks the normal miRNA-mediated regulation of target mRNAs, resulting in developmental defects in Arabidopsis.

Arabidopsis↗

PubMed2006

Expression and function of human steroid receptor RNA activator in prostate cancer cells: role of endogenous hSRA protein in androgen receptor-mediated transcription.

Steroid receptor RNA activator (SRA) was first isolated as a steroid receptor co-activator that functioned as an RNA transcript. Later, we demonstrated that SRA needs to be translated in order to co-activate androgen receptor (AR). Here, we showed that three isoforms of human SRA enhanced AR activities. Small interfering RNA against SRA suppressed AR activities in PC-3 cells transfected with pSG5AR and in LNCaP cells that have an endogenous mutated-AR. Western blot showed that SRA protein was expressed at a higher level in PC-3 than in LNCaP cells, suggesting that SRA may be related to hormone-independent growth of prostate cancer.

Base Sequence↗

PubMedDate not supplied

Renal pathology in WAGR syndrome.

The Wilms' tumor-aniridia-genital anomalies-mental retardation (WAGR) syndrome is associated with an increased risk for developing Wilms' tumor. A right nephrectomy was performed following the diagnosis of Wilms' tumor in a 2-year-old girl with WAGR syndrome and chromosome 11, del 11p13. Pathologic examination revealed intralobar nephrogenic rests and a peripelvic multicystic mass, sharply delineated from the adjacent typical intralobar nephrogenic rests and renal parenchyma, which may represent a cystic Wilms' tumor (cystic partially differentiated nephroblastoma). We studied the expression of the H19 gene by in-situ hybridization performed on paraffin sections of the kidney. H19 is an imprinted maternally-expressed gene that is not translated to protein and functions as a regulatory RNA molecule. It is tightly linked with the paternally-imprinted gene of insulin-like growth factor 2. While IGF2 presumably plays a role in tumorigenesis of Wilms' tumor, H19 is not expressed in the majority of Wilms' tumors. The expression of H19 in the intralobar nephrogenic rests was found to be prominent in the component of the blastema and markedly reduced with differentiation to tubular structures similar to the fetal kidney. The differential diagnosis of hyperplastic intralobar nephrogenic rests from a small Wilms' tumor arising in intralobar nephrogenic rests is difficult. Complete understanding of the chain of molecular events occurring in the evolution of Wilms' tumors may lead to the development of tumor markers to be used on paraffin sections and so help in the differential diagnosis of hyperplasia versus malignant transformation.

Child, Preschool↗

PubMed2004

MicroRNA control of PHABULOSA in leaf development: importance of pairing to the microRNA 5' region.

MicroRNAs (miRNAs) are approximately 22-nucleotide noncoding RNAs that can regulate gene expression by directing mRNA degradation or inhibiting productive translation. Dominant mutations in PHABULOSA (PHB) and PHAVOLUTA (PHV) map to a miR165/166 complementary site and impair miRNA-guided cleavage of these mRNAs in vitro. Here, we confirm that disrupted miRNA pairing, not changes in PHB protein sequence, causes the developmental defects in phb-d mutants. In planta, disrupting miRNA pairing near the center of the miRNA complementary site had far milder developmental consequences than more distal mismatches. These differences correlated with differences in miRNA-directed cleavage efficiency in vitro, where mismatch scanning revealed more tolerance for mismatches at the center and 3' end of the miRNA compared to mismatches to the miRNA 5' region. In this respect, miR165/166 resembles animal miRNAs in its pairing requirements. Pairing to the 5' portion of the small silencing RNA appears crucial regardless of the mode of post-transcriptional repression or whether it occurs in plants or animals, supporting a model in which this region of the silencing RNA nucleates pairing to its target.

Alleles↗

PubMed2005

A systematic search for new mammalian noncoding RNAs indicates little conserved intergenic transcription.

BACKGROUND: Systematic identification and functional characterization of novel types of noncoding (nc)RNA in genomes is more difficult than it is for protein coding mRNAs, since ncRNAs typically do not possess sequence features such as splicing or translation signals, or long open reading frames. Recent "tiling" microarray studies have reported that a surprisingly larger proportion of mammalian genomes is transcribed than was previously anticipated. However, these non-genic transcripts often appear to be low in abundance, and their functional significance is not known. RESULTS: To systematically search for functional ncRNAs, we designed microarrays to detect 3,478 intergenic and intronic sequences that are conserved between the human, mouse, and rat genomes, and that score highly by other criteria that characterize ncRNAs. We probed these arrays with total RNA isolated from 16 wild-type mouse tissues. Among 55 candidates for highly-expressed novel ncRNAs tested by northern blotting, eight were confirmed as small, highly-and ubiquitously-expressed RNAs in mouse. Of the eight, five were also detected in rat tissues, but none were detected at appreciable levels in human tissues or cultured cells. CONCLUSION: Since the sequence and expression of most known coding transcripts and functional ncRNAs is conserved between human and mouse, the lack of northern-detectable expression in human cells and tissues of the novel mouse and rat ncRNAs that we identified suggests that they are not functional or possibly have rodent-specific functions. Our results confirm that relatively little of the intergenic sequence conserved between human, mouse and rat is transcribed at high levels in mammalian tissues, possibly suggesting a limited role for transcribed intergenic and intronic sequences as independent functional elements.

Animals↗

PubMed1991

Pretranslational mechanisms determine the type of potassium channels expressed in the rat skeletal and cardiac muscles.

We have cloned a cDNA (RMK2) coding for a Shaker type delayed rectifier K+ channel from a rat skeletal muscle cDNA library. The clone encodes a putative protein of 602 amino acids, identical with a rat brain K+ channel Kv1 (Swanson, R., Marshall, R., Smith, J. S., Williams, J. B., Boyle, M. B., Folander, K., Luneau, C. J., Antanavage, J., Oliva, C., Burhow, S. A., Bennet, C., Stein, R. B., and Kaczmarek, L. K. (1990) Neuron 4, 929-939). Northern blot analysis showed that RMK2 is expressed in skeletal and cardiac muscle. RNase protection analysis showed that the 3'-noncoding regions of the brain, cardiac, and skeletal muscle RMK2 transcripts are identical. Cloning of the gene confirmed that the protein is encoded by a single exon (Swanson et al. (1990) Neuron 4, 929-939). We expressed RMK2 in Xenopus oocytes and showed that it encodes noninactivating delayed rectifier K+ channels, resistant to block by external tetraethylammonium, with a small unitary conductance of 8.0 picosiemens. Coinjection of RMK2 and RCK1 (RMK1) (Baumann, A., Grupe, A., Ackermann, A., and Pongs, O. (1988) EMBO J. 7, 2457-2463; Koren, G., Liman, E. R., Logothetis, D. E., Nadal-Ginard, B., and Hess, P. (1990) Neuron 4, 39-51) into Xenopus oocytes resulted in the expression of currents that have tetraethylammonium inhibition curves that differ from the linear combination of inhibition curves of the two types expressed individually. Thus, RMK2 and RCK1 (RMK1) can form heteromultimers. RNA blot hybridization analysis revealed that the RMK2 transcript is developmentally regulated in a different manner in the rat skeletal muscle, ventricle, and atrium.

Aging↗

PubMed1990

Molecular biology of macrophage colony-stimulating factor.

In this chapter we have described one of the more complex hemopoietic factors, M-CSF. The single-copy M-CSF gene is almost 21 kb in length and is arranged into 10 exons and 9 introns. Expression of the gene at the RNA level is heterogeneous, and several species of M-CSF mRNA have been found in human and murine cells and tissues. In human cells the different mRNAs arise from alternative splicing of the nuclear RNA precursor in both coding and noncoding regions. This results in mRNAs encoding two distinct M-CSF proteins, 256 and 554 amino acids in length. In murine cells only a 552-amino-acid form has been found thus far. All forms of M-CSF have a 32-amino-acid signal peptide and a 23-amino-acid hydrophobic region near the carboxy-terminus, which resembles a transmembrane domain. A large portion of the carboxy-terminal end, including the hydrophobic region, is not found in the mature protein. Thus, the primary translation product of M-CSF is a prepropolypeptide, with processing occurring at both amino- and carboxy-terminal ends. The exact size of the mature protein is still somewhat in doubt, but deletion mutagenesis from the carboxy-terminal end indicates that the protein may be as small as 150 amino acids and still be functional. Site-directed mutagenesis has also shown that the first seven cysteines in the mature molecule are probably necessary for biological activity, whereas the next two cysteine residues are not. In spite of the heavy glycosylation found in the native protein, removal of the N-linked glycosylation signals does not seem to affect activity to any great degree. The M-CSF gene and its receptor, C-FMS, are tightly linked on the long arm of chromosome 5, a unique finding in the ligand/receptor field. This region also contains the genes for GM-CSF, IL-3, ECGF, and the receptor for PDGF. A similar situation may exist on chromosome 11 of the mouse. The close linkage of these factors and receptors is the probable cause for the disorders of hemopoiesis that arise when deletions occur in this area. The preceding discussion has shown how quickly the area of M-CSF molecular biology has advanced in the past 2-3 years. A great deal of effort is now being directed toward expressing M-CSF at high levels in a variety of prokaryotic and eukaryotic systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1998

Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus.

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.

Animals↗