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PubMed1995

The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus.

The sequences of the 3'-noncoding regions (NCR) of 12 strains of tick-borne encephalitis (TBE) virus were analyzed and found to vary in length from 350 to approximately 750 nucleotides. The size heterogeneity is restricted to a variable region following the stop codon, whereas the most 3'-terminal 350 nucleotides form a highly conserved core element containing several potentially important sequence motifs and secondary structure elements. A homoadenosine tract previously thought to form the 3'-terminus of some TBE virus strains was now shown to be an internal part of the variable region of certain strains. The strains included in this study were isolated from both humans and ticks over a time period of more than 40 years at various locations throughout the entire endemic area of TBE virus, but there was no correlation between these parameters and the observed lengths of the 3'-NCRs. Identity data calculated from common 3'-NCR sequences and also from short sections of the open reading frame indicated that coding and noncoding sequences were linked during evolution, but the lengths of the 3'-NCRs were independent of these relationships. These observations together with detailed analyses and alignments of the sequences suggest that the variable region was originally acquired through duplication and recombination events, but--much more recently during evolution--various portions of this region were lost again, resulting in the now observed heterogeneous 3'-NCRs.

Animals↗
PubMed1992

Evolution of the H3 influenza virus hemagglutinin from human and nonhuman hosts.

The nucleotide and amino acid sequences of 40 influenza virus hemagglutinin genes of the H3 serotype from mammalian and avian species and 9 genes of the H4 serotype were compared, and their evolutionary relationships were evaluated. From these relationships, the differences in the mutational characteristics of the viral hemagglutinin in different hosts were examined and the RNA sequence changes that occurred during the generation of the progenitor of the 1968 human pandemic strain were examined. Three major lineages were defined: one containing only equine virus isolates; one containing only avian virus isolates; and one containing avian, swine, and human virus isolates. The human pandemic strain of 1968 was derived from an avian virus most similar to those isolated from ducks in Asia, and the transfer of this virus to humans probably occurred in 1965. Since then, the human viruses have diverged from this progenitor, with the accumulation of approximately 7.9 nucleotide and 3.4 amino acid substitutions per year. Reconstruction of the sequence of the hypothetical ancestral strain at the avian-human transition indicated that only 6 amino acids in the mature hemagglutinin molecule were changed during the transition between an avian virus strain and a human pandemic strain. All of these changes are located in regions of the molecule known to affect receptor binding and antigenicity. Unlike the human H3 influenza virus strains, the equine virus isolates have no close relatives in other species and appear to have diverged from the avian viruses much earlier than did the human virus strains. Mutations were estimated to have accumulated in the equine virus lineage at approximately 3.1 nucleotides and 0.8 amino acids per year. Four swine virus isolates in the analysis each appeared to have been introduced into pigs independently, with two derived from human viruses and two from avian viruses. A comparison of the coding and noncoding mutations in the mammalian and avian lineages showed a significantly lower ratio of coding to total nucleotide changes in the avian viruses. Additionally, the avian virus lineages of both the H3 and H4 serotypes, but not the mammalian virus lineages, showed significantly greater conservation of amino acid sequence in the internal branches of the phylogenetic tree than in the terminal branches. The small number of amino acid differences between the avian viruses and the progenitor of the 1968 pandemic strain and the great phenotypic stability of the avian viruses suggest that strains similar to the progenitor strain will continue to circulate in birds and will be available for reintroduction into humans.

Amino Acid Sequence↗
PubMed1988

Sequence and structure at the genome 3' end of the U2-strain of tobacco mosaic virus, a histidine-accepting tobamovirus.

The primary sequence of the 3' noncoding region of U2-TMV RNA was determined. A structural model was proposed based on chemical and enzymatic structure mapping as well as on analyses of nuclease protection by aminoacyl-tRNA-synthetase. The model agrees with those proposed for TMV "vulgare" RNA and confirms their general validity for the tobamoviruses. The RNA appears to have a tRNA-like, L-shaped structure at the 3' terminus, linked to a quasi-continuous double-helical stalk, with five pseudoknots involved in the formation of the whole structure. However, the structure of U2-TMV RNA is less stringently conserved than the 3' termini of "vulgare" and other histidine-accepting tobamoviruses. This difference is reflected in the kinetics of aminoacylation of the RNA.

Base Sequence↗
PubMed1992

Nucleotide sequence and genetic map of cowpea severe mosaic virus RNA 2 and comparisons with RNA 2 of other comoviruses.

We report the nucleotide sequence of cowpea severe mosaic comovirus (CPSMV) genomic RNA 2. The molecule is composed of 3732 nucleotide (nt) residues, exclusive of the polyadenylate at the 3' end. Only one of the six reading frame registers has a long open reading frame, from nt 255 to nt 3260 in the polarity of encapsidated RNA and corresponding to a polyprotein of 1002 amino acid residues (aa). As has been reported for other comoviruses, a second in-frame AUG, at nt position 531, apparently also initiates translation, at least in vitro. Multiple alignments of the deduced CPSMV polyprotein aa sequence with those of bean pod mottle comovirus (BPMV), cowpea mosaic comovirus (CPMV), and red clover mottle comovirus (RCMV) were consistent with a similar size for each of the three genes: the putative movement protein, beginning at the second in-frame AUG, the large coat protein (L), and the small coat protein. Identical nucleotide sequences in the terminal noncoding regions of RNA 2 of the four viruses are limited to 9 nt at the 5' end and the 3' polyadenylate. However, extensive similarities in sequence and potential structure were found. For all three genes and the 5' untranslated region, CPSMV and BPMV are more similar to each other than either is to CPMV or RCMV, the last two being similar to each other. Observed similarities predict that both cleavage sites in the CPSMV RNA 2 polyprotein are at glutamine-serine dipeptides. A sequence of 16 aa at the amino terminus of L, determined by automated Edman degradation, matched a region of the deduced aa sequence in the polyprotein and is consistent with cleavage at the predicted glutamine-serine dipeptide.

Amino Acid Sequence↗
PubMed1999

Absence of hepatitis G virus within liver tissue of patients undergoing liver transplantation for cryptogenic cirrhosis.

BACKGROUND: Epidemiological studies have detected up to a 9% incidence of hepatitis G (HGV)-RNA in patients with acute and chronic liver disease of unknown etiology. We sought to clarify the role of HGV as a causative agent in cryptogenic cirrhosis by analyzing archival liver tissue for HGV-RNA in patients undergoing orthotopic liver transplantation. METHODS: Using a computer database, we identified 54 patients who underwent orthotopic liver transplantation for cryptogenic cirrhosis. After using rigorous serologic and histopathologic screening guidelines, 20 patients were studied, 7 of whom had concurrent hepatocellular carcinoma (HCC). RNA was extracted from archival paraffin-embedded liver tissue; HGV sequences were amplified by nested reverse transcription-polymerase chain reaction using primers designed from the 5' noncoding region. RESULTS: HGV-RNA was absent from all 20 liver specimens, including those 7 with HCC. Beta-actin RNA, used as a positive control for cellular RNA, was isolated from all 20 liver specimens, including the 7 with HCC. CONCLUSIONS: Utilizing a highly sensitive reverse transcription-polymerase chain reaction assay for HGV-RNA, we were unable to detect HGV-RNA within the livers of patients with cryptogenic cirrhosis or in the HCC arising within them. This lends further evidence to HGV infection not being a cause of cryptogenic cirrhosis and not being associated with the development of HCC in cryptogenic cirrhosis.

Actins↗
PubMed1992

Genetic variation in vivo and proposed functional domains of the 5' noncoding region of poliovirus RNA.

Poliovirus has a single-stranded RNA genome of about 7,440 nucleotides (nt) with an unusually long 750-nt noncoding region in the 5' end (5'NCR). Several regulatory functions have been assigned to the 5'NCR. We sequenced the 5'NCRs of 33 wild-type 3 poliovirus strains to study the range and distribution of naturally occurring sequence variations. In this regard, the 5'NCR can be divided into a conserved part (nt 1 to 650) and a hypervariable part (nt 651 to 750). In the conserved part, altogether 234 unevenly distributed nucleotide positions (36%) showed variation. When these positions were plotted against the predicted secondary-structure models, it was found that the existence of most of the proposed stem-loop structures was supported by extensive structure-conserving substitutions in the stems. Regions with conserved sequences, as well as mutational hot spots, were observed. The hypervariable part of the 5'NCR varied up to 56% between the strains studied. The A + U percentage was significantly higher than in the conserved part. The number of AUG codons varied between 5 and 15 in the conserved part of the 5'NCR, while none was found in the hypervariable part. These results provide information that can be used in site-directed mutagenesis and other approaches targeted to reveal the functional domains of the 5'NCR.

Base Sequence↗
PubMed1991

Structure and organization of the hepatitis C virus genome isolated from human carriers.

Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis, which often develops into malignant chronic diseases, including liver cirrhosis and hepatocellular carcinoma. We have cloned from human carriers overlapping cDNAs (9,416 bp) covering the entire coding region of the HCV genome. The latter encodes a 3,010-amino-acid polyprotein. In addition, there are 332 and 54 bases of 5' and 3' noncoding sequences, respectively. Our HCV strain has a 77% nucleic acid identity to the HCV strain cloned by workers at Chiron Corporation. The hydrophobicity profile of the putative polyprotein is similar to those of flaviviruses, but it has limited amino acid homology to polyproteins of flaviviruses and other viruses, indicating that HCV is at most distantly related to flaviviruses.

Amino Acid Sequence↗
PubMed1998

Hepatitis G virus infection in screened Chinese blood donors.

BACKGROUND AND OBJECTIVES: To determine the prevalence of the recently identified hepatitis G virus (HGV)/GBV-C in screened Chinese paid blood donors. MATERIALS AND METHODS: Two hundred and seventy-nine plasma samples were tested for HGV RNA by RT-PCR with nested primers from the 5'-noncoding region of GBV-C. All samples were obtained from plasma or blood bags that had been screened twice by routine selection tests (ALT, HBsAg, Anti-HCV, anti-HIV, and syphilis) and were available for clinical use. RESULTS: HGV RNA was detected in 2 (4%) of 50 paid plasma donors from the Beijing Red Cross Blood Center, 1 (2%) of 50 paid blood donors from Taiyuan, and 9 (5%) of 179 paid blood donors from Hebei, a total HGV detection rate of 4.3% (12/279). CONCLUSIONS: Our data suggest that HGV infection is relatively frequent even in screened donors, at least in paid screened donors, although larger-scale studies are required.

Blood Donors↗
PubMedDate not supplied

RNA transcription from immobilized DNA templates.

We describe an RNA transcription protocol based on the multiple reuse of solid-phase synthetic DNA templates. The templates are assembled onto streptavidin-coated agarose beads via a single 5'-terminal biotin located on the noncoding template strand. Transcription occurs in an aqueous buffered suspension containing solid-phase DNA, dissolved enzyme (T7 RNA polymerase), and nucleoside triphosphate substrates (NTPs). A direct comparison of solution and solid-phase templates under standard transcription conditions reveals similar initial reaction rates and overall yields. Immobilized templates store stably for periods of several months and are easily recovered by mild centrifugation. We demonstrate the successive reuse of these templates throughout 15 rounds of transcription. The templates remained active, although an incremental decay in transcription was observed beyond five rounds. Template activity was partially restored by supplementing the support-bound oligonucleotide with fresh coding-strand DNA. These findings indicate that multiple reuse of template is a viable strategy for reducing the amount of DNA template required in RNA transcription.

Autoradiography↗
PubMed1990

Mapping of mutations associated with neurovirulence in monkeys infected with Sabin 1 poliovirus revertants selected at high temperature.

Poliovirus type 1 neurovirulence is difficult to analyze because of the 56 mutations which differentiate the neurovirulent Mahoney strain from the attenuated Sabin strain. We have isolated four neurovirulent mutants which differ from the temperature-sensitive parental Sabin 1 strain by only a few mutations, using selection for temperature resistance: mutant S(1)37C1 was isolated at 37.5 degrees C, S(1)38C5 was isolated at 38.5 degrees C, and S(1)39C6 and S(1)39C10 were isolated at 39.5 degrees C. All four mutants had a positive reproductive capacity at supraoptimal temperature (Rct+ phenotype). Mutant S(1)37C1 induced paralysis in two of four cynomolgus monkeys, and the three other mutants induced paralysis in four of four monkeys. The lesion score increased from the S(1)37C1 mutant to the S(1)39 mutants. To map the mutations associated with thermoresistance and neurovirulence, we sequenced all regions in which the Sabin 1 genome differs from the Mahoney genome. The S(1)37C1 mutant had one mutation in the 5' noncoding region and another in the 3' noncoding region. Mutant S(1)38C5 had these mutations plus another mutation in the 3D polymerase gene. The S(1)39 mutants had three additional mutations in the capsid protein region. The mutations were located at positions at which the Sabin 1 and Mahoney genomes differ, except for the mutation in the 5' noncoding region. The noncoding-region mutations apparently confer a low degree of neurovirulence. The 3D polymerase mutation, which distinguishes S(1)38C5 and S(1)39 mutants from S(1)37C1, is probably responsible for the high neurovirulence of S(1)38C5 and S(1)39 mutants. The capsid region mutations may contribute to the neurovirulence of the S(1)39 mutants, which was the highest among the mutants.

Animals↗
PubMed1991

Identification and characterization of a new base substitution in the vaccine strain of Sabin 3 poliovirus.

The complete RNA sequence of Sabin 3 (LED3) used in vaccine in the United States has been determined. The LED3 Sabin 3 sequence contains the attenuating mutations at bases 472 and 2034 but differs from that published by Stanway et al. (Nucleic Acids Res., 11, 5629-5643, 1983) at two other base positions, 2493 and 6061. The change at base 6061 is silent and does not affect amino acid composition. The other base, a C at position 2493, is contained in the viral capsid protein VP1 and predicts a new Sabin 3 specific amino acid change of a threonine instead of an isoleucine at amino acid 6 of the protein [corrected]. Reversion of this base to that present in the pathogenic progenitor strain, Leon, is observed to occur after replication of vaccine virus in the gut of primary vaccines and in nervous tissue of neurovirulence test monkeys. Passage conditions have been identified that lead to the reversion of base 2493 as well as the reversion of the attenuated base to the parental base (Leon) at position 472 in the 5' noncoding region. The observation that these two bases delta position are found to revert during passage suggests that there is a selective advantage for virus containing the parental bases at these positions.

Animals↗
PubMed1993

Nucleotide sequence of a Singapore isolate of zucchini yellow mosaic virus coat protein gene revealed an altered DAG motif.

A cDNA clone of zucchini yellow mosaic virus (ZYMV) RNA was mapped to the 3' terminal region. The nucleotide sequence revealed a single open reading frame of 1035 nucleotides followed by a 3' noncoding region of 215 nucleotides. The putative protease cleavage site for the release of coat protein (CP) was deduced to be between Glu-Ser (at amino acid position 66-67), which would result in a protein of 279 amino acids. This non-aphid-transmissible Singapore isolate of ZYMV showed a change of DAG to GAG triplet near the N-terminal of the CP. The CP gene was expressed as a protein fused to the beta-galactosidase in Escherichia coli and as an unfused protein in Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1995

Identification of hepatitis C virus genotypes among hospitalized patients in British Columbia, Canada.

The distribution pattern of hepatitis C virus (HCV) genotypes among patients from British Columbia has been investigated by reverse transcription polymerase chain reaction (PCR) amplification of the 5' noncoding region (NCR) and direct DNA sequencing of the PCR product. The sequence data obtained from the British Columbia isolates were compared with HCV sequences for the NCR obtained from GenBank for the construction of a phylogenetic tree using the GDE program, version 2.2. The data show that in British Columbia there are 7 subtypes distributed among major genotypes 1, 2, and 3. The study also shows a new subtype of genotype 1 that represents 29% of the analyzed cases.

Base Sequence↗
PubMed2003

Structure and function analysis of the poliovirus cis-acting replication element (CRE).

The poliovirus cis-acting replication element (CRE) templates the uridylylation of VPg, the protein primer for genome replication. The CRE is a highly conserved structural RNA element in the enteroviruses and located within the polyprotein-coding region of the genome. We have determined the native structure of the CRE, defined the regions of the structure critical for activity, and investigated the influence of genomic location on function. Our results demonstrate that a 14-nucleotide unpaired terminal loop, presented on a suitably stable stem, is all that is required for function. These conclusions complement the recent analysis of the 14-nucleotide terminal loop in the CRE of human rhinovirus type 14. The CRE can be translocated to the 5' noncoding region of the genome, at least 3.7-kb distant from the native location, without adversely influencing activity, and CRE duplications do not adversely influence replication. We do not have evidence for a specific interaction between the CRE and the RNA-binding 3CD(pro) complex, an essential component of the uridylylation reaction, and the mechanism by which the CRE is coordinated and orientated during the reaction remains unclear. These studies provide a detailed overview of the structural determinants required for CRE function, and will facilitate a better understanding of the requirements for picornavirus replication.

Base Sequence↗
PubMed2001

[Detection and partial nucleotide sequence analysis of hepatitis G virus in sera of patients with hepatitis in Guangzhou].

OBJECTIVE: To investigate hepatitis G virus (HGV) infection in hepatitis patients and the gene type of HGV in Guangzhou. METHODS: Reverse transcriptase-nested polymerase chain reaction(RT-nested PCR) method using primers locating in noncoding region of HGV genome was used for detecting HGV RNA in sera of hepatitis patients and the amplified products were directly sequenced. RESULTS: 25 cases were HGV RNA positive in 251 sera of acute and chronic hepatitis(9.96%), of which 4 cases were positive in 56 sera with hepatitis non A-E(7.14%), 8 cases were positive in 77 sera with hepatitis B (10.39%), 13 cases were positive in 118 sera with hepatitis C(11.17%). The identity of nucleotide sequence was 98.0% between 2 Guangzhou HGV isolates, both were 91.1% with American isolate, 81.7% and 84.2% respectively between 2 Guangzhou isolates and African isolate. CONCLUSION: HGV infection exists in hepatitis patients, 2 Guangzhou HGV isolates may belong to the same gene type and both of them have a high homology with American HGV isolate.

Base Sequence↗
PubMed1987

Variations in template protection by the RNA polymerase II transcription complex during the initiation process.

Preinitiation complexes (complex 0) or complexes which either made 2 or an average of 10 phosphodiester bonds (complexes 2 and 10, respectively) were assembled in vitro on the adenovirus 2 major late promoter. Each of the complexes was digested extensively with DNase I; the protected DNAs were purified and hybridized in a series of end-labeled oligonucleotides homologous to sequences on the coding or noncoding strands near the initiation site. The hybrids were then extended with reverse transcriptase to map the extent of template protection conferred by proteins in the complex. The downstream protection edge revealed by this approach was approximately +30, +25, and +35 for complexes 0, 2, and 10, respectively. We subsequently found that the apparent inward movement of the downstream protection boundary on initiation could be produced by satisfying the energy requirement for transcription initiation (i.e., by treating with ATP or dATP). The downstream boundary change occurred as rapidly as we could perform the test (less than 60 s) and was not blocked by alpha-amanitin. DNAs from trimmed complexes 0, 2, or 10 all supported extension to a single upstream edge at about position -42. Upstream protection was stable in the preinitiation complex, but when postinitiation complexes were incubated for extended periods, protection of the entire upstream region was lost. This decay of upstream protection, like the movement of the downstream boundary, was found to result from exposure to ATP or dATP. Unlike the downstream boundary movement, however, the upstream change was relatively slow; about 15 min was required to lose one-half of the protection.

Adenosine Triphosphate↗
PubMed1993

Partial characterization of hepatitis A viruses from three intermediate passage levels of a series resulting in adaptation to growth in cell culture and attenuation of virulence.

Viruses from passages 9/10, 21, and 32 of a serially passaged human isolate of hepatitis A virus, strain HM-175, were partially sequenced and compared for their abilities to grow in cell cultures and to cause disease in primates. Viruses from all passages grew more efficiently in cell culture than did the wild-type virulent virus from which they were derived, and all displayed some degree of attenuation of virulence for primates. Within the 5' noncoding region and the 2B2C region of the HAV genome, passage 9/10 virus differed in sequence from wild-type at a single and novel position in the 2C gene, while the sequence of the passage 32 virus was almost identical to that of a fully attenuated passage 35 virus. Passage 21 viruses were found to consist of a mixture of viruses which included all but two of the 13 mutations present in the sequenced regions of the virus from passage 32.

Animals↗
PubMed1997

Molecular characterisation of the 3'-end of the astrovirus genome.

We have sequenced the 3'-end of the RNA genomes of 14 serotyped and 12 untyped isolates of human astrovirus. The sequences, which include all 8 serotypes, were used to predict secondary structures, postulate possible functional domains, reveal conserved regions suitable for nucleic acid amplification and perform phylogenetic analysis. The final nucleotides of the capsid protein precursor gene and the adjacent 3'-noncoding region were highly conserved and, except for 35 nucleotides with homology to a sequence in the 3'-end of a coronavirus RNA genome, unique to astrovirus family. This confirms that the 3'-end is a suitable target for universal and specific detection of astrovirus RNA. For the deduced 72 C-terminal amino acids of the capsid protein precursor, distances between the serotypes were found to vary from 0.1 substitution per site between serotypes 3 and 7 to more than one substitution per site between serotype 4 and the other serotypes. Different isolates of the same serotype were closely related, which indicates that the presently used type-specific antibodies differentiate between phylogenetically distinct groups. RNA secondary structures with minimal free energy were predicted using computer programs. Comparative sequence analysis verified the significance of certain of the predicted structural elements.

Base Sequence↗