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)

RNA sequences of five flaviviruses were detected by a modified polymerase chain reaction (PCR) that incorporated a reverse transcriptase and RNase inhibitor. Oligonucleotide primer pairs were synthesized to amplify sequences from St. Louis encephalitis (SLE), Japanese encephalitis (JBE), yellow fever (YF), dengue 2 (DEN-2), and dengue 4 (DEN-4) viruses. The amplified products were visualized as bands of appropriate size on ethidium bromide-stained agarose gels. The identity of these products was confirmed by restriction endonuclease cleavage to generate fragments of predicted lengths. The reverse-transcriptase PCR (RT-PCR) successfully amplified flavivirus sequences from cell cultures, frozen brain tissue, and formalin-fixed, paraffin-embedded brain tissue. The reactions were highly specific, and the method compared favorably to two conventional assays of viral infectivity. RT-PCR followed by PCR with nesting primers (N-PCR) was 1,000-fold more sensitive in detecting virus than classical infectivity titration by intracerebral inoculation of suckling mice and nearly 1,000-fold more sensitive than amplification of virus in cell culture followed by inoculation of mice.
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PMID:Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. 171 65

Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acids residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.
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PMID:Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5. 764 75

Reverse transcriptase polymerase chain reaction (RT PCR) was utilized to observe the complementary DNA (cDNA) synthesis from the 10723 base dengue-2 virus template by in vitro reverse transcription. The PCR product amplified from 5'-end of the genome (PCR primers N1A-E1) indicates the completeness of the cDNA synthesis because the cDNA primer D8B was located at 3'-end and the cDNA synthesized encompassed the entire RNA template. The integrity of the dengue virus RNA was also determined by the cDNA primer extension from 3'-end and the PCR amplification at various regions from 5'-end of the RNA template. In conclusion, RT PCR could be applied to monitor cDNA synthesis by in vitro reverse transcription and measure the integrity of viral RNA template. Moreover, it is demonstrated that at least 10.7 kilobases of the cDNA could be synthesized from dengue-2 virus RNA by in vitro reverse transcription.
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PMID:Monitoring the cDNA synthesis of dengue-2 virus by RT PCR. 773 Apr 37

We have developed an assay using flavivirus RNA-dependent RNA polymerase to test the inhibitory activity of potential antiviral agents. The effects of antiviral agents on RNA synthesis were examined in this assay using extracts of Vero cells infected with dengue virus type 2 or Kunjin virus. Several different classes of known polymerase inhibitors were tested. The synthesis of double-stranded replicative form RNA was inhibited in a dose-dependent fashion in the presence of the polyoxometalate HPA-23 [(NH4)18(NaW21Sb9O86)17].14 H2O and several structurally related compounds.
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PMID:Use of a flavivirus RNA-dependent RNA polymerase assay to investigate the antiviral activity of selected compounds. 799 77

An assay for flavivirus RNA-dependent RNA polymerase activity in vitro was established using extracts of Vero cells infected with dengue virus type 2 (DEN-2) or Kunjin virus (KUN). RNA synthesis was initiated on a template of viral replicative form (RF) and RF was converted to the replicative intermediate (RI). The RNA-dependent RNA polymerase complex of DEN-2 utilised either DEN-2 or KUN RF as template, and similarly the KUN polymerase complex utilised either DEN-2 or KUN RF template. In addition, antibodies against the nonstructural proteins NS3 and NS5 inhibited the conversion of RF to RI, indicating that NS3 and NS5 are involved in viral RNA replication.
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PMID:Synthesis of dengue virus RNA in vitro: initiation and the involvement of proteins NS3 and NS5. 841 88

Each of the four serotypes of dengue viruses is responsible for a spectrum of illnesses that range from nonspecific febrile syndrome with good prognosis to dengue haemorrhagic fever or dengue shock syndrome. Definite diagnosis of dengue is provided by the detection of virus in acute-phase sera of patients. Virus isolation can be accomplished with mosquito cell lines or mosquito inoculations. However, these methods are time consuming and labour intensive. The reverse-transcriptase polymerase chain reaction (RT-PCR) provides a potential means of rapid diagnosis but requires specialised facilities and equipment and is expensive. Therefore a rapid, simple, sensitive, and economical method for direct detection of viral antigens in viraemic sera is needed for clinical and epidemiological investigations. An amplified fluorogenic enzyme-linked immunosorbent assay (F-ELISA) is described for the detection and identification of dengue-3 viruses in serum specimens. This assay utilizes biotinylated mouse IgG antibody directed against dengue antigens captured by anti-dengue monoclonal antibody coated onto polystyrene microplate wells. It takes advantage of the high affinity of biotin for the multivalent binding sites of streptavidin-labelled beta-galactosidase, and combines the amplification effect of biotin-streptavidin interaction with the high sensitivity of fluorogenic detection methods. Following optimisation of the procedure by reducing non-specific binding of proteins and enhancing the specific binding of antigens, F-ELISA was tested on 259 sera submitted routinely to our laboratory for confirmation of dengue diagnosis. The sensitivity of the F-ELISA was 90%, the specificity was 99% and the agreement rate was 98% between F-ELISA and virus isolation results.
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PMID:Rapid and sensitive streptavidin-biotin amplified fluorogenic enzyme-linked immunosorbent-assay for direct detection and identification of dengue viral antigens in serum. 855 Dec 57

The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.
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PMID:Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity. 860 61

Dengue viruses infect more than 100 million people each year and cause serious clinical manifestations. It is important to understand the replication of these viruses so that therapeutic and/or prophylactic agents may be designed. Dengue virus type 2 nonstructural proteins NS-5 and NS-3 were produced by in vitro transcription and translation of cloned genes. Both proteins possessed RNA-dependent RNA polymerase activity as measured by their ability to convert purified replicative form (RF) RNA to replicative intermediate (RI). The recombinant proteins, however, required one or more cellular protein(s) for their activity. Examination of NS-3 protein sequence revealed heretofore unnoticed sequence similarities with other polymerases.
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PMID:Conversion of dengue virus replicative form RNA (RF) to replicative intermediate (RI) by nonstructural proteins NS-5 and NS-3. 945 98

The dengue virus NS5 RNA-dependent RNA polymerase has been detected in the nucleus of virus-infected mammalian cells. We demonstrate here for the first time using in vitro and in vivo assay systems that the 37-amino-acid linker interdomain of NS5 (residues 369 to 405) contains a nuclear localization sequence (NLS) which is capable of targeting b-galactosidase to the nucleus. Further, we show that the linker is recognized by subunits of the NLS-binding importin complex with an affinity similar to that of the bipartite NLS of the retinoblastoma protein and, in analogous fashion to proteins such as the SV40 large tumor antigen, contains a functional protein kinase CK2 phosphorylation site (threonine 395). Interestingly, this site appears to inhibit NS5 nuclear targeting, probably through a cytoplasmic retention mechanism. The linker may have an important role in targeting NS5 to the nucleus in a regulated manner during the dengue virus infectious cycle.
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PMID:The 37-amino-acid interdomain of dengue virus NS5 protein contains a functional NLS and inhibitory CK2 site. 1020 52

The family Flaviviridae contains three genera: Hepacivirus, Flavivirus, and Pestivirus. Worldwide, more than 170 million people are chronically infected with Hepatitis C virus and are at risk of developing cirrhosis and/or liver cancer. In addition, infections with arthropod-borne flaviviruses (such as dengue fever, Japanese encephalitis, tick-borne encephalitis, St. Louis encephalitis, Murray Valley encephalitis, West Nile, and yellow fever viruses) are emerging throughout the world. The pestiviruses have a serious impact on livestock. Unfortunately, no specific antiviral therapy is available for the treatment or the prevention of infections with members of the Flaviviridae. Ongoing research has identified possible targets for inhibition, including binding of the virus to the cell, uptake of the virus into the cell, the internal ribosome entry site of hepaciviruses and pestiviruses, the capping mechanism of flaviviruses, the viral proteases, the viral RNA-dependent RNA polymerase, and the viral helicase. In light of recent developments, the prevalence of infections caused by these viruses, the disease spectrum, and the impact of infections, different strategies that could be pursued to specifically inhibit viral targets and animal models that are available to study the pathogenesis and antiviral strategies are reviewed.
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PMID:Perspectives for the treatment of infections with Flaviviridae. 1062 92


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