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)

The plant bromoviruses and animal nodaviruses are distinct groups of positive strand RNA viruses that have proven to be useful models for RNA replication studies. Bromoviruses encode two large proteins required for RNA replication: 1a contains domains implicated in helicase and capping functions, and 2a contains a central polymerase-like domain. Using immunoprecipitation and far-western blotting, we have now shown that 1a and 2a form a specific complex in vitro and have mapped the interacting domains. Molecular genetic data implicate the 1a-2a complex in RNA replication and suggest that it supports coordinate action of the putative helicase, polymerase, and capping domains. The locations of the interacting 1a and 2a domains have implications for replication models and the evolution of virus genomes bearing homologous replication genes in fused vs. divided forms. For the nodavirus Flock house virus (FHV), a true RNA replicase has been isolated that carries out complete, highly active replication of added FHV RNA, producing newly synthesized positive strand RNA in predominantly ssRNA form. Positive strand RNA synthesis in this FHV cell-free system is strongly dependent on the addition of any of several glycerophospholipids. Positive strand RNA synthesis depends on the complete glycerophospholipid structure, including the polar head group and diacyl glycerol lipid portion, and is strongly influenced by acyl chain length.
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PMID:Protein-protein interactions and glycerophospholipids in bromovirus and nodavirus RNA replication. 803 45

The paramyxovirus large protein (L) and phosphoprotein (P) are both required for viral RNA-dependent RNA polymerase activity. Previous biochemical experiments have shown that L and P can form a complex when expressed from cDNA plasmids in vivo. In this report, L and P proteins of the paramyxovirus simian virus 5 (SV5) were coexpressed in HeLa T4 cells from cDNA plasmids, and L-P complexes were examined. To identify regions of the SV5 L protein that are required for L-P complex formation, 16 deletion mutants were constructed by mutagenesis of an SV5 L cDNA. Following coexpression of these L mutants with cDNA-derived P and radiolabeling with 35S-amino acids, cell lysates were analyzed for stable L-P complexes by a coimmunoprecipitation assay and by sedimentation on 5 to 20% glycerol gradients. Mutant forms of L containing deletions that removed as much as 1,008 residues from the C-terminal half of the full-length 2,255-residue L protein were detected in complexes with P by these two assays. In contrast, large deletions in the N-terminal half of L resulted in proteins that were defective in the formation of stable L-P complexes. Likewise, L mutants containing smaller deletions that individually removed N-terminal regions which are conserved among paramyxovirus and rhabdovirus L proteins (domain I, II, or III) were also defective in stable interactions with P. These results suggest that the N-terminal half of the L protein contains sequences important for stable L-P complex formation and that the C-terminal half of L is not directly involved in these interactions. SV5-infected HeLa T4 cells were pulse-labeled with 35S-amino acids, and cell extracts were examined by gradient sedimentation. Solubilized L protein was detected as an approximately 8 to 10S species, while the P protein was found as both a approximately 4S form (approximately 85%) and a species that cosedimented with L (approximately 15%). These data provide the first biochemical evidence in support of a simple domain structure for an L protein of the nonsegmented negative-sense RNA viruses. The results are discussed in terms of a structural model for the L protein and the interactions of L with the second viral polymerase subunit P.
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PMID:Mapping of a region of the paramyxovirus L protein required for the formation of a stable complex with the viral phosphoprotein P. 803 85

The RNA-dependent RNA polymerase (RdRp) of foxtail mosaic virus (FMV) was partially purified from infected leaves of Chenopodium quinoa. The membrane fraction of crude plant extracts contained most of the FMV RdRp activity. Additional purification was obtained by solubilization of the RdRp using KCl and dodecyl-sucrose and by centrifugation through a glycerol gradient. The RNA template endogenous to RdRp preparations could be removed using micrococcal nuclease but the resulting fraction was unable to copy added template purified from FMV virions. However, supplementation of fractions containing RdRp activity with FMV RNA resulted in a significant decrease in the level of RNA synthesis. This effect was specific to potexviral RNAs since a similar interference was also observed with clover yellow mosaic virus RNA but not with brome mosaic virus RNA or yeast RNA. RNA transcripts corresponding to various regions of the FMV genome were tested for their ability to inhibit RNA synthesis on endogenous template. The simultaneous presence of both 5' and 3' terminal regions of the viral genome was necessary to interfere with RNA synthesis suggesting that this inhibition resulted from competition for the binding of component(s) of the RdRp complex.
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PMID:Partial purification and characterization of foxtail mosaic potexvirus RNA-dependent RNA polymerase. 824 92

Plasma cholesterol levels increase after birth, and to a greater extent in breast-fed versus formula-fed infants. This increase is believed to be due to the high fat and cholesterol content of the infant diet, but little is known about the effects of early diet on the expression of proteins involved in regulating cholesterol metabolism. This study examined changes in the expression of hepatic proteins regulating cholesterol metabolism during development. Newborn piglets were fed sow milk or one of four formulas for 18 days. The formulas had similar levels of palmitic acid (16:0) as in milk, supplied as palm olein oil with 16:0 esterified predominantly to the sn-1,3 position or as synthesized triglyceride (TG) with 16:0 esterified mainly to the sn-2 position of glycerol, each with no cholesterol (<0.10 mmol/L) or 0.65 mmol/L cholesterol added. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of mRNA levels was used to assess the effects of diet on hepatic hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, low-density lipoprotein (LDL) receptor, and 7alpha-hydroxylase (C7H). LDL receptor mRNA levels showed no appreciable difference between milk- and formula-fed piglets. However, the levels of HMG-CoA reductase and C7H mRNA were higher (P < .05) in all formula-fed versus milk-fed piglets, irrespective of the formula TG source or cholesterol content. The lower levels of HMG-CoA reductase and C7H mRNA in milk-fed piglets were accompanied by higher (P < .05) plasma total, high-density lipoprotein (HDL), and apolipoprotein (apo) B-containing cholesterol. These studies show that the levels of hepatic HMG-CoA reductase and C7H mRNA, but probably not LDL receptor mRNA, are altered by early diet.
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PMID:Early diet influences hepatic hydroxymethyl glutaryl coenzyme A reductase and 7alpha-hydroxylase mRNA but not low-density lipoprotein receptor mRNA during development. 944 Apr 72

Cucumber mosaic virus (CMV) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic RNAs. The very 3' ends of each of the genomic RNAs can fold into a tRNA-like structure. Based on the structural analysis of the 3' tRNA-like structure of the brome mosaic virus (BMV), we superimposed and redrew the 3' tRNA-like structure of CMV. We homogenized virus infected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra-centrifugation three times to get detergent solubilized membrane bound fractions. We accidentally found that these fractions were enriched with a host-encoded RNA-dependent RNA polymerase (RdRp) activity. Similar activity could also be found in other plants tested. Alternately, the membrane bound fraction could be simply precipitated by low speed centrifugation (3,000 g) and high speed ultra-centrifugation (40,000 g). The pellet was then suspended in a detergent-containing buffer, after which 25%-55% glycerol gradient fractionation was performed. Activity was tested through the incorporation of [alpha-32P]UTP using endogenous CMV RNAs as templates on each fraction collected. It was found that most of the fractions contained the viral-encoded RNA-dependent RNA polymerase. The products of RdRp reaction were found to have a double-stranded from through further analysis of the RNase protection assay.
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PMID:The preparation of RNA-dependent RNA polymerase complex from virus infected plants. 961 71

Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.
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PMID:Characterization of soluble hepatitis C virus RNA-dependent RNA polymerase expressed in Escherichia coli. 988 74

Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from pooled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5BDeltaCT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays. NS5BDeltaCT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 mM. Interestingly, Mg2+, at concentrations up to 20 mM, did not support the GBV-B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 microM. Higher concentrations of monovalent salts (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory. NS5BDeltaCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology ( approximately 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.
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PMID:RNA-dependent RNA polymerase activity encoded by GB virus-B non-structural protein 5B. 1097 21

RNA-dependent RNA polymerase (RdRp) activity was detected in the crude microsomal fraction of rice cultured cells that contain a 14 kbp double-stranded RNA (dsRNA). RdRp activity is maximal in the presence of all four nucleotide triphosphates and Mg2+ ion and is resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D and alpha-amanitin). RdRp activity increases approximately 2.5-fold in the presence of 0.5% deoxycholate. Treatment of purified microsomal fraction with proteinase K plus deoxycholate suggests that the RdRp enzyme complex with its own 14 kb RNA template is located in vesicles. The RdRp enzyme complex was solubilized with Nonidet P-40 and purified by glycerol gradient centrifugation, then exogenous RNA templates were added. Results indicate that exogenous dsRNA reduces RNA synthesis from the endogenous 14 kb RNA template.
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PMID:RNA-dependent RNA polymerase activity associated with endogenous double-stranded RNA in rice. 1123 May 74

Alfalfa mosaic virus (AMV) RNAs 1 and 2 encode the replicase proteins P1 and P2, respectively, whereas RNA 3 encodes the movement protein and the coat protein (CP). When RNAs 1 and 2 were transiently expressed from a T-DNA vector (R12 construct) by agroinfiltration of Nicotiana benthamiana, the infiltrated leaves accumulated minus-strand RNAs 1 and 2 and relatively small amounts of plus-strand RNAs. In addition, RNA-dependent RNA polymerase (RdRp) activity could be detected in extracts of the infiltrated leaves. After transient expression of RNAs 1 and 2 with the 3'-untranslated regions (UTRs) of both RNAs deleted (R1Delta/2Delta construct), no replication of RNAs 1 and 2 was observed, while the infiltrated leaves supported replication of RNA 3 after inoculation of the leaves with RNA 3 or expression of RNA 3 from a T-DNA vector (R3 construct). No RdRp activity could be isolated from leaves infiltrated with the R1Delta/2Delta construct, although P1 and P2 sedimented in a region of a glycerol gradient where active RdRp was found in plants infiltrated with R12. RdRp activity could be isolated from leaves infiltrated with constructs R1Delta/2 (3'-UTR of RNA 1 deleted), R1/2Delta (3'-UTR of RNA 2 deleted), or R1Delta/2Delta plus R3. This demonstrates that the 3'-UTR of AMV RNAs is required for the formation of a complex with in vitro enzyme activity. RNAs 1 and 2 with the 3'-UTRs deleted were encapsidated into virions by CP expressed from RNA 3. This shows that the high-affinity binding site for CP at the 3'-termini of AMV RNAs is not required for assembly of virus particles.
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PMID:Role of the 3'-untranslated regions of alfalfa mosaic virus RNAs in the formation of a transiently expressed replicase in plants and in the assembly of virions. 1141 11

The NS5B gene, cloned from Classical swine fever virus (CSFV) genome, was expressed in porcine kidney cells PK-15, natural host of CSFV. In purifying cytoplasmic extracts from these cells by means of different concentrations of salt, glycerol and detergent four fractions, namely crude supernatant (SC) and different purified supernatants (S1, S2 and S3) were obtained. Using Western blot analysis the NS5B protein was found in all these fractions, showing that it was soluble in both higher and lower concentrations of salt, glycerol and detergent. The NS5B protein present in the four different fractions exhibited RNA-dependent RNA polymerase (RdRp) activity, but it was unable to complete the whole process of RNA synthesis. Site-directed mutation analysis showed that Thy216 and Cyt228 were essential for RNA synthesis while Cyt219 was not, suggesting that CSFV RdRp was template-specific. We conclude that initiation of RNA synthesis by CSFV RdRp includes also template priming.
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PMID:RNA-dependent RNA polymerase activity of Classical swine fever virus NS5B protein expressed in natural host cells. 1452 73


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