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 complete nucleotide sequence of apple stem grooving virus (ASGV) genome has been determined. The genome is 6496 nucleotides in length excluding a 3'-terminal poly(A) tail and contains two overlapping open reading frames (ORFs). ORF1 begins at nucleotide position 37 and is terminated at position 6341, encoding a protein with a molecular weight of 241 kDa. ORF2, which is in a different reading frame within ORF1, begins at position 4788 and can encode a 36-kDa protein. The 241-kDa protein contains two consensus sequences associated with the RNA-dependent RNA polymerase and the NTP-binding helicase. Comparisons of amino acid sequences around these conserved motifs with other RNA viruses revealed that ASGV has extensive similarities with apple chlorotic leaf spot, tymo-, carla-, and potexviruses, and is a member of the sindbis-like supergroup. ASGV coat protein is found to be located in the C-terminal region of the 241-kDa polyprotein. The 36-kDa protein encoded by ORF2 contains the consensus sequence Gly-Asp-Ser-Gly found in the active site of several cellular and viral serine proteases.
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PMID:The nucleotide sequence of apple stem grooving capillovirus genome. 141 30

Grapevine fanleaf nepovirus (GFLV) has a bipartite plus-sense RNA genome. Its structural and functional proteins originate from polyprotein maturation by at least one virus-encoded proteinase. Here we describe the cloning of the 24-kDa proteinase cistron located between the virus-linked protein (VPg) and the RNA-dependent RNA polymerase cistron in GFLV RNA1 (nucleotides 3966 to 4622). Proteinase expressed from this clone is able to cleave GFLV polyprotein P2 in order to produce the coat protein and a 66-kDa protein which is further processed to the 38-kDa presumed movement protein. The GFLV 24-kDa proteinase sequence contains sequence similarities with other nepovirus and comovirus proteinases, particularly at the level of the conserved domains corresponding to the hypothetical catalytic triad and to the substrate-binding pocket (amino acids 192 to 200). Site-directed mutagenesis of residues His43, Glu87, and Leu197 abolished proteinase activity. Inactivation of the enzyme is also observed if the catalytic residue Cys179 was substituted by isoleucine, but replacement by a serine at the same position produced a mutant with an activity identical to that of native proteinase. All our data show that GFLV cysteine proteinase presents structure similarities to the proteinases of cowpea mosaic virus and potyviruses but is most closely related to trypsin.
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PMID:Effects of site-directed mutagenesis on the presumed catalytic triad and substrate-binding pocket of grapevine fanleaf nepovirus 24-kDa proteinase. 151 63

RNA-dependent RNA polymerases contain a highly conserved region of amino acids with a core segment composed of the amino acids YGDD which have been hypothesized to be at or near the catalytic active site of the molecule. Six mutations in this conserved YGDD region of the poliovirus RNA-dependent RNA polymerase were made by using oligonucleotide site-directed DNA mutagenesis of the poliovirus cDNA to substitute A, C, M, P, S, or V for the amino acid G. The mutant polymerase genes were expressed in Escherichia coli, and the purified RNA polymerases were tested for in vitro enzyme activity. Two of the mutant RNA polymerases (those in which the glycine residue was replaced with alanine or serine) exhibited in vitro enzymatic activity ranging from 5 to 20% of wild-type activity, while the remaining mutant RNA polymerases were inactive. Alterations in the in vitro reaction conditions by modification of temperature, metal ion concentration, or pH resulted in no significant differences in the activities of the mutant RNA polymerases relative to that of the wild-type enzyme. An antipeptide antibody directed against the wild-type core amino acid segment containing the YGDD region of the poliovirus polymerase reacted with the wild-type recombinant RNA polymerase and to a limited extent with the two enzymatically active mutant polymerases; the antipeptide antibody did not react with the mutant RNA polymerases which did not have in vitro enzyme activity. These results are discussed in the context of secondary-structure predictions for the core segment containing the conserved YGDD amino acids in the poliovirus RNA polymerase.
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PMID:Enzymatic activity of poliovirus RNA polymerase mutants with single amino acid changes in the conserved YGDD amino acid motif. 165 2

The nucleotide sequence of the genomic RNA1, 7342 nucleotides (nt) of grapevine fanleaf virus strain F13 (GFLV-F13) has been determined from cDNA clones. The complete sequence contained only one long open reading frame (ORF) of 6852 nucleotides extending from nucleotide 243 to 7101. The putative polyprotein encoded by this ORF is 2284 amino acids in length with an Mr of 253K. The location of genome-linked protein and comparison of the primary structure of the 253K polyprotein to that of other closely related viral proteins of the picronavirus-like family allows the proposal of a scheme for the genetic organization of GFLV-F13 RNA1. The primary structure of the polyprotein includes a putative RNA-dependent RNA polymerase of 92K and a cysteine protease of 25K. This protease shares not only major structural homologies, particularly in the substrate-binding pocket, with the trypsin-like serine proteases of other picorna-like viruses, but also their specificity in terms of cleavage. The large region of Mr 133K upstream of the VPg was found to contain at least two domains, one of which could be easily aligned with the NTP-binding sequence pattern and another which may have the characteristics of a protease cofactor. Thus, the 253K protein possesses the same general genetic organization as the corresponding protein of other picorna-like viruses.
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PMID:Complete nucleotide sequence and genetic organization of grapevine fanleaf nepovirus RNA1. 165 53

The amino acid sequence of the polyprotein deduced from the nucleotide sequence of the Japanese hepatitis C virus genome (N. Kato et al. (1990) Proc. Natl. Acad. Sci. USA 87, 9524-9528) indicated that this virus is a member of a new class of positive-stranded RNA viruses. Several domains of this polyprotein also showed weak homology with those of flaviviruses and pestiviruses including the chymotrypsin-like serine proteinase, NTPase and RNA-dependent RNA polymerase.
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PMID:Molecular structure of the Japanese hepatitis C viral genome. 184 88

Proteolytic processing of poliovirus polyprotein is carried out by the products of two viral genes, 2A and 3C. 2A protease catalyzes cleavage of the polyprotein of type 1 poliovirus at two sites, one a cis cleavage at the 2A N-terminus and the other a trans cleavage within the 3D polymerase. In addition to polyprotein cleavage activity, 2A protease also indirectly induces cleavage of the p220 component of the cap-binding protein complex, which results in selective inhibition of host protein synthesis. Molecular genetic and biochemical analyses of 2A protease were performed to test its putative homology to small trypsin-like serine proteases and to examine the roles of individual amino acids in the reaction mechanism of 2A protease. A recombinant plasmid containing poliovirus 1C, 1D, and 2A gene sequences was expressed in a cell-free transcription/translation system, resulting in synthesis of a precursor protein that underwent efficient self-processing and produced mature 2A protease. To identify residues involved in the catalytic center and/or substrate-binding loops, we generated a series of 2A mutants by site-specific mutagenesis of this plasmid. Mutants were then expressed in vitro and tested for autocatalytic cis cleavage activity, trans cleavage of the 1D/2A junction, and trans-activation of p220-specific protease. Our data suggest that the conserved His20, Asp38, and Cys109 residues recently proposed to be equivalent to the catalytic triad of known serine proteases may comprise the catalytic triad of 2A protease. Surprisingly, Asp38 could be replaced with glutamic acid and retain autocatalytic function. Other amino acid substitutions at Tyr88, Tyr89, and Thr124 suggested that these residues lie in loops involved in substrate binding. Biochemical studies with protease inhibitors indicate that 2A protease activity is blocked by inhibitors specific for serine and cysteine proteases. Overall, the results are consistent with the hypothesis that 2A proteinase is structurally similar to the trypsin-like family of serine proteases with the substitution of cysteine 109 as the active site nucleophile.
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PMID:Identification of essential amino acid residues in the functional activity of poliovirus 2A protease. 185 Sep 21

Poliovirus-specific RNA-dependent RNA polymerase (3Dpol) was purified to apparent homogeneity. A single polypeptide of an apparent molecular weight of 63,000 catalyzes the synthesis of dimeric and monomeric RNA products in response to the poliovirion RNA template. Analysis of purified 3Dpol by two-dimensional electrophoresis showed multiple forms of 3Dpol, suggesting posttranslational modification of the protein in virus-infected cells. The two major forms of 3Dpol appear to have approximate pI values of 7.1 and 7.4. Incubation of purified 3Dpol with calf intestinal phosphatase resulted in almost complete disappearance of the pI 7.1 form and a concomitant increase in the intensity of the pI 7.4 form of 3Dpol. Addition of 32P-labeled Pi during infection of HeLa cells with poliovirus resulted in specific labeling of 3Dpol and 3CD, a viral protein which contains the entire 3Dpol sequence. Both 3Dpol and 3CD appear to be phosphorylated at serine residues. Ribosomal salt washes prepared from both mock- and poliovirus-infected cells contain phosphatases capable of dephosphorylating quantitatively the phosphorylated form (pI 7.1) of 3Dpol.
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PMID:Multiple isoelectric forms of poliovirus RNA-dependent RNA polymerase: evidence for phosphorylation. 255 40

1. The RNA-dependent RNA polymerase from Halobacterium cutirubrum was purified to electrophoretic homogeneity. 2. It requires a single-stranded molecule of RNA or polyribonucleotide as template. 3. Nearest-neighbour analyses of the products formed on random poly(A,U) or alternating poly(A-U) templates and base analysis of the product of synthesis directed by wheat-germ RNA prove that the template is copied accurately. 4. The enzyme initiates new chains with purine ribonucleoside triphosphates. 5. Sucrose-density-gradient analysis of the product indicates that it has a size distribution similar to that of the template. 6. Preliminary amino acid analysis of the RNA-dependent polymerase shows that it contains much less serine than either of the subunits of H. cutirubrum DNA-dependent RNA polymerase. 7. The RNA-dependent enzyme is unable to substitute for either subunit of the DNA-dependent polymerase, and both the latter are devoid of RNA-dependent activity.
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PMID:Purification and properties of the ribonucleic acid-dependent ribonucleic acid polymerase from Halobacterium cutirubrum. 463 91

The phosphoprotein P of human respiratory syncytial virus (RSV) was expressed in eukaryotic cells in phosphorylated form. Site-directed mutagenesis of the recombinant protein established Ser232 as the major site of phosphorylation in vivo. Phosphorylation of bacterially made P protein in vitro by purified casein kinase II (CKII) resulted in the phosphorylation of Ser237, whereas mainly Ser232 was phosphorylated by a crude cell extract. The P kinase activity in the cell extract exhibited properties characteristic of CKII. While the Ser232,237 to Ala double mutant was nearly completely defective for phosphorylation and transcription, phosphorylation at Ser232, through the use of appropriate P mutant or kinase, activated P protein. Phosphorylation of Ser237 restored activity only to the extent it facilitated phosphorylation of Ser232. Phosphate groups of P protein in RSV-infected cells were highly stable; inhibitors of protein serine phosphatases had no effect on the intracellular turnover of the phosphates. Highly purified viral polymerase L was transcriptionally active but devoid of P protein kinase activity. Thus, CKII-mediated phosphorylation of Ser232 appears to be the primary regulator of P protein activity while phosphorylation of Ser237 may be involved in a modulatory role under certain conditions.
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PMID:Phosphorylation of Ser232 directly regulates the transcriptional activity of the P protein of human respiratory syncytial virus: phosphorylation of Ser237 may play an accessory role. 749 65

The poliovirus RNA-dependent RNA polymerase (3Dpol) contains a region of homology centered around the amino acid motif YGDD (amino acids 326 to 329), which has been postulated to be involved in the catalytic activity of the enzyme. Previous studies from this laboratory have used oligonucleotide site-directed mutagenesis to substitute the tyrosine amino acid at this motif with other amino acids (S. A. Jablonski and C. D. Morrow, J. Virol. 67:373-381, 1993). The viruses recovered with 3Dpol genes with a methionine mutation also contained a second mutation at amino acid 108 resulting in a glutamic acid-to-aspartic acid change (3D-E-108 to 3D-D-108) in the poliovirus RNA polymerase. On the basis of these results, we suggested that the amino acid at position 108 might interact with the YGDD region of the poliovirus polymerase. To further investigate this possibility, we have constructed a series of constructs in which the poliovirus RNA polymerases contained a mutation at amino acid 108 (3D-E-108 to 3D-D-108) as well as a mutation in which the tyrosine amino acid (3D-Y-326) was substituted with cysteine (3D-C-326) or serine (3D-S-326). The mutant 3Dpol polymerases were expressed in Escherichia coli, and in vitro enzyme activity was analyzed. Enzymes containing the 3D-D-108 mutation with the wild-type amino acid (3D-Y-326) demonstrated in vitro enzyme activity similar to that of the wild-type enzyme containing 3D-E-108. In contrast, enzymes with the 3D-C-326 or 3D-S-326 mutation had less in vitro activity than the wild type. The inclusion of the second mutation at amino acid 3D-D-108 did not significantly affect the in vitro activity of the polymerases containing 3D-C-326 or 3D-S-326 mutation. Transfections of poliovirus cDNAs containing the substitution at amino acid 326 with or without the second mutation at amino acid 108 were performed. Consistent with previous findings, we found that transfection of poliovirus cDNAs containing the 3D-C-326 or 3D-S-326 mutation in 3Dpol did not result in the production of virus. Surprisingly, transfection of the poliovirus cDNAs containing the 3D-D-108/C-326 double mutation, but not the 3D-D-108/S-326 mutation, resulted in the production of virus. The virus obtained from transfection of polio-virus cDNAs containing 3D-D-108/C-326 mutation replicated with kinetics similar to that of the wild-type virus. RNA sequence analysis of the region of the 3Dpol containing the 3D-C-326 mutation revealed that the codon for cysteine (UGC) reverted to the codon for tyrosine (UAC). The results of these studies establish that under the appropriate conditions, poliovirus has the capacity to revert mutations within the YGDD amino acid motif of the poliovirus 3Dpol gene and further strengthen the idea that interaction between amino acid 108 and the YGDD region of 3Dpol is required for viral replication.
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PMID:An aspartic acid at amino acid 108 is required to rescue infectious virus after transfection of a poliovirus cDNA containing a CGDD but not SGDD amino acid motif in 3Dpol. 749 45


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