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PubMed1990

Nucleotide sequence of segment S9 of the genome of rice gall dwarf virus.

DNA complementary to the ninth largest (S9) of the 12 genome segments of rice gall dwarf virus (RGDV) was cloned and its sequence was determined. It is 1202 nucleotides in length and contains one open reading frame which extends for 969 nucleotides from nucleotide 26. It encodes a polypeptide of 323 amino acids with an Mr of 35,560. The dinucleotide sequence at the 5' end and the trinucleotide sequence at the 3' end of the plus strand, 5' GG--GAU 3', which are present in the RNA of both wound tumour virus (WTV) and rice dwarf virus (RDV), were also found in RGDV genome segment S9. The nucleotide sequences in the noncoding region at the 5' terminus and in the 15 nucleotides at the 3' terminus, which form an imperfect inverted repeat of 10 bp together with the 5' terminus, are approximately 70% homologous with those of the WTV genome segment S9, but only 30% and 50% homologous with the respective termini of RDV S9.

Amino Acid Sequence↗
PubMed1994

Consistent selection of mutations in the 5'-untranslated region of oral poliovirus vaccine upon passaging in vitro.

We have previously found that upon passaging type 3 oral poliovirus vaccine (OPV) in cell cultures the proportion of revertants at nucleotide 472 rapidly increases [Chumakov et al.: Proceedings of the National Academy of Sciences of the United States of America 88:199-203 1991]. Systematic study on the accumulation of these revertants showed that it was dependent on the multiplicity of infection and the temperature at which virus was grown. Revertants at position 472 of type 3 OPV accumulated faster in vaccines derived from Sabin Original (SO) substrain than from RNA-plaque purified (RSO) substrain. The rate of accumulation of 472-C revertants differed among cell lines and was higher in overgrown cell cultures suggesting that host factors are involved in the selection of mutants. We also found that accumulation of mutants occurred in vitro at position 480 in type 1 and position 481 in type 2 OPV, making the selection for revertants in domain F of the 5'-noncoding region a general phenomenon for all three Sabin strains. Assessment of the abundance of these mutants may be used for evaluation of the quality of OPV lots.

Base Sequence↗
PubMed1989

Chromatin structure and transcriptional regulation of human papillomavirus type 18 DNA in HeLa cells.

Mapping analysis of the nucleosomal organization of integrated human papillomavirus type 18 (HPV18) DNA in HeLa cells reveals a very prominent nuclease-hypersensitive site within the viral noncoding regulatory region that harbors transcriptional control sequences and coincides with most of the 5' ends of the cytoplasmic early mRNAs. Moreover, it is shown that the conserved coamplified 5' cellular flank, common to all HPV18 copies in HeLa cells and located close to the virus-cell integration site, also contains several distinct hypersensitive sites, accessible not only to DNase I but also to restriction enzymes. Nuclear run-on analysis in isolated HeLa nuclei demonstrates the occurrence of nascent transcripts covering the cellular flank (the late and the viral noncoding regulatory region), indicating that a cellular promoter, marked by the hypersensitive sites, cooperates with the viral control region in generating the HPV18 transcripts. Cycloheximide treatment of HeLa cells results in a reduction of the cytoplasmic steady-state level of the 3.5-kb mRNA corresponding to the viral E6, E7, and parts of the E1 open reading frames (ORFs), whereas the expression of the 1.6-kb transcript corresponding only to the E6 and E7 ORFs is not influenced. Nuclear run-on analysis carried out after the cycloheximide chase reveals that the distribution of nascent transcripts spanning the viral E6, E7, and parts of the E1 region is substantially decreased. In contrast to this finding, an even, pronounced increase of the elongation rate of those transcripts, which cover the cellular flank, the late and the viral noncoding regulatory region was noted indicating a different involvement of regulatory factors in the activity of both promoters.

Blotting, Southern↗
PubMed1996

Determination of the 5' and 3' terminal noncoding sequences of the bi-segmented genome of the avibirnavirus infectious bursal disease virus.

Terminal sequences of the bi-segmented dsRNA genome of 3 different strains of infectious bursal disease virus (IBDV) were analyzed by the rapid amplification of cDNA 5' ends (5'RACE) procedure. Both segments are 85% homologous in a 32-nucleotide sequence comprising the 5' end, whereas the 3' end has a conserved pentamer. Comparison to published terminal sequences of other IBDV strains revealed high conservation between the two segments but more serotype-specific nucleotide changes (5 on segment A and 3 on segment B) in the 5' noncoding region compared to the 3' noncoding region (none on segment A and 1 on segment B).

Animals↗
PubMed1991

Mutations responsible for adaptation of hepatitis A virus to efficient growth in cell culture.

Chimeric genomes of hepatitis A virus strain HM-175 were constructed from cDNA clones of the wild-type virus and its cell culture-adapted variant. RNA transcribed in vitro from each construct was assayed for infectivity by transfection of cultured cells. RNA transcribed from the wild-type cDNA clone was minimally infectious and produced virus that grew inefficiently in vitro, whereas that transcribed from certain chimeric genomes consistently produced virus that grew efficiently in cultured cells. Mutations in the P2 region were found to be necessary for efficient virus growth in vitro, while mutations in the 5' noncoding region imparted a conditional enhancement of growth in vitro.

Animals↗
PubMed1981

In vivo transcription of the 5'-terminal extracistronic region of vesicular stomatitis virus RNA.

In vivo transcription and polyadenylation at the junction of the L cistron and the 5'-terminal extracistronic region of vesicular stomatitis virus RNA was investigated. Annealing of 5'32P-labeled RNA representing the 5'-terminal noncoding 77 nucleotides of vesicular stomatitis virus genomic RNA to L gene mRNA resulted in specific duplex formation. Two specific RNase T1- and RNase A resistant duplexes, 66 and 77 nucleotides long, bound to oligodeoxythymidylic acid cellulose. The specific sizes of the duplexes and their selection by oligodeoxythymidylic acid cellulose chromatography demonstrated that they were covalently linked to the polyadenylic acid tail of L gene mRNA. These data strongly suggest that the viral polymerase polyadenylates L gene mRNA in vivo by using the stretch of seven uridine residues at the end of the L cistron and that the polymerase can resume transcribing the 5'-terminal extracistronic region, resulting in a covalent linkage of the transcript to the polyadenylic acid tail of L gene mRNA.

Genes↗
PubMed2005

Identification and characterization of human cytomegalovirus-encoded microRNAs.

MicroRNAs (miRNAs) are an extensive class of noncoding genes that regulate gene expression through posttranscriptional repression. Given the potential for large viral genomes to encode these transcripts, we examined the human cytomegalovirus AD169 genome for miRNAs using a bioinformatics approach. We identified 406 potential stem-loops, of which 110 were conserved between chimpanzee cytomegalovirus and several strains of human cytomegalovirus. Of these conserved stem-loops, 13 exhibited a significant score using the MiRscan algorithm. Examination of total RNA from human cytomegalovirus-infected cells demonstrated that 5 of the 13 predicted miRNAs were expressed during infection. These studies demonstrate that human cytomegalovirus encodes multiple conserved miRNAs and suggest that human cytomegalovirus may utilize an miRNA strategy to regulate cellular and viral gene function.

Base Sequence↗
PubMed1998

Effects of defined mutations in the 5' nontranslated region of rubella virus genomic RNA on virus viability and macromolecule synthesis.

The 5' end of the genomic RNA of rubella virus (RUB) contains a 14-nucleotide (nt) single-stranded leader (ss-leader) followed by a stem-and-loop structure [5'(+)SL] (nt 15 to 65), the complement of which at the 3' end of the minus-strand RNA [3'(-)SL] has been proposed to function as a promoter for synthesis of genomic plus strands. A second intriguing feature of the 5' end of the RUB genomic RNA is the presence of a short (17 codons) open reading frame (ORF) located between nt 3 and 54; the ORF encoding the viral nonstructural proteins (NSPs) initiates at nt 41 in an alternate translational frame. To address the functional significance of these features, we compared the 5'-terminal sequences of six different strains of RUB, with the result that the short ORF is preserved (although the coding sequence is not conserved) as is the stem part of both the 5'(+)SL and 3'(-)SL, while the upper loop part of both structures varies. Next, using Robo302, an infectious cDNA clone of RUB, we introduced 31 different mutations into the 5'-terminal noncoding region, and their effects on virus replication and macromolecular synthesis were examined. This mutagenesis revealed that the short ORF is not essential for virus replication. The AA dinucleotide at nt 2 and 3 is of critical importance since point mutations and deletions that altered or removed both of these nucleotides were lethal. None of the other mutations within either the ss-leader or the 5'(+)SL [and accordingly within the 3'(-)SL], including deletions of up to 15 nt from the 5'(+)SL and three different multiple-point mutations that lead to destabilization of the 5'(+)SL, were lethal. Some of the mutations within both ss-leader and the 5'(+)SL resulted in viruses that grew to lower titers than the wild-type virus and formed opaque and/or small plaques; in general mutations within the stem had a more profound effect on viral phenotype than did mutations in either the ss-leader or upper loop. Mutations in the 5'(+)SL, but not in the ss-leader, resulted in a significant reduction in NSP synthesis, indicating that this structure is important for efficient translation of the NSP ORF. In contrast, viral plus-strand RNA synthesis was unaffected by the 5'(+)SL mutations as well as the ss-leader mutations, which argues against the proposed function of the 3'(-)SL as a promoter for initiation of the genomic plus-strand RNA.

Animals↗
PubMed1990

Detection of hepatitis C virus RNA by a two-stage polymerase chain reaction with two pairs of primers deduced from the 5'-noncoding region.

The 5'-noncoding region of hepatitis C virus (HCV) genomes is highly conserved. A two-stage polymerase chain reaction (PCR), involving two pairs of primers deduced from the 5'-noncoding region of the HCV genome, was developed for a sensitive and specific detection of HCV RNA. The first stage of PCR was performed for 35 cycles with primers capable of multiplying fragments of 221 base pairs. PCR products in samples negative for HCV RNA were subjected to the second stage of PCR for 30 cycles with primers located internal to those employed in the first stage of PCR. The two-stage PCR detected up to 10 chimpanzee infectious doses/ml of HCV, and HCV RNA in 11 (92%) of 12 sera from patients with chronic non-A, non-B hepatitis without detectable antibodies to HCV by a commercial assay kit. Primers from the 5'-noncoding region of the HCV genome would be suitable for detecting HCV RNA by PCR, since the other regions of the HCV genome diverge extensively in sequence because of its nature as an RNA virus.

Animals↗
PubMed2002

High prevalence of GB virus C/hepatitis G virus genotype 3 among autochthonous Venezuelan populations.

GB virus C or hepatitis G virus (GBV-C/HGV) is highly prevalent among population groups at risk of parenterally transmitted viral agents, but it has also a worldwide distribution in other non-risk population groups. GBV-C/HGV RNA and antibodies against its envelope protein (anti-E2 Abs) were found in 3/86 (3%) and 7/89 (8%) of biomedical science personnel (BSP), in 31/453 (7%) and 37/200 (19%) of blood donors (BD), and in 6/64 (9%) and 26/59 (44%) of hemodialysis patients (HD) from Caracas, Venezuela. A significant gradient of GBV-C/HGV exposure (anti-E2 Abs and/or GBV-C/HGV RNA) was found between BSP (lowest prevalence), BD, and HD (P < 0.001). GBV-C/HGV RNA and anti-E2 Abs were also found in 2/69 (2.9%) and 2/44 (4.5%) of individuals from a rural community, in 9/162 (5.5%) and 2/40 (5%) of West Amerindians, and in 14/56 (25%) and 4/53 (7.5%) of South Amerindians. Socioeconomic and cultural factors may have contributed to the relatively high risk of exposure to GBV-C/HGV in BD and Amerindians. Whereas GBV-C/HGV genotypes 1 (n = 1), 2 (n = 6), and 3 (n = 22) were present in Venezuela, only the Asiatic genotype 3 was found infecting Amerindians and rural populations (n = 16). Genotype assignment based on the 5' noncoding region of the GBV- C/HGV genome was corroborated in some isolates by genetic analysis of the E2 region. This report confirms the circulation of the Asiatic genotype of GBV-C/HGV among Amerindians, suggesting an old origin of GBV-C/HGV. This might be associated with the apparently low pathogenesis of this virus.

Adolescent↗
PubMed1982

Genetic alterations of RNA leukemia viruses associated with the development of spontaneous thymic leukemia in AKR/J mice.

T1-oligonucleotide fingerprinting and mapping were used to study the expression of RNA leukemia viruses in leukemic and preleukemic AKR/J mice, with techniques designed to minimize the loss or inadvertent selection of viruses in vitro before biochemical analysis. In leukemic animals, complex mixtures of ecotropic and mink-tropic viruses were expressed. Unique but similar polytropic virus-like genomes were present in each tumor isolate. In preleukemic mice, viral isolates from the thymus that were grown on NIH3T3 fibroblasts contained genomes with non-Akv polytropic virus-related oligonucleotides. This phenomenon was not evident in fingerprints of viruses from the spleen and bone marrow of the same animals. Remarkably, the non-Akv oligonucleotides located in the 3' portion of the P15E gene, the U3 noncoding region, and the 5' part of the gp70 gene were often expressed independently. Our results suggest the following. (i) Recombinant viruses can be detected in the thymuses of young preleukemic AKR mice and increase in relative abundance with age. (ii) During in vivo generation of the recombinant leukemogenic viruses, the selection of polytropic virus-related sequences in the 3' part of p15E and the U3 region and the 5' portion of gp70 occurs independently. (iii) Independent biological properties encoded in the gp70 and p15E regions of env of the recombinant viruses may mediate viral selection or leukemogenicity. (iv) The leukemogenic polytropic viruses of AKR/J mice arise via genetic recombination involving at least three endogenous viral sequences.

Animals↗
PubMed1993

Nucleotide sequence and genetic organization of peanut clump virus RNA 2 and partial characterization of deleted forms.

The complete nucleotide sequence of peanut clump virus RNA 2 has been determined by characterization of cloned cDNA and by direct RNA sequencing. The sequence is 4503 residues in length and contains six long open reading frames (ORFs). The cistron for the coat protein (ORF 1) is located nearest the 5' end of the RNA and is separated from the 5' extremity by a noncoding region of 389 nucleotides. ORF 2 starts one nucleotide upstream of the first residue of the UGA termination codon of the coat protein cistron and encodes a polypeptide of 39300 M(r). Four additional ORFs with the potential to encode polypeptides with approximate M(r)'s of 51,000 (ORF 3), 13,800 (ORF 4), 9400 (ORF 5), and 6800 (ORF 6) are found on the 3' proximal half of the RNA. ORFs 3, 4, and 5 resemble the Triple Gene Block found in potexviruses, carlaviruses, a hordeivirus, and a furovirus. The 276 nucleotide 3' noncoding region of RNA 2 displays extensive sequence homology with the 3' noncoding region of RNA 1. Several peanut clump virus isolates propagated on Nicotiana benthamania contain RNA 2's significantly shorter than that of the type isolate. Partial characterization of two such isolates reveals that their RNA 2's have undergone deletions within ORF 2.

Amino Acid Sequence↗
PubMed1983

Endogenous avian retroviruses contain deficient promoter and leader sequences.

A sensitive and quantitative biological assay has been utilized to measure the ability of the exogenous and endogenous avian retroviral long terminal repeats (LTR) to promote gene expression in avian cells. This assay has revealed that the exogenous virus RAV-2 LTR is approximately equal to 10-fold more active than the LTRs of endogenous viruses RAV-0, ev-1, and ev-2. The endogenous viral LTRs show approximately equal activity. Upstream flanking cellular or viral sequences have no significant modulating effect on gene expression in our assay. Unexpectedly, we have detected and localized an additional defect outside of the LTR in the 5' noncoding leader sequence of ev-1 that further decreases gene expression relative to RAV-0 by approximately equal to 10-fold.

Animals↗
PubMed1998

Nucleotides in the panhandle structure of the influenza B virus virion RNA are involved in the specificity between influenza A and B viruses.

Influenza A and B viruses share common sequences and potentially similar panhandle structures in the terminal noncoding regions of virion RNA (vRNA). Interesting differences exist, however, in the number of conserved nucleotides at the 5' and 3' ends of the vRNAs, in base pairs constituting the panhandle duplex, and the length of uridine stretch (U stretch) juxtaposed to the RNA duplex. To analyse the contribution of these signals to the specificity between the two viruses, a transient ribonucleoprotein transfection method was used for the expression of the chloramphenicol acetyltransferase (CAT) reporter gene flanked by the noncoding nucleotides derived from influenza B vRNA. While the base pairing in the RNA duplex was primarily important for template activity, mismatch mutations G11 x G12' and C12 x A13' in the terminal RNA duplex region were utilized by influenza B virus, whereas these mutations were detrimental for influenza A virus. Different activity profiles were observed in the length preference of the RNA duplexes: maximum template activity was observed with 11 base pairs for influenza B virus, and 8 base pairs for influenza A virus. When the mutants with various lengths of U stretch were tested, highest CAT activities were observed with 5 to 7 uridine residues in influenza A virus, whereas in influenza B virus the activity was drastically decreased with 7 uridine residues. We suggest that the specific interaction of influenza virus RNA polymerase with these noncoding cis-acting signals in transcription of the RNA genome, along with unique coding strategies adopted by influenza B virus, has contributed to the divergence of these two closely related viruses.

Animals↗
PubMed1990

Polyomavirus late pre-mRNA processing: DNA replication-associated changes in leader exon multiplicity suggest a role for leader-to-leader splicing in the early-late switch.

Polyomavirus late mRNAs contain at their 5' ends multiple, tandem repeats of a 57-base noncoding sequence, the late leader, whose sequence appears only once in the viral genome. Pre-mRNA molecules are processed by a pathway that includes the splicing of late leader exons to each other in giant, multigenome-length precursors which are the result of inefficient transcription termination. We have devised a method involving reverse transcription and the polymerase chain reaction to determine the number of tandem late leader units on polyomavirus late RNA molecules. Using this technique, we have shown that each class of late viral mRNA (mVP1, mVP2, and mVP3) consists of molecules with between 1 and 12 tandem leader units at their 5' ends. Importantly, single-leader RNAs are underrepresented in both the cytoplasm and the nucleus, suggesting that single-leader primary transcripts are preferentially degraded in the nucleus. In addition, the average number of leaders on late RNAs increases in the presence of DNA replication. Taken together with previous work from our laboratory, the results presented here are consistent with a model for the control of late gene expression at the level of RNA splicing and stability which is in turn controlled by the efficiency of transcription termination.

Animals↗
PubMed1995

Molecular characterization of the 11th RNA segment from human group C rotavirus.

The complete nucleotide sequence of genome segment 11 from the noncultivatable, human group C rotavirus (Bristol strain) was determined. Comparison of the nucleotide sequence of the segment termini with the consensus 5' and 3' terminal noncoding sequences of the human group C rotavirus genome revealed characteristic 5' and 3' sequences. Human group C rotavirus genome segment 11 is 613 bp long and encodes a single open reading frame of 450 nucleotides (150 amino acids) starting at nucleotide 39 and terminating at nucleotide 489, leaving a long 3' untranslated region of 124 nucleotides. The predicted translation product has a calculated molecular weight of 17.7 kD and contains four potential N-linked glycosylation sites. No significant homologies to other viral proteins were found in database searches. Hydropathy analysis predicted the human group C rotavirus genome segment 11 translation product has a hydrophilic carboxy terminus (amino acids 54-150) and a hydrophobic amino terminus (amino acids 1-53) that can be further subdivided into three short hydrophobic sequences--H1, H2, and H3. These features are analogous to the integral membrane glycoprotein NSP4 encoded by group A rotavirus gene 10.

Amino Acid Sequence↗
PubMed1987

Kinetic analysis of T7 RNA polymerase-promoter interactions with small synthetic promoters.

Specific interactions between T7 RNA polymerase and its promoter have been studied by a simple steady-state kinetic assay using synthetic oligonucleotide promoters that produce a short five-base message. A series of promoters with upstream lengths extending to promoter positions -19, -17, -14, and -12 show that promoters extending to -19 and -17 produce very specific transcripts with initiation rate constant Kcat = 50 min-1 and a Michaelis constant Km = 0.02 microM, indicating that the consensus sequence to position -17 is sufficient for maximum promoter usage. Shortening the upstream region of the promoter to -14 substantially increases Km (0.3 microM) but does not significantly reduce the maximum velocity (kcat = 30 min-1). Finally, truncation of the promoter at position -12 results in extremely low levels of specific transcription. The coding and noncoding strands appear to make different contributions to promoter recognition. Although the double-stranded promoter of upstream length -12 is very poor as a transcription template, extension of only the noncoding strand to -17 very significantly improves both Kcat and Km. In contrast, extension of only the coding strand results in no significant improvement. Substitution of an AT base pair at position -10 by CG (as found in T3 RNA polymerase promoters) produces a 10-fold increase in Km, with little effect on Kcat. Comparison of two promoters containing a base pair mismatch at this site (AG or CT) demonstrates that promoter recognition is very sensitive to the nature of the base on the noncoding strand and is only slightly affected by the presence of a mismatch created by a wrong base in the coding strands.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

A determinant of disease symptom severity is located in the 3'-terminal noncoding region of the RNA of a plant virus.

Inoculation of Nicotiana tabacum plants with RNA transcribed in vitro from a variant (pXBS8) of a cloned full-length DNA copy of tobacco vein mottling virus (TVMV) RNA resulted in attenuation of the vein mottling and blotching symptoms typically produced by transcripts of cloned wild-type cDNA (pXBS7). Similar amounts of virus were detected by ELISA (using anti-TVMV coat protein serum) in systemically infected leaves of plants inoculated with pXBS7 or pXBS8 transcripts. pXBS8 was shown to contain a 58-nucleotide segment in the sequence corresponding to the 3'-terminal untranslated region of TVMV RNA that was not present in pXBS7. This segment resulted in the appearance in pXBS8 transcripts of four adjacent direct repeats of a 14-nucleotide sequence, AUAAUUAUAUAUAU, that is present in the 3'-untranslated region of TVMV RNA, with two additional nucleotides (AU) between the first and second repeats. Insertion of restriction fragments containing the segment into pXBS7 and inoculation of plants with transcripts of the chimeric construct (pXBS78) resulted in the attenuated-symptom phenotype and was not accompanied by a reduced accumulation of virus in the plant as determined by ELISA and Northern blot analysis. When the extra nucleotides were removed from the variant clone, symptoms induced by transcripts of the cDNA (pXBS87) resembled those induced by wild-type transcripts. The results indicate that a noncoding region of the genome can have a direct effect on the induction of disease symptoms by an RNA virus.

Base Sequence↗