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PubMed2000

Translation by ribosome shunting on adenovirus and hsp70 mRNAs facilitated by complementarity to 18S rRNA.

Translation initiation on eukaryotic mRNAs involves 40S ribosome association with mRNA caps (m(7)GpppN), mediated by initiation factor eIF4F. 40S eukaryotic ribosomes and initiation factors undergo 5' scanning to the initiation codon, with no known role for complementarity between eukaryotic 18S rRNA and the 5' noncoding region of mRNAs. We demonstrate that the 5' noncoding region of human adenovirus late mRNAs, known as the tripartite leader, utilizes a striking complementarity to 18S rRNA to facilitate a novel form of translation initiation referred to as ribosome shunting, in which 40S ribosomes bind the cap and bypass large segments of the mRNA to reach the initiation codon. Related elements are also shown to promote ribosome shunting in adenovirus IVa2 intermediate phase mRNA during virus infection and in human heat shock protein 70 (hsp70) mRNA for selective translation during heat shock. The importance of mRNA complementarity to 18S rRNA suggests that ribosome shunting may involve either specific RNA structural features or a prokaryotic-like interaction between mRNA and rRNA.

Adenoviruses, Human↗
PubMed1991

Oral poliovirus vaccine in the United States: molecular characterization of Sabin type 3 after replication in the gut of vaccinees.

Derivatives of Sabin 3 shed from recipients of oral poliovirus vaccine in the United States (U.S.) were examined for genetic changes identified in strains excreted by vaccinees in the United Kingdom [U.K.; Evans et al., 1985; Cammack et al., 1988, Macadam et al., 1989]. Among the eight primary vaccinees studied, the duration of excretion and molecular evolution of type 3 strains varied greatly. The period of virus excretion after vaccination ranged from as few as 2 days to as many as 36 days. Nucleotide sequence analysis of viral RNAs extracted from shed virus indicated that only fifty percent of the vaccinees exclusively excreted strains in which the attenuating mutation at nucleotide 472 in the 5' noncoding region of the genome had reverted from uracil (U) to cytosine (C), the nucleotide found in neurovirulent strains. Compared to the wild-type Leon strain, the low activity of stool isolate KW4 in a complete monkey neurovirulence test demonstrated that presence of C at 472 does not render a type 3 strain pathogenic. Conversely, an isolate was identified which efficiently replicated in monkey nervous tissue and maintained the attenuated U at 472. Oligonucleotide fingerprinting and sequence analysis of viral RNAs from stool isolates indicated that one vaccinee (KW) eventually excreted intertypic recombinant strains consistent with those reported in the U.K. studies. Unique to this study, one vaccinee (KS) excreted nonrecombinant virus possessing U at 472 for up to 21 days. The significance of the KS strain profile in relation to differences in the U.S. vaccine compared to the vaccine distributed in the U.K. and other countries is discussed.

Animals↗
PubMed1994

Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants.

In order to study cell tropism and attenuation of hepatitis A virus (HAV), the genome of HAV wild-type GBM and two cell culture-adapted variants, GBM/FRhK and GBM/HFS, were cloned and sequenced after amplification by reverse transcriptase-PCR. During virus cultivation, the HAV variant GBM/FRhK had a strict host range for FRhK-4 cells, in contrast to GBM/HFS, which can be grown in HFS and FRhK-4 cells. The HAV variant GBM/HFS was shown to be attenuated when inoculated into chimpanzees (B. Flehmig, R. F. Mauler, G. Noll, E. Weinmann, and J. P. Gregerson, p. 87-90, in A. Zuckerman, ed., Viral Hepatitis and Liver Disease, 1988). On the basis of this biological background, the comparison of the nucleotide sequences of these three HAV GBM variants should elucidate differences which may be of importance for cell tropism and attenuation. The comparison of the genome between the GBM wild type and HAV wild types HM175 (J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987) and HAV-LA (R. Najarian, O. Caput, W. Gee, S. J. Potter, A. Renard, J. Merryweather, G. Van Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985) showed a 92 to 96.3% identity, whereas the identity was 99.3 to 99.6% between the GBM variants. Nucleotide differences between the wild-type and the cell culture-adapted variants, which were identical in both cell culture-adapted GBM variants, were localized in the 5' noncoding region; in 2B, 3B, and 3D; and in the 3' noncoding region. Our result concerning the 2B/2C region confirms a mutation at position 3889 (C-->T, alanine to valine), which had been shown to be of importance for cell culture adaptation (S. U. Emerson, C. McRill, B. Rosenblum, S. M. Feinstone, and R. H. Purcell, J. Virol. 65:4882-4886, 1991; S. U. Emerson, Y. K. Huang, C. McRill, M. Lewis, and R. H. Purcell, J. Virol. 66:650-654, 1992), whereas other mutations differ from published HAV sequence data and may be cell specific. Further comparison of the two cell culture-adapted GBM variants showed cell-specific mutations resulting in deletions of six amino acids in the VP1 region and three amino acids in the 3A region of the GBM variant GBM/FRhK.

Adaptation, Biological↗
PubMed1989

The full-length nucleotide sequences of the virulent Trinidad donkey strain of Venezuelan equine encephalitis virus and its attenuated vaccine derivative, strain TC-83.

Nucleotide sequence analysis of cDNA clones covering the entire genomes of Trinidad donkey (TRD) Venezuelan equine encephalitis (VEE) virus and its vaccine derivative, TC-83, has revealed 11 differences between the genomes of TC-83 virus and its parent. One nucleotide substitution and a single nucleotide deletion occurred in the 5'- and 3'-noncoding regions of the TC-83 genome, respectively. The deduced amino acid sequences of the nonstructural polypeptides of the two viruses differed only in a conservative Ser(TRD) to Thr(TC-83) substitution in nonstructural protein (nsP) three at amino acid position 260. The two silent mutations (one each in E1 and E2), one amino acid substitution in the E1 glycoprotein, and five substitutions in the E2 envelope glycoprotein of TC-83 virus were reported previously (B.J.B. Johnson, R.M. Kinney, C.L. Kost, and D.W. Trent, 1986, J. Gen. Virol. 67, 1951-1960). The genome of TRD virus was 11,444 nucleotides long with a 5'-noncoding region of 44 nucleotides. The carboxyl terminal portion of VEE nsP3 contained two peptide segments (7 and 34 amino acids long) that were repeated with high fidelity. The open reading frame of the nonstructural polyprotein was interrupted by an in-frame opal termination codon between nsP3 and nsP4, as has been reported for Sindbis, Ross River, and Middelburg viruses. The deduced amino acid sequences of the VEE TRD nsP1, nsP2, nsP3, and nsP4 polypeptides showed 60-66%, 57-58%, 35-44%, and 73-71% identity with the aligned sequences of the cognate polypeptides of Sindbis and Semliki Forest viruses, respectively. The lack of homology in the nsP3 of the viruses is due to sequence variation in the carboxyl terminal half of this polypeptide.

Amino Acid Sequence↗
PubMed1995

Enzymatic properties of overexpressed HBV-mevalonate kinase fusion proteins and mevalonate kinase proteins in the human hepatoma cell line PLC/PRF/5.

We have previously reported a case of integration of HBV sequences in the 5'-noncoding region of the gene for mevalonate kinase (Mk) (EC.2.7.1.36) in the human hepatoma cell line PLC/PRF/5, resulting in overexpression of viral-cellular fusion transcripts and enhanced intracellular levels of the enzyme. Here, we present an evaluation of the functionalities of Mk and HBV/Mk fusion proteins derived from viral-cellular fusion- and Mk-transcripts, some of which lack 156 bp in the Mk coding region as a result of a differential splicing process. cDNA clones with a full-length Mk-ORF produce proteins which can metabolize mevalonate to its monophosphorylated form. Our results suggest that the enhanced and inappropriate expression of Mk may lead to increased metabolism of mevalonate and phosphorylation of hitherto unknown cellular proteins. This consequence of HBV-DNA insertion could thus be related to the activation of proteins that may be relevant in oncogenesis.

Alternative Splicing↗
PubMed1992

Importance of primer selection for the detection of hepatitis C virus RNA with the polymerase chain reaction assay.

We compared four primer sets from conserved regions of the hepatitis C virus (HCV) genome for their ability to detect HCV RNA in a "nested" cDNA polymerase chain reaction assay on sera from 114 anti-HCV antibody-positive individuals from around the world. The different primer sets had equivalent sensitivity, detecting less than 1 chimpanzee ID50 (dose that infects 50%) when tested against reference strain H of HCV. We tested equal amounts of RNA extracted from the serum of each individual with the four primer sets. The set derived from two highly conserved domains within the 5' noncoding (NC) region of the HCV genome, which also share significant similarity with Pestivirus 5' NC sequences, was the most effective at detecting HCV RNA. All samples positive for HCV RNA with any other primer set were also positive with the primer set from the 5' NC region, and the latter was at least 3 times more likely to detect HCV infection than a primer set from within the nonstructural protein 3-like gene region (P less than 0.001). We had no false positive results in greater than 500 negative controls interspersed among the test samples. The 5' NC region primer set detected HCV-specific RNA, verified by high-stringency Southern blot hybridization and DNA sequencing, in 100% of 15 acute and 33 chronic non-A, non-B hepatitis patients from the United States, Europe, and Asia and 10 hepatocellular carcinoma patients from Africa and Asia that tested negative for the hepatitis B virus-encoded surface antigen. In conclusion, use of an appropriate primer set is crucial for detecting HCV RNA in the serum of infected individuals.

Base Sequence↗
PubMed1998

Prevalence of hepatitis C and G virus infection in chronic hemodialysis patients.

An RNA virus designated hepatitis G virus (HGV) has been recently identified in patients with acute and chronic liver disease. HGV is transfusion transmissible, it has global distribution, and it is present in the volunteer blood donor population in the United States. One hundred sixty patients undergoing maintenance hemodialysis at the University of Miami-affiliated unit were evaluated. There were 99 men and 61 women ranging in age from 22 to 80 years. Sixty percent had a history of blood transfusion, 6% had a history of drug abuse, and 9% were infected with the human immunodeficiency virus. HGV-RNA was detected by reverse-transcriptase polymerase chain reaction with amplification of two independent regions (5'-nontranslated region and NS5a coding region). Detection of digoxigenin-labeled amplification products with specific capture probes to the coding and noncoding regions was performed with the Enzymun-test DNA on an ES-300 Immunoassay System (Boehringer-Mannheim, Mannheim, Germany). Hepatitis C antibodies were measured with anti-hepatitis C virus enzyme-linked immunosorbent third-generation assays and hepatitis C virus RNA by reverse-transcriptase polymerase chain reaction. There were 32 (20%) patients with detectable HGV RNA with both primer pairs. Because of possible mutations, the HGV virus may be detectable only with one primer pair. We considered the latter as indeterminate: 12 had detectable levels to the NS5a region only, seven to the 5'-nontranslated region, and six had borderline results. Detectable and indeterminate samples were confirmed by repeat measurements in a new blood sample. Seven of 24 (29%) patients with detectable hepatitis C virus RNA had coexisting HGV with one or both HGV primer pairs (four with both and three with one). Five patients were hepatitis B surface antigen positive and HGV negative. We conclude that HGV infection is prevalent in our dialysis patients. The clinical significance of HGV infection remains to be established.

Adult↗
PubMed1993

[Importance of primer selection in 5'NC region for the detection of hepatitis C virus RNA by polymerase chain reaction].

We compared two primer sets (A: 167bp, B: 269bp) derived from highly conserved domains within the 5' noncoding region (5'NC) of the hepatitis C virus (HCV) genome for their ability to detect HCV-RNA in a nested cDNA polymerase chain reaction assay (nested-PCR) in sera from 31 patients suspected of having HCV infection. Seventeen (54%) of 31 patients were positive for HCV-RNA with both primer sets. Using primer set A, 14(93%) of 15 samples with positive and 3(19%) of 16 samples with negative anti-HCV antibody test gave positive results for HCV-RNA. With primer set B, 15(100%) of 15 antibody positive samples and 2(13%) of 16 negative samples were positive for HCV-RNA. One antibody negative sample from a patient with alcoholic liver cirrhosis was positive for HCV-RNA only with primer set A. Another sample with positive antibody test, from a patient with chronic renal failure, was positive for HCV-RNA only with primer set B. A combination of more than one set of primers directed to the highly conserved 5'NC region, as well as proper selection of the exact nucleotide sequences, are important in improving the detection rate of HCV-RNA by PCR in serum of infected patients.

Base Sequence↗
PubMed1996

Poly(rC) binding protein 2 binds to stem-loop IV of the poliovirus RNA 5' noncoding region: identification by automated liquid chromatography-tandem mass spectrometry.

The 5' noncoding region of poliovirus RNA contains an internal ribosome entry site (IRES) for cap-independent initiation of translation. Utilization of the IRES requires the participation of one or more cellular proteins that mediate events in the translation initiation reaction, but whose biochemical roles have not been defined. In this report, we identify a cellular RNA binding protein isolated from the ribosomal salt wash of uninfected HeLa cells that specifically binds to stem-loop IV, a domain located in the central part of the poliovirus IRES. The protein was isolated by specific RNA affinity chromatography, and 55% of its sequence was determined by automated liquid chromatography-tandem mass spectrometry. The sequence obtained matched that of poly(rC) binding protein 2 (PCBP2), previously identified as an RNA binding protein from human cells. PCBP2, as well as a related protein, PCBP1, was over-expressed in Escherichia coli after cloning the cDNAs into an expression plasmid to produce a histidine-tagged fusion protein. Specific interaction between recombinant PCBP2 and poliovirus stem-loop IV was demonstrated by RNA mobility shift analysis. The closely related PCBP1 showed no stable interaction with the RNA. Stem-loop IV RNA containing a three nucleotide insertion that abrogates translation activity and virus viability was unable to bind PCBP2.

Amino Acid Sequence↗
PubMed1998

Determination of the nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 9 and its expression in BmN4 cells.

Cloning and sequencing of segment 9 of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strains H and I were performed. The segment consisted of 1,186 bp harboring 5' and 3' noncoding regions and an open reading frame from positions 75 to 1037, encoding a protein with 320 amino acids, termed NS5. Comparison of the nucleotide sequences of NS5 for the two strains indicated 37 point differences resulting in only six amino acid replacements. Homology search showed that NS5 has localized similarities to human poliovirus RNA-dependent RNA polymerase and human rotavirus NS26. By Western blot analysis, NS5 was found in BmCPV-infected midgut cells, but not in polyhedra or virus virions, and was mainly detectable in the nucleus in BmCPV-infected BmN4 cells. Immunoblot analysis with anti-NS5 and antipolyhedrin antibodies displayed marked differences in the period of expression of NS5 and polyhedrin: the polyhedrin molecule was first detected 2 or 3 days after infection with BmCPV, whereas the expression of NS5 was initiated within a few hours. In addition, the level of polyhedrin increased as the infection developed, whereas the amount of NS5 remained essentially constant. When segment 9 was expressed with a baculovirus expression system, the resulting NS5 protein possessed the ability to bind to the double-stranded RNA genome. These results suggest that NS5 is expressed in early stages of infection and contributes to regulation of genomic RNA function.

Amino Acid Sequence↗
PubMed1999

Recombination in Tula hantavirus evolution: analysis of genetic lineages from Slovakia.

To examine the evolution of Tula hantavirus (TUL), carried by the European common vole (Microtus arvalis and M. rossiaemeridionalis), we have analyzed genetic variants from Slovakia, the country where the virus is endemic. Phylogenetic analysis (PHYLIP) based on either partial (nucleotides [nt] 441 to 898) or complete N-protein-encoding sequences divided Slovakian TUL variants into two main lineages: (i) strains from eastern Slovakia, which clustered with Russian strains, and (ii) strains from western Slovakia situated closer to those from the Czech Republic. We found genetic diversity of 19% between the two groups and 4% within the western Slovakian TUL strains. Phylogenetic analysis of the 3' noncoding region (3'-NCR), however, placed the eastern Slovakian strains closer to those from western Slovakia and the Czech Republic, with a greater distance to the Russian strains, suggesting a recombinant nature of the S segment in the eastern Slovakian TUL lineage. A bootscan search of the S-segment sequences of TUL strains revealed at least two recombination points in the S sequences of eastern Slovakian TUL strains (nt 400 to 415 and around 1200) which agreed well with the pattern of amino acid substitutions in the N protein and deletions/insertions in the 3'-NCR of the S segment. These data suggest that homologous recombination events occurred in the evolution of hantaviruses.

3' Untranslated Regions↗
PubMed2002

[Amplification of hepatitis C virus 5' untranslated region gene by RACE and its secondary structure analysis].

OBJECTIVE: To obtain very end full-length cDNA of hepatitis C virus (HCV) 5' untranslated region (5' UTR), and analyse its primary and secondary structure. METHODS: By reverse transcription-nested polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP), a patient infected with genotype 2a HCV was found. Total RNA isolated from the serum as template, the cDNA of 5' noncoding region was amplified using rapid amplification of cDNA ends methods (RACE), the fragments were recombined by A-T clone strategy, the recombinants were confirmed by RFLP and PCR then sequenced. Secondary structures were analysed by RNA draw. RESULTS: Very end full-length cDNA of 2a genotype HCV 5' UTR was obtained by RACE. In five clones obtained, three contained full-length 5' UTR cDNA, and A21G, G170A, T222C, T247C, C339T substitutions were found compared with HC-J6. he homologies with HCV-1,HC-J6,HC-C2, HC-J8 were 93.6%-94.4%, 92.1%-93.0%, 98.8%-99.7%, 96.2%-96.5%, respectively; however, the substitutions did not alter the secondary structure. Two out of five clones were deleted to have 53 and 144 bases at 5' terminus of HCV 5' UTR, respectively. CONCLUSIONS: RACE is rapid and effective, works well to obtain very end of virus genome. With that, Authors obtained full-length cDNA of genotype 2a of HCV 5' UTR. There are genes deleted at 5' terminus circulated in hepatitis C patients.

5' Untranslated Regions↗
PubMed1995

TA1, a highly conserved oncofetal complementary DNA from rat hepatoma, encodes an integral membrane protein associated with liver development, carcinogenesis, and cell activation.

Hepatocellular carcinoma is characterized by changes in gene expression associated with cell growth and differentiation. Cell surface antigenic changes have also been described based on differential antibody reactivity between normal and neoplastic liver. We obtained a novel tumor-associated cDNA designated TA1 on the basis of its differential expression between hepatoma cells and normal liver. Sequence analysis predicted a 723-base pair open reading frame with the deduced amino acid sequence encoding an integral membrane protein containing multiple hydrophobic transmembrane domains. Database searches revealed TA1 as the likely rat homologue of E16, a recently cloned human cDNA associated with lymphocyte activation. Although noncoding sequences diverged significantly, the 95% conservation of the predicted proteins between species strongly suggests an important, although as yet undefined, function in normal cells. TA1 transcripts were detected in normal adult rat tissues including testes, brain, ovary, spleen, mammary gland, and uterus with the highest steady-state expression in placenta. Although no expression was detected in normal liver, all rat hepatomas examined expressed an abundant 3.2-kilobase transcript. TA1 expression was closely associated with progression in this tumor model and suggests this molecule, originally linked to cell activation, also plays a role in the malignant phenotype.

Amino Acid Sequence↗
PubMed1992

Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction.

A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected by idiopathic dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackie-virus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several enterovirus species (replicase gene, 5' noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothiocyanate extraction from tissue stored frozen at -80 degrees C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated by electrophoresis on a 10% polyacrylamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of 10(5) to 10(6) cells starting from 1 microgram total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1.

Adult↗
PubMed1998

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Dengue↗
PubMed1997

Influence of the 5' noncoding region of hepatitis A virus strain GBM on its growth in different cell lines.

Previous sequence analysis of consecutive passages of the hepatitis A virus (HAV) strain GBM/WT in human embryonic kidney cells (HEK cells), human embryonic lung fibroblasts (HFS cells) and in FRhK-4 cells (foetal rhesus monkey kidney cells) pointed to a host cell dependent cell culture adaptation of GBM/WT in HFS cells involving mutations in the 5' noncoding region (5'NCR). Multiple nucleotide changes occurred in the 5'NCR of the GBM genome after the cell line used for virus passage was changed from HEK cells to HFS cells. In contrast, no mutations in the 5'NCR occurred during the first 20 passages of GBM/WT in FRhK-4 cells. In order to analyse the influence of the 5'NCR on host cell specific adaptation of HAV strain GBM in different cell cultures, GBM/HM175 chimeras were constructed which contained 5'NCRs from different GBM variants by replacing the 5'NCR of the infectious clone pHAV/7. Parallel transfection assays in FRhK-4 and HFS cells, performed with transcripts from the chimeric GBM/HM175 constructs, showed that the 5'NCR of the GBM variant GBM/HFS is essential for virus growth in HFS cells. The GBM/HM175 chimeric RNA, which contained the 5'NCR of GBM/HFS, exclusively, was able to produce infectious virus after transfection of HFS cells. The growth of the different GBM/HM175 chimeras in FRhK-4 cells, in contrast, did not seem to be strongly influenced by a specific sequence of the 5'NCR.

Animals↗
PubMed2001

Sequence comparison and secondary structure analysis of the 3' noncoding region of flavivirus genomes reveals multiple pseudoknots.

Sequences of 191 flavivirus RNAs belonging to four sero-groups were used to predict the secondary structure of the 3' noncoding region (3' NCR) directly upstream of the conserved terminal hairpin. In mosquito-borne flavivirus RNAs (n = 164) a characteristic structure element was identified that includes a phylogenetically well-supported pseudoknot. This element is repeated in the dengue and Japanese encephalitis RNAs and centers around the conserved sequences CS2 and RCS2. In yellow fever virus RNAs that contain one CS2 motif, only one copy of this pseudoknotted structure was found. The conserved pseudoknotted element is absent from the 3' NCR of tick-borne virus RNAs, which altogether adopt a secondary structure that is very different from that of mosquito-borne virus RNAs. The strong conservation of the pseudoknot in mosquito-borne flavivirus RNAs implies a stronger relationship between these viruses than concluded from previous secondary structure analyses. The role of the (tandem) pseudoknots in flavivirus replication is discussed.

Base 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↗