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)

Cloning and sequencing of segment 9 of Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) strains H and I were performed. The segment consisted of 1,186 bp harboring 5' and 3' noncoding regions and an open reading frame from positions 75 to 1037, encoding a protein with 320 amino acids, termed NS5. Comparison of the nucleotide sequences of NS5 for the two strains indicated 37 point differences resulting in only six amino acid replacements. Homology search showed that NS5 has localized similarities to human poliovirus RNA-dependent RNA polymerase and human rotavirus NS26. By Western blot analysis, NS5 was found in BmCPV-infected midgut cells, but not in polyhedra or virus virions, and was mainly detectable in the nucleus in BmCPV-infected BmN4 cells. Immunoblot analysis with anti-NS5 and antipolyhedrin antibodies displayed marked differences in the period of expression of NS5 and polyhedrin: the polyhedrin molecule was first detected 2 or 3 days after infection with BmCPV, whereas the expression of NS5 was initiated within a few hours. In addition, the level of polyhedrin increased as the infection developed, whereas the amount of NS5 remained essentially constant. When segment 9 was expressed with a baculovirus expression system, the resulting NS5 protein possessed the ability to bind to the double-stranded RNA genome. These results suggest that NS5 is expressed in early stages of infection and contributes to regulation of genomic RNA function.
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PMID:Determination of the nucleotide sequence of Bombyx mori cytoplasmic polyhedrosis virus segment 9 and its expression in BmN4 cells. 962 Oct 35

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 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

Sequence motifs within the non-structural protein NS5 or NS5B of the members of the family Flaviviridae suggest that this protein is the RNA-dependent RNA polymerase. This protein has now been expressed in various in vitro systems and used in polymerase assays. To understand the role of the RNA polymerase in RNA replication, this review will examine not only the polymerase protein but also the other proteins in the RNA replication complex. To date, several groups have investigated the interaction of these proteins both in vitro and in vivo and also the interaction of these proteins with the RNA signals at the 3' terminus of the RNA. Infectious clones and replicons containing the non-structural proteins have now been generated and these will be useful tools in understanding the processes of initiation and elongation of both positive and negative RNA synthesis.
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PMID:Flaviviridae polymerase and RNA replication. 1060 40

Extracellular ATP can function as a glial trophic factor as well as a neuronal transmitter. In astrocytes, mitogenic signalling by ATP is mediated by metabotropic P(2Y) receptors that are linked to the extracellular signal regulated protein kinase (Erk) cascade, but the types of P(2Y) receptors expressed in astrocytes have not been defined and it is not known whether all P(2Y) receptor subtypes are coupled to Erk by identical or distinct signalling pathways. We found that the P(2Y) receptor agonists ATP, ADP, UTP and 2-methylthioATP (2MeSATP) activated Erk and its upstream activator MAP/Erk kinase (Mek). cRaf-1, the first kinase in the Erk cascade, was activated by 2MeSATP, ADP and UTP but, surprisingly, cRaf-1 was not stimulated by ATP. Furthermore, ATP did not activate B-Raf, the major isoform of Raf in the brain, nor other Mek activators such as Mek kinase 1 (MekK1) and MekK2/3. Reverse transcriptase-polymerase chain reaction (RT - PCR) studies using primer pairs for cloned rat P(2Y) receptors revealed that rat cortical astrocytes express P(2Y(1)), a receptor subtype stimulated by ATP and ADP and their 2MeS analogues, as well as P(2Y(2)) and P(2Y(4)), subtypes in rats for which ATP and UTP are equipotent. Transcripts for P(2Y(6)), a pyrimidine-preferring receptor, were not detected. ATP did not increase cyclic AMP levels, suggesting that P(2Y(11)), an ATP-preferring receptor, is not expressed or is not linked to adenylyl cyclase in rat cortical astrocytes. These signal transduction and RT - PCR experiments reveal differences in the activation of cRaf-1 by P(2Y) receptor agonists that are inconsistent with properties of the P(2Y(1)), P(2Y(2)) and P(2Y(4)) receptors shown to be expressed in astrocytes, i.e. ATP=UTP; ATP=2MeSATP, ADP. This suggests that the properties of the native P(2Y) receptors coupled to the Erk cascade differ from the recombinant P(2Y) receptors or that astrocytes express novel purine-preferring and pyrimidine-preferring receptors coupled to the ERK cascade.
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PMID:P(2Y) purinoceptor subtypes recruit different mek activators in astrocytes. 1069 92

The NS5 protein of the flavivirus Kunjin (KUN) contains conserved sequence motifs characteristic of RNA-dependent RNA polymerase (RdRp) activity. To investigate this activity in vitro, recombinant NS5 proteins with C-terminal (NS5CHis) and N-terminal (NS5NHis) hexahistidine tags were produced in baculovirus-infected insect cells and purified to near homogeneity by nickel affinity chromatography. Purified NS5CHis exhibited RdRp activity with both specific (9 kb KUN replicon) and non-specific (8.3 kb Semliki Forest virus replicon) RNA templates; this activity did not require the presence of additional viral and/or cellular cofactors. RdRp activity of purified NS5NHis protein was reduced in comparison to NS5CHis, while purified NS5NHis incorporating a GDD-->GVD mutation within the polymerase active site (NS5GVD) lacked RdRp activity. RNase A digestion of the RdRp reaction products indicated that they were double-stranded and of a similar size to the KUN replicative form produced in Vero cells, thus demonstrating that the KUN NS5 protein has an intrinsic, albeit low and non-specific RdRp activity in vitro, similar to that reported for recombinant RdRp of other flaviviruses. However, in contrast to RNA polymerases of other Flavivirus species, purified KUN NS5 polymerase produced a single, full-length replicon RNA product, thus demonstrating efficient processivity.
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PMID:Expression and purification of enzymatically active recombinant RNA-dependent RNA polymerase (NS5) of the flavivirus Kunjin. 1116 16

The dengue virus RNA-dependent RNA polymerase, NS5, and the protease/helicase, NS3, are multidomain proteins that have been shown to interact both in vivo and in vitro. A hyperphosphorylated form of NS5 that does not interact with NS3 has been detected in the nuclei of virus-infected cells, presumably as the result of the action of a functional nuclear localization sequence within the interdomain region of NS5 (residues 369-405). In this study, it is shown by using the yeast two-hybrid system that the C-terminal region of NS3 (residues 303-618) interacts with the N-terminal region of NS5 (residues 320-368). Further, it is shown that this same region of NS5 is also recognized by the cellular nuclear import receptor importin-beta. The interaction between NS5 and importin-beta and competition by NS3 with the latter for the same binding site on NS5 were confirmed by pull-down assays. The direct interaction of importin-beta with NS5 has implications for the mechanism by which this normally cytoplasmic protein may be targetted to the nucleus.
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PMID:A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, NS3. 1125 77

Our research is directed towards enhancing the understanding of the molecular biology of dengue virus replication with the ultimate goal being to develop novel antiviral strategies based on preventing critical inter- or intra-molecular interactions required for the normal virus life cycle. The viral RNA-dependent RNA polymerase (NS5) and the viral helicase (NS3) interaction offers a possible target for inhibitors to bind and prevent replication. In this study the yeast-two hybrid system was used to show that a small region of NS5 interacts with NS3, and also with the cellular nuclear transport receptor importin-beta. Furthermore, intramolecular interaction between the two putative domains of NS5 can also be detected by the yeast two-hybrid assay. We have also modified the colony lift assay for the beta-galactosidase reporter activity in intact yeast cells which reflects the strength of interaction between two proteins to a microtiter plate format. This assay offers a unique opportunity to screen for small molecule compounds that block physiologically important interactions.
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PMID:Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets. 1134 63

We recently developed a model for flavivirus infection in mice and hamsters using the Modoc virus (MODV), a flavivirus with no known vector (P. Leyssen, A. Van Lommel, C. Drosten, H. Schmitz, E. De Clercq, and J. Neyts, 2001, Virology 279, 27-37). We now present the coding and noncoding sequence of MODV. The Modoc virus genome was determined to be 10,600 nucleotides in length with a single open reading frame extending from nucleotides 110 to 10,234, encoding 3374 amino acids. The deduced gene order of the single open reading frame is C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5, which is exactly the same as that of the mosquito- and tick-borne flaviviruses. It is flanked by a 5'- and 3'-untranslated region (UTR) of 109 and 366 nucleotides, respectively. Alignment of the MODV amino acid sequence with that of 20 other flaviviruses revealed several regions with high sequence similarity corresponding to functionally important domains (e.g., the serine protease/helicase/NTPase of NS3 and the methyltransferase/RNA-dependent RNA polymerase of NS5) and conserved sites for proteolytic cleavage by viral and cellular proteases. Phylogenetic analysis of the entire coding region confirmed the classification of MODV within the flaviviruses with no known vector, which is in agreement with previous findings based on partial NS5 sequences. A detailed comparative analysis of the putative folding patterns of the 5'- and 3'-UTR of MODV and of the tick- and mosquito-borne viruses was carried out. Structural elements in the 5'- and 3' UTR of MODV that are preserved among vector-borne flaviviruses were noted and so were structural elements distinguishing the MODV UTRs from mosquito-borne and tick-borne flaviviruses. Also the putative secondary structure of circularized MODV RNA is presented.
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PMID:Complete genome sequence, taxonomic assignment, and comparative analysis of the untranslated regions of the Modoc virus, a flavivirus with no known vector. 1185 6

Viruses represent an attractive system with which to study the molecular basis of mRNA capping and its relation to the RNA transcription machinery. The RNA-dependent RNA polymerase NS5 of flaviviruses presents a characteristic motif of S-adenosyl-L-methionine-dependent methyltransferases at its N-terminus, and polymerase motifs at its C-terminus. The crystal structure of an N-terminal fragment of Dengue virus type 2 NS5 is reported at 2.4 A resolution. We show that this NS5 domain includes a typical methyltransferase core and exhibits a (nucleoside-2'-O-)-methyltransferase activity on capped RNA. The structure of a ternary complex comprising S-adenosyl-L-homocysteine and a guanosine triphosphate (GTP) analogue shows that 54 amino acids N-terminal to the core provide a novel GTP-binding site that selects guanine using a previously unreported mechanism. Binding studies using GTP- and RNA cap-analogues, as well as the spatial arrangement of the methyltransferase active site relative to the GTP-binding site, suggest that the latter is a specific cap-binding site. As RNA capping is an essential viral function, these results provide a structural basis for the rational design of drugs against the emerging flaviviruses.
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PMID:An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. 1203 88


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