Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA clone derived from genomic RNA of hog cholera virus (HCV) was identified using an oligonucleotide complementary to the RNA encoding a hexapeptide from the putative RNA-dependent RNA polymerase of the closely related bovine viral diarrhea virus (BVDV). This clone served as a probe for screening different size-selected cDNA libraries. After molecular cloning and nucleotide sequencing the HCV genome was shown to consist of 12,284 nucleotides containing one long open reading frame. Sequence comparison revealed a high degree of homology between HCV and BVDV genomic RNAs. With respect to HCV the genome of BVDV contains an insertion coding for 90 amino acids.
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PMID:Molecular cloning and nucleotide sequence of the genome of hog cholera virus. 276 66

Based on genome analysis of the RNA-dependent RNA polymerase region, it has been proposed that human caliciviruses (HuCV) can be classified into at least three genogroups: genogroup I is represented by Norwalk virus (NV), genogroup II by Snow Mountain agent (SMA) and genogroup III by HuCV/Sapporo/82/Japan (HuCV/Sa/82/J) virus. HuCV/Sa/82/J strain is genetically unique and more closely related to animal caliciviruses than are other known HuCVs, such as NV and SMA. HuCV/Sa/82/J strain was detected in four outbreaks of HuCV gastroenteritis occurring between 1977 and 1982 in an infant home in Sapporo. The HuCVs detected from these four outbreaks all showed a typical "Star of David" configuration by electron microscopy (EM), and they were identical antigenically and genetically. This strain has also been detected in other prefectures in Japan, as well as in the USA, UK, Saudi Arabia and Kenya. Seroepidemiological studies have shown a worldwide distribution of this virus, including Japan, USA, UK, Southeast Asia, Canada, China and Kenya. This virus has been circulating in Sapporo for at least 19 years (1977-1995). HuCV/Sa/82/J strain is thought to be one of the common causes of viral gastroenteritis worldwide. The HuCV/Sa/82/J strain has been detected mainly in infants. Age-related prevalence of antibody to this strain also shows that infections commonly occur in children less than 5 years old, although viruses in the NV and SMA genogroups commonly infect adults. The pattern of acquisition of antibodies to strain HuCV/Sa/82/J is similar to that of other common viral infections. HuCV/Sa/82/J strain is unique virologically and clinically among caliciviruses.
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PMID:The epidemiology of human calicivirus/Sapporo/82/Japan. 901 23

Nonstructural protein 5B (NS5B) of bovine viral diarrhea virus (BVDV) contains sequence motifs that are predictive of an RNA-dependent RNA polymerase activity. We describe the expression and purification of the BVDV NS5B protein derived from an infectious cDNA clone of BVDV (NADL strain). BVDV NS5B protein was active in an in vitro RNA polymerase assay using homopolymeric RNA or BVDV minigenomic RNA templates. The major product was a covalently linked double-stranded molecule generated by a "copy-back" mechanism from the input template RNA. In addition, a nucleotide-nonspecific and template-independent terminal nucleotidyl transferase activity was observed with the BVDV NS5B preparation.
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PMID:Identification and characterization of an RNA-dependent RNA polymerase activity within the nonstructural protein 5B region of bovine viral diarrhea virus. 976 90

Recombinant RNA-dependent RNA polymerases have been reported to synthesize RNAs by extending from the 3' hydroxyl of a template or an oligonucleotide primer. De novo initiation has not been reported. Establishment of such an assay would facilitate the analysis of the initiation requirements and allow the testing of antiviral compounds specifically targeting initiation. Using chemically synthesized RNAs and DNAs, we demonstrate that the recombinant RNA-dependent RNA polymerase (NS5B) of bovine viral diarrhea virus initiates de novo RNA synthesis. Nucleotides required for efficient initiation of RNA synthesis and for stable interaction with NS5B were identified.
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PMID:De novo initiation of RNA synthesis by a recombinant flaviviridae RNA-dependent RNA polymerase. 988 12

Four bovine viral diarrhea virus type 2 (BVDV-2) pairs consisting of cytopathogenic (cp) and noncp BVDV-2 were isolated during an outbreak of mucosal disease. Comparative sequence analysis showed that the four noncp BVDV-2 isolates were almost identical. For the cp BVDV-2 isolates, viral subgenomic RNAs were shown by Northern blot to have a length of about 8 kb, which is about 4.3 kb shorter than the genome of noncp BVDV. Cytopathogenicity and the expression of NS3 were both strictly correlated to the presence of viral subgenomic RNAs. By reverse transcription-PCR, Southern blot analysis, and nucleotide sequencing, a set of 11 unique subgenomes was identified with up to 5 different subgenomes isolated from one animal. To our knowledge, this is the first report on isolation of a set of pestiviral subgenomes from individual animals. Common features of the BVDV-2 subgenomic RNAs include (i) deletion of most of the genomic region encoding the structural proteins, as well as the nonstructural proteins p7 and NS2, and (ii) insertion of cellular (poly)ubiquitin coding sequences. Three subgenomes also comprised 15 to 75 nucleotides derived from the 5' part of the NS2 gene. Comparisons of the obtained nucleotide sequences revealed that the different BVDV-2 subgenomes evolved from the respective noncp BVDV-2 by RNA recombination. The presence of short regions of sequence similarity at several crossing-over sites suggests that base pairing between the nascent RNA strand and the acceptor RNA template facilitates template switching of the BVDV RNA-dependent RNA polymerase.
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PMID:Nonhomologous RNA recombination in bovine viral diarrhea virus: molecular characterization of a variety of subgenomic RNAs isolated during an outbreak of fatal mucosal disease. 1036 14

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDeltaCT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (DeltaCT179 and DeltaCT218) that lacked RdRp activities.
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PMID:Mutational analysis of bovine viral diarrhea virus RNA-dependent RNA polymerase. 1055 28

The virus-encoded RNA-dependent RNA polymerase (RdRp), which is required for replication of the positive-strand RNA genome, is a key enzyme of members of the virus family Flaviviridae. By using heterologously expressed proteins, we demonstrate that the 77 kDa NS5B protein of two pestiviruses, bovine viral diarrhoea virus and classical swine fever virus, and the 100 kDa NS5 protein of the West Nile flavivirus possess RdRp activity in vitro. As originally shown for the RdRp of hepatitis C virus, RNA synthesis catalysed by the pestivirus and flavivirus enzymes is strictly primer-dependent in vitro. Accordingly, initiation of RNA polymerization on homopolymeric RNAs and heteropolymeric templates, the latter with a blocked 3'-hydroxyl group, was found to be dependent on the presence of complementary oligonucleotide primer molecules. On unblocked heteropolymeric templates, including authentic viral RNAs, the RdRps were shown to initiate RNA synthesis via intramolecular priming at the 3'-hydroxyl group of the template and 'copy-back' transcription, thus yielding RNase-resistant hairpin molecules. Taken together, the RdRps of different members of the Flaviviridae were demonstrated to exhibit a common reactivity profile in vitro, typical of nucleic acid-polymerizing enzymes.
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PMID:The RNA-dependent RNA polymerases of different members of the family Flaviviridae exhibit similar properties in vitro. 1057 50

We report here the discovery of a small molecule inhibitor of pestivirus replication. The compound, designated VP32947, inhibits the replication of bovine viral diarrhea virus (BVDV) in cell culture at a 50% inhibitory concentration of approximately 20 nM. VP32947 inhibits both cytopathic and noncytopathic pestiviruses, including isolates of BVDV-1, BVDV-2, border disease virus, and classical swine fever virus. However, the compound shows no activity against viruses from unrelated virus groups. Time of drug addition studies indicated that VP32947 acts after virus adsorption and penetration and before virus assembly and release. Analysis of viral macromolecular synthesis showed VP32947 had no effect on viral protein synthesis or polyprotein processing but did inhibit viral RNA synthesis. To identify the molecular target of VP32947, we isolated drug-resistant (DR) variants of BVDV-1 in cell culture. Sequence analysis of the complete genomic RNA of two DR variants revealed a single common amino acid change located within the coding region of the NS5B protein, the viral RNA-dependent RNA polymerase. When this single amino acid change was introduced into an infectious clone of drug-sensitive wild-type (WT) BVDV-1, replication of the resulting virus was resistant to VP32947. The RNA-dependent RNA polymerase activity of the NS5B proteins derived from WT and DR viruses expressed and purified from recombinant baculovirus-infected insect cells confirmed the drug sensitivity of the WT enzyme and the drug resistance of the DR enzyme. This work formally validates NS5B as a target for antiviral drug discovery and development. The utility of VP32947 and similar compounds for the control of pestivirus diseases, and for hepatitis C virus drug discovery efforts, is discussed.
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PMID:Mechanism of action of a pestivirus antiviral compound. 1086 40

Ca2+ plays a key role in many pathological processes, including viral infections. Rotavirus, the major etiological agent of viral gastroenteritis in children and young animals, provides a useful model to study a number of Ca2+ dependent virus-cell interactions. Rotavirus entry, activation of transcription, morphogenesis, cell lysis, particle release, and the distant action of viral proteins are Ca2+ dependent processes. In the extracellular medium, Ca2+ stabilizes the structure of the viral capsid. During entry into the cell the low cytoplasmic Ca2+ concentration induced the solubilization of the outer protein layer of the capsid and transcriptase activation. Viral protein synthesis modifies Ca2+ homeostasis which, in turn, favours viral morphogenesis and induces cell death. The generation of diarrhea is a multifactorial process involving Ca2+ dependent secretory processes of mediators and water and electrolytes, as well as the induction of cell death in the different cell types that compose the intestinal epithelium. The discovery of the non-structural viral protein NSP4 as a viral enterotoxin and the possible participation of the enteric nervous system in the pathogenesis of diarrhea represent significant advances in its understanding. Ca2+ also plays a role in the replication cycles and pathogenesis of other viral diseases such as poliovirus, Coxsackie virus, cytomegalovirus, vaccinia and measles virus and HIV.
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PMID:Role of Ca2+in the replication and pathogenesis of rotavirus and other viral infections. 1102 Mar 76

The recombinant RNA-dependent RNA polymerase of the bovine viral diarrhea virus specifically requires a cytidylate at the 3' end for the de novo initiation of RNA synthesis (C. C. Kao, A. M. Del Vecchio, and W. Zhong, Virology 253:1-7, 1999). Using RNAs containing nucleotide analogs, we found that the N3 and C4-amino group at the initiation cytidine were required for RNA synthesis. However, the ribose C2'-hydroxyl of the initiating cytidylate can accept several modifications and retain the ability to direct synthesis. The only unacceptable modification is a protonated C2'-amino group. Quite strikingly, the recognition of the functional groups for the initiation cytidylate and other template nucleotides are different. For example, a C5-methyl group in cytidine can direct RNA synthesis at all template positions except at the initiation cytidylate and C2'-amino modifications are tolerated better after the +11 position. When a 4-thiouracil (4sU) base analog that allows only imperfect base pairing with the nascent RNA is placed at different positions in the template, the efficiency of synthesis is correlated with the calculated stability of the template-nascent RNA duplex adjacent to the position of the 4sU. These results define the requirements for the specific interactions required for the initiation of RNA synthesis and will be compared to the mechanisms of initiation by other RNA-dependent and DNA-dependent RNA polymerases.
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PMID:Template nucleotide moieties required for de novo initiation of RNA synthesis by a recombinant viral RNA-dependent RNA polymerase. 1104 75


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