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 association of an RNA-dependent RNA polymerase activity with virions of pike fry rhabdovirus has been demonstrated by both in vitro and in vivo studies. The temperature optimum for the in vitro assay is around 20 C, although enzyme activity can be observed at 4 C. Preparations of pike fry virus possess a glycoprotein, a membrane protein, a nucleoprotein, an L protein, and a phosphoprotein, as well as an RNA of about 3.8 times 10-6 mol wt. A protein kinase activity has been found associated with virus preparations. In vitro RNA product analyses indicate that the virus-associated enzyme functions principally as a transcriptase synthesizing viral-complementary, heteropolymeric RNA.
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PMID:RNA polymerase associated with virions of pike fry rhabdovirus. 116 3

The phosphoprotein (P) and the large protein (L) constitute the RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV). We show that phosphate-free P protein expressed in bacteria is transcriptionally inactive when reconstituted with L protein and viral N-RNA template free of cellular protein kinase. Phosphorylation of P protein by a cellular kinase(s) was essential for transcription as well as for further phosphorylation by an L-associated kinase, the two kinases acting in a sequential (cascade) manner. Phosphate groups introduced by cell kinase were stable, whereas those due to L kinase underwent a turnover which was coupled to ongoing transcription. We present a model for the phosphorylation pathway of P protein and propose that continued phosphorylation and dephosphorylation of P protein may represent a transcriptional regulatory (on-off) switch of nonsegmented negative-strand RNA viruses.
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PMID:Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation. 130 93

Ribonucleoprotein complexes isolated from measles virus-infected HeLa cells contained an RNA-dependent RNA polymerase activity that catalyzed the incorporation of ribonucleotides into ribonucleic acid. The ribonucleoprotein complexes were composed of measles virus nucleoprotein, phosphoprotein, and a large protein, as well as viral RNA. The kinetics of RNA synthesis at different temperatures, time intervals, and protein, ribonucleotide, and mono- and divalent cation concentrations were analyzed. Enzyme activity was maximum at 4 h at 25 degrees C in the presence of 100 mM Na+-2.5 mM Mg2+-1 mM ribonucleotides. Actinomycin D and alpha-amanitin had no effect on the enzyme activity. Addition of cytoplasmic extracts from uninfected HeLa cells to the reaction mixture did not increase the incorporation of ribonucleotides into RNA. The in vitro synthesized RNAs were characterize by slot blot analysis and quantitated by densitometer scanning. All mRNAs coding for the structural proteins of measles virus were synthesized. Nucleoprotein RNA was the most abundant species made, followed by phosphoprotein, hemagglutinin, fusion protein, matrix protein, and large-protein RNAs. The system described here resulted in the first efficient transcription of measles virus RNA and analysis of products.
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PMID:Characterization of in vitro transcription and transcriptional products of measles virus. 366 51

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 RNA-dependent RNA polymerase of human respiratory syncytial (RS) virus was expressed in a functional form from a cDNA clone. Coexpression of the viral polymerase (L) protein, phosphoprotein (P), and nucleocapsid (N) protein allowed us to develop a system for expression and recovery of replicable RS virus RNA entirely from cDNA clones. cDNA clones of the N, P, and L genes were constructed in pGEM-based expression plasmids and shown to direct expression of the appropriate polypeptides. Two types of RS virus genomic RNA analogs were expressed from an intracellular transcription plasmid that directed the synthesis of RNAs with defined 5' and 3' ends. One analog included the authentic 5' and 3' termini of the genome, and the second contained the authentic 5' terminus and its complement at the 3' terminus as found in copyback defective interfering RNAs of other negative-strand RNA viruses. Both types of genomic analogs were encapsidated and replicated in cells expressing the RS virus N, P, and L proteins. Omission of any of the three viral proteins abrogated replication, thereby defining the N, P, and L proteins as the minimal trans-acting proteins required for RNA replication. This system has the advantages that expression occurs at a level sufficient to allow direct biochemical analysis of the products of RNA replication and that neither the use of reporter genes nor wild-type RS helper virus is required. These features allow analysis of both cis- and trans-acting factors involved in the control of replication of RS virus RNA.
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PMID:Functional cDNA clones of the human respiratory syncytial (RS) virus N, P, and L proteins support replication of RS virus genomic RNA analogs and define minimal trans-acting requirements for RNA replication. 788 88

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

We have mapped the genome of lettuce necrotic yellows virus (LNYV), the type member of the genus cytorhabdovirus of the family Rhabdoviridae. We have cloned and sequenced all intergenic regions and the 3' leader and 5' trailer of the negative-sense, single-stranded RNA genome of LNYV. The LNYV genome appears to contain six genes, the five expected genes coding for the virion proteins, and a sixth gene of unknown function, as for sonchus yellow net virus (SYNV), a member of the genus nucleorhabdovirus. The proposed LNYV genomic map is 3'-N-4a-4b-M-G-L-5', where N is the nucleocapsid protein gene; 4a and 4b are two genes, one of which codes for the proposed phosphoprotein P and the other for a putative protein of unknown function; M is the proposed matrix protein gene; G is the proposed glycoprotein gene; and L is the proposed transcriptase gene. The different LNYV intergenic regions have highly conserved consensus sequences, which could be divided into three components: the sequences corresponding to the 3' end of the mRNAs, intergenic sequences of variable length, and the sequences corresponding to the 5' end of the mRNAs. A leader sequence of 84 nucleotides (nt) at the 3' end of the LNYV genomic RNA preceeded the N gene. A trailer sequence of 187 nt at the 5' end of the genomic RNA followed the L gene. A comparison between LNYV leader and trailer sequences revealed complementary 3' and 5' ends, which could give rise to a putative "panhandle" structure with a two bases overhang in the leader sequence. We have compared these sequences to the corresponding sequences of SYNV as well as to vesicular stomatitis virus (VSV) and rabies virus (RV), the type members of the vesiculovirus and lyssavirus genera, respectively, of animal rhabdoviruses. Homologies were found in the intergenic regions between LNYV, SYNV, VSV, and RV, at the 3' ends of the mRNAs. LNYV intergenic sequences were of variable lengths, as were those found in RV. The consensus sequences found at the 5' ends of LNYV mRNAs differed from the highly conserved consensus transcription start sequence UUGU/A found in SYNV, VSV, and RV. Conserved sequences were also found in the first 30 nt of the leader and the last 30 nt of the trailer, between LNYV, SYNV, VSV, and RV.
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PMID:Genomic organization of lettuce necrotic yellows rhabdovirus. 817 30

Infectious human respiratory syncytial virus (RSV) was produced by the intracellular coexpression of five plasmid-borne cDNAs. One cDNA encoded a complete positive-sense version of the RSV genome (corresponding to the replicative intermediate RNA or antigenome), and each of the other four encoded a separate RSV protein, namely, the major nucleocapsid N protein, the nucleocapsid P phosphoprotein, the major polymerase L protein, or the protein from the 5' proximal open reading frame of the M2 mRNA [M2(ORF1)]. RSV was not produced if any of the five plasmids was omitted. The requirement for the M2(ORF1) protein is consistent with its recent identification as a transcription elongation factor and confirms its importance for RSV gene expression. It should thus be possible to introduce defined changes into infectious RSV. This should be useful for basic studies of RSV molecular biology and pathogenesis; in addition, there are immediate applications to the development of live attenuated vaccine strains bearing predetermined defined attenuating mutations.
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PMID:Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5' proximal open reading frame of the M2 mRNA in gene expression and provides a capability for vaccine development. 852 4

Measles virus (MV) expresses at least 3 proteins from the phosphoprotein (P) cistron. Alternative translation initiation directs synthesis of the C protein from the +1 reading frame, while so-called RNA editing generates a second population of mRNAs which express the V protein from the -1 reading frame which lies within and overlaps the larger P reading frame. While the P protein has been demonstrated to be an essential cofactor for the L protein in the formation of active transcriptase complexes, the functions of the V and C proteins remain unknown. In order to investigate these functions, we have expressed the MV P, V and C proteins as GST fusions in E. coli for affinity purification and use in an in vitro binding assay with other viral and cellular proteins. The P protein was found to interact with L, NP, and with itself. These interactions were mapped to the carboxy-terminal half of the protein which is absent in the V protein. In contrast, both the V and C proteins failed to interact with any other viral proteins, but were each found to interact specifically with one or more cellular proteins. Appropriate aspects of these results were confirmed in vivo using the yeast two-hybrid system. These observations suggest that the V and C proteins may be involved in modulation of the host cellular environment within MV-infected cells. Such activity would be distinct from their previously proposed role in the possible down-regulation of virus-specific RNA transcription and replication.
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PMID:Protein interactions entered into by the measles virus P, V, and C proteins. 857 62

The recent demonstration that bone sialoprotein (BSP) can be detected in human breast cancer tissue by immunoperoxidase suggests that this phosphoprotein is ectopically expressed by malignant mammary epithelial cells. Its detection in human breast cancer cells raises questions about its potential role(s) during breast cancer progression. Because BSP is secreted and is present in the serum, the positivity of breast cancer cells for BSP could have been the result of an uptake of the circulating phosphoprotein by the cells rather than of an intrinsic expression. We examined the expression of BSP at both the protein and mRNA levels in nine human breast cancer samples as well as in three human breast cancer cell lines (MCF-7, T47-D, and MDA-MB-231) using immunohistochemistry, flow cytometric analysis, immunoblot, and reverse-transcriptase PCR. BSP was detected at both protein and mRNA levels in human breast cancer tissue and in the three human breast cancer cell lines. Using a specific polyclonal anti-BSP antibody, we showed by both fluorescence-activated cell sorter analysis and immunohistochemistry experiments that all of the human breast cancer cell lines studied express BSP. This was localized at the cell surface and in the cytosol of the estrogen receptor-positive MCF-7 and T47-D cell lines, whereas it was detected only in the cytosol of the estrogen receptor-negative MDA-MB-231 cells. Using the same polyclonal anti-BSP antibody, we were able to identify an approximately 97-kd band on total protein extracts from the three cell lines by immunoblotting. Reverse-transcriptase PCR reactions using specific oligonucleotides performed on total RNA of nine human breast cancer biopsy samples and the three cell lines demonstrated the presence of BSP mRNA in all of the samples examined. This study is the first demonstration that human malignant breast epithelial cell lines express BSP at the protein and mRNA levels. Our study identified MCF-7, T47-D, and MDA-MB-231 cells as useful models for the examination of the molecular mechanisms involved in the ectopic expression of BSP in breast malignant lesions.
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PMID:Detection of bone sialoprotein in human breast cancer tissue and cell lines at both protein and messenger ribonucleic acid levels. 876 20


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