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At least 235 records · Page 13Linked to original sources
PubMed1991

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage.

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.

Base Sequence↗
PubMed1986

Molecular cloning of cDNA from hepatitis A virus strain HM-175 after multiple passages in vivo and in vitro.

Hepatitis A virus (HAV) strain HM-175 was passaged six times in marmosets, 59 times in cell culture and purified from infected cell culture supernatant fluid. The viral RNA was extracted, copied into cDNA and the cDNA:RNA hybrids were cloned into the PstI site of plasmid pBR322. The cDNA clones were authenticated by hybridization to RNA extracted from HAV-infected cells and clones representing the 3' end of the genome were identified using a previously authenticated cDNA clone. The clones represented all but 29 bases of the HAV genome. They were compared to HAV strain HM-175 cDNA cloned from viral RNA after three passages in marmosets on the basis of restriction endonuclease mapping and DNA sequencing. No differences were found in either the presence or absence of restriction endonuclease sites using 33 different restriction enzymes. Sequencing of cDNA representing bases 29 to 1002 of the HAV genome revealed eight base changes all of which were within the 5' noncoding region.

Animals↗
PubMed1987

Characterization of cloned measles virus mRNAs by in vitro transcription, translation, and immunoprecipitation.

A cDNA library was constructed from poly(A)+ RNA prepared from VERO cells infected with the Edmonston strain of measles virus. Clones corresponding to five major viral-specific transcripts were identified by northern blot hybridization analysis. Probes prepared from these five clones detected an additional five minor viral RNA transcripts. The sizes and hydridization patterns of these minor RNA species are consistent with their being bicistronic transcripts arising from a viral genomic template with the gene order 3'..NP-9/C-M-F-HA-(L)..5'. To assess the coding capacity of these cDNA clones they were inserted into pSP64, transcribed in vitro, the RNA was translated in reticulocyte lysates, and the protein was immunoprecipitated with specific antisera. From this analysis the genes for NP, P/C, M, F, and HA were identified. In vitro translation of natural mRNAs and SP6 transcripts of cDNAs consistently produced smaller polypeptides that appear to be initiated at internal AUGs. The relative abundance of these various cell-free translation products reflects the probability of translational initiation at the various in-frame AUGs. The patterns observed suggest that other factors besides sequences immediately flanking the AUGs have a significant effect on the selection of translational initiation sites. An increase in translational efficiency of the F transcript was achieved by removing 450 bases of the G-C-rich 5' noncoding region.

Animals↗
PubMed1998

Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein.

The full-length dengue virus NS3 protein has been successfully expressed as a 94-kDa GST fusion protein in Escherichia coli. Treatment of the purified fusion protein with thrombin released a 68-kDa protein which is the expected molecular mass for the DEN1 NS3 protein. The identity of this protein was confirmed by Western blotting using dengue virus antisera. Two related activities of the recombinant NS3 protein were characterized, which were the binding of the protein to the 3'-noncoding region of the dengue virus RNA genome and NTPase activity. We demonstrated using a band shift assay that the DEN1 NS3 protein could form a complex with the stem-loop structure in the 3'-noncoding region (3'-NCR), although sites outside the stem-loop may also participate in binding. Using various unlabeled homopolymeric and heteropolymeric RNAs as competitors for binding, it was further shown that the DEN1 NS3 protein exhibits preferential binding to a 94-nt RNA transcript from the 3'-NCR of the dengue virus. The NTPase activity of the recombinant DEN1 NS3 protein was characterized using a thin-layer chromatography assay. We found that the DEN1 NS3 protein possesses some aspects of NTPase activity, which are distinct from those found in other flaviviruses. Although the NS3 protein was able to utilize all four ribonucleoside triphosphates as its substrates, the NS3 protein showed a distinct preference for purine triphosphates (i.e., ATP and GTP). The addition of poly(U) did not stimulate NTPase activity in DEN1 NS3 protein, which contrasts with the reports for other flaviviral NS3 proteins. However, NTPase activity was specifically stimulated by the viral NS5 protein, which was manifested by a more than twofold increase in the rate of ATP hydrolysis and a 25% increase in the yield of ADP at the end of a 120-min reaction. These data suggest that the NTPase activity of the NS3 protein may be regulated by the viral NS5 protein during virus replication.

Adenosine Triphosphatases↗
PubMed1997

cDNA sequencing of the 5' noncoding region (5' NCR) to determine hepatitis C genotypes in patients with chronic hepatitis C.

Reports suggest that response to interferon-alpha therapy is influenced by both hepatitis C viral genotype and titer. Our aim was to determine if direct, automated, cycle sequencing of the PCR product from an HCV RNA detection assay could be used to reliably determine HCV genotype. In addition, the approach was used to determine the HCV genotype distribution in our patient population and to learn if there was a correlation between HCV genotype and RNA titer that could be used to predict response to treatment. In all 143 consecutive patients were tested for both HCV RNA titer and genotype. Automated, cycle sequencing of PCR product was highly effective and failed to yield a genotype in only 3 (2%) patients. The distribution of HCV genotypes was: 1a (40%), 1b (39%), 2a (2%), 2b (6%), 3a (4%). There were significant differences in the median HCV RNA titers between genotypes 1, 2, and 3. High HCV RNA titers >4.4 x 10(6) copies/ml were only seen in genotype 1. However, the HCV RNA level should not be used as a surrogate marker of genotype because of a significant overlap of titers within the genotypes.

Base Sequence↗
PubMed1993

Comparison of nucleic acid hybridization and nucleic acid amplification using conserved sequences from the 5' noncoding region for detection of bovine viral diarrhea virus.

Primers and probes derived from conserved sequences located in the 5' noncoding region of pestiviruses were evaluated for detection of bovine viral diarrhea virus. With these reagents, hybridization and polymerase chain reaction tests detected 62 of 90 and 90 of 90 bovine viral diarrhea virus isolates, respectively. A quick lysis method for preparing RNA for use in polymerase chain reaction amplification also was evaluated.

Animals↗
PubMed2006

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes.

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.

Acetylation↗
PubMed1997

Two functional complexes formed by KH domain containing proteins with the 5' noncoding region of poliovirus RNA.

The 5' noncoding region of the poliovirus genome contains RNA structures important for replication and translation. Here we show that two closely related cellular poly(rC) binding proteins (PCBP1 and PCBP2) bind to the terminal cloverleaf structure and facilitate the interaction of the viral protein 3CD (the uncleaved precursor of the protease-polymerase). In addition, these cellular proteins bind to stem-loop IV of the internal ribosomal entry site. The proteins are cytoplasmic and largely associated with ribosomes; they appear to dimerize in solution and to form heterodimers when binding to stem-loop IV. Initiation of viral translation in Xenopus oocytes is strongly inhibited by co-injection of specific antibodies directed against PCBP1 or PCBP2, indicating that the poly(rC) binding proteins may facilitate this process. Furthermore, PCPB-depleted HeLa extracts translate poliovirus RNA inefficiently and the activity is partially restored by addition of recombinant PCBP proteins.

Animals↗
PubMed1999

Is hepatitis G/GB virus-C virus hepatotropic? Detection of hepatitis G/GB virus-C viral RNA in liver and serum.

The recently identified hepatitis G virus (HGV, also named GB virus-C, GBV-C) appears to have similarities to hepatitis C virus and other flaviviridae. To better understand its clinical significance and hepatotropism, we collected liver tissue and matched serum samples from 56 patients undergoing liver transplantation. HGV/GBV-C RNA was detected by reverse transcription-nested PCR, using primers from the relatively conserved 5' noncoding region of the genome to detect HGV/GBV-C RNA and the amount was semiquantitatively estimated by serial 10-fold endpoint dilution. The presence and amount of HCV RNA was estimated by the same methodology. Seventeen patients (30%) had HGV/GBV-C RNA detectable either in liver or in serum, including two of three with cryptogenic liver disease. Interestingly, 5 of 17 (29%) patients had HGV/GBV-C RNA in serum but not liver, even with repeated testing of hepatic RNA from different portions of the liver. Furthermore, the titer of HGV/GBV-C RNA was significantly lower in liver than in serum in most samples (mean log titer, 1.33 vs. 2.56, P < 0.05). In contrast, all 21 patients with HCV RNA in serum also had the virus detectable in liver. In five patients coinfected with HCV and HGV/GBV-C, the mean titer of HCV RNA in liver was higher than that in serum (log titer, 2.8 vs. 3.0, P > 0.05). Thus, our results suggest that HGV/GBV-C is probably not hepatotropic and may replicate predominantly in sites other than the liver. These findings brings into question the role of HGV in causing significant liver disease.

5' Untranslated Regions↗
PubMed1993

Nucleotide sequence and in vitro translation of the coat protein gene of cymbidium mosaic virus.

The nucleotide sequence of the coat protein gene of cymbidium mosaic virus (CyMV) has been determined from a cDNA clone that encompassed the 3'-region of the virus genome. The 0.75 kb coat protein open reading frame (ORF) codes for a 24kD polypeptide. The coat protein coding region was separated from the 3'-poly(A) terminal of the viral genome by an 80 base noncoding region. The derived amino acid sequence of this ORF showed considerable homology with other potexvirus coat protein sequences, notably with narcissus mosaic virus (NMV), potato virus X (PVX), and white clover mosaic virus (WClMV). In vitro expression of the CyMV coat protein gene was carried out by T7 RNA polymerase on the linearized cDNA clone template, followed by in vitro translation of the purified transcript in wheat germ extract. One of the translated products was found to comigrate with the coat protein of the virus at about 24 kD, as determined by NaDodSO4-PAGE. This translated product was also found to crossreact with antibodies raised against CyMV.

Amino Acid Sequence↗
PubMed1993

Patterns of antibodies to hepatitis C virus in patients with chronic non-A, non-B hepatitis and their relationship to viral replication and liver disease.

Patients with hepatitis C virus infection may have circulating antibodies to various structural and nonstructural antigens of the virus. To assess whether the antibody profile is related to epidemiological or clinical features of chronic infection or to viral replication, sera from 172 consecutive patients with biopsy-proven chronic non-A, non-B hepatitis were studied for antibodies to nonstructural and structural hepatitis C virus antigens and for serum hepatitis C virus RNA with the polymerase chain reaction using primers derived from the 5' noncoding region. Three subgroups could be identified on the basis of their seroreactivity to hepatitis C virus: 133 cases (77.3% [group A]) were positive on first- and second-generation assays and had antibodies to C100-3 and to C22, C33c or both identified on recombinant immunoblot assay; 23 cases (13.4% [group B]) were positive only on second-generation assay and reacted with C22, C33c or both but not with C100-3; and 26 cases (9.3% [group C]) were negative for all hepatitis C virus antibodies. Mean age and sex distributions were similar among the three groups; a history of transfusion was more frequent among cases in group B (p = 0.06). These patients also had the highest serum aminotransferase values (p = 0.001). Liver histological studies showed active necroinflammatory changes in 69.2% of patients in group A and 52.2% of those in group B but only in 25% of cases in group C. Serum hepatitis C virus RNA was frequently detected in patients of groups A and B, independent of their recombinant immunoblot assay profiles.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic Disease↗
PubMed1993

Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples.

A method based on nested polymerase chain reaction was developed for the detection of enteroviral genomes in biological samples. By taking advantage of the conserved 5' noncoding region of the enteroviral RNA, two sets of primers were utilized, enabling the detection either of a broad range of enteroviruses or of group B coxsackieviruses only. The sensitivity of the method is close to the detection of single molecules of viral RNA in as much as 1 mg of tissue sample. A preliminary study showed the usefulness of this technique for the analysis of endomyocardial biopsy samples from patients with idiopathic dilated cardiomyopathy and myocarditis.

Base Sequence↗
PubMed1994

Detection of hepatitis C virus RNA in hemodialysis patients.

The clinical significance of the high prevalence of antibodies to hepatitis C virus (HCV) in dialysis patients remains undefined. In order to assess the relationship between seropositivity and potential infectivity, 63 patients undergoing maintenance hemodialysis were evaluated between April and May 1990. The mean duration of maintenance hemodialysis was 45 mo (range, 13 to 144). Eighty-two percent (52 of 63) had received blood transfusions, and 16% (10 of 63) had a history of iv drug abuse. Serum samples were analyzed by HCV-cDNA polymerase chain reaction; antibodies to HCV structural (core) and nonstructural regions NS3 and NS4 were determined by enzyme immunoassay. Specimens repeatedly reactive for anti-HCV and HCV-RNA-positive samples were tested by HCV MATRIX dot immunoblot assay and HBV-DNA PCR. Twenty-five percent (16 of 63) were anti-HCV-positive. Of the 16 anti-HCV-positive patients, HCV-RNA was detected in 5 (31%) with the NS3 primers and in 12 (75%) with 5'-noncoding primers. Among the anti-HCV-negative patients, HCV-RNA was detected in 2 (4.3%) of 47 patients. Eleven of the 18 patients with HCV infection (anti-HCV and/or HCV-RNA-positive) had evidence of additional present or past viral infections (human immunodeficiency virus and/or hepatitis B virus). In summary, HCV-RNA is present in at least 75% of anti-HCV-positive patients, suggesting that they may be infectious. The detection of HCV-RNA in anti-HCV-negative patients may indicate early or chronic HCV infection not detected by current antibody assays or the inability of these patients to mount or sustain a significant antibody response.

Adult↗
PubMed1997

The hepatitis C virus: overview.

Our knowledge of hepatitis C virus (HCV) dates only from 1975, when non-A, non-B hepatitis was first recognized. It was not until 1989 that the genome of the virus was first cloned and sequenced, and expressed viral antigens used to develop serological assays for screening and diagnosis. HCV is in a separate genus of the virus family Flaviviridae. It is a spherical enveloped virus of approximately 50 nm in diameter. Its genome is a single-stranded linear RNA molecule of positive sense and consists of a 5' noncoding region, a single large open reading frame, and a 3' noncoding region. The open reading frame encodes at least three structural and six nonstructural proteins. The genome is characterized by significant genetic heterogeneity, based on which HCV isolates can be classified into six major genotypes and more than 50 subtypes. Even individual isolates of HCV are genetically heterogeneous (quasispecies diversity). Genetic heterogeneity of HCV is greatest in the amino-terminal end of the second envelope protein (hypervariable region 1). This region may represent a neutralization epitope that is under selective pressure from the host's humoral immune response. Infection with HCV proceeds to chronicity in more than 80% of cases, and even recovery does not protect against subsequent re-exposure to the virus. The development of a broadly protective vaccine against HCV will therefore require a better understanding of the molecular biology and immune response to this virus.

Animals↗
PubMed1979

Sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA.

The sequence of 1000 nucleotides at the 3' end of tobacco mosaic virus RNA has been determined. The sequence contains the entire coat protein cistron as well as regions to its left and right. Sequence characterization was by conventional methods for use with uniformly 32P labeled RNA complemented by newer methods for in vitro 5' and 3' 32P end-labeling of RNA and its subsequent rapid analysis. The noncoding region separating the coat protein cistron from the 3' terminus is 204 residues long and may be folded into a clover-leaf-type secondary structure. The distribution of termination codons to the left of the coat protein cistron suggests that the end of the adjacent cistron is separated from the beginning of the coat protein cistron by only two nucleotides. The subgenomic viral coat protein mRNA was isolated from infected tissue and shown to be capped. The nontranslated sequence separating the cap from the AUG initiation codon is 9 residues long and thus overlaps a portion of the adjacent cistron on the genome RNA.

Base Sequence↗
PubMed1997

Sequence characterization of the 5' noncoding region of GB virus C/hepatitis G virus.

The nucleotide sequences of the 5' noncoding region of the GB virus C/hepatitis G virus (GBV-C/HGV) were determined in 18 isolates from the United States. Two genotypes have been classified based on the sequence heterogeneity within the 5' noncoding region of GBV-C/HGV. The most distantly related isolates between the two genotypes were 84.6% identical. Sequence identity of the isolates within a genotype was 95-99%. The 5' noncoding region of this virus contains four highly conserved domains. These conserved elements would facilitate the selection of optimal primers for the sensitive detection of GBV-C/HGV RNA by PCR. In addition, they suggest a crucial role for this region in viral replication and/or gene expression. Detection of genotypic variation among GBV-C/HGV infected individuals may provide further insight into the possible pathogenicity and into the transmission of the virus.

Base Sequence↗
PubMed1989

Leader-to-leader splicing is required for efficient production and accumulation of polyomavirus late mRNAs.

Polyomavirus late mRNA molecules contain multiple, tandem copies of a noncoding 57-base "late leader" exon at their 5' ends. This exon is encoded only once in the genome. Leader multiplicity arises from leader-leader splicing in giant primary transcripts, which are the result of multiple circuits of the viral genome by RNA polymerase II. We have been interested in learning more about the role of the leader exon in late viral gene expression. We recently showed that an abbreviated-leader mutant virus (ALM) with a 9-base leader exon is nonviable (G. R. Adami and G. G. Carmichael, Nucleic Acids Res. 15:2593-2610, 1987) and has a severe defect in both late pre-mRNA splicing and stability. However, a mutant virus with a different, substituted leader sequence of 51 nucleotides (SLM/MP8) is viable and has no apparent defects. Here we examined further the role of the late leader exon in late pre-mRNA processing. When the leader exon length was gradually reduced from 51 nucleotides to 9 nucleotides in a series of mutants, RNA splicing and stability defects were coupled. In this system there was a minimum exon size of between 33 and 27 nucleotides. Next, a number of mutations were introduced into the 3' splice site which precedes the late leader. Such mutations blocked leader-leader splicing. Surprisingly, they also interfered with leader-mVP1 body splicing and resulted in unstable primary transcripts. Thus, polyomavirus leader-leader splicing appears to be important for the efficient accumulation of late viral mRNA molecules.

Animals↗