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)

An RNA-dependent RNA polymerase activity has been found copurifying with measles virus infectivity and complement-fixing antigen in three Vero cell-grown variants of measles virus: the attenuated Edmonston B strain, the natural non-attenuated Edmonston strain, and a subacute sclerosing panencephalitis isolate, IP-3. Incubation of purified measles virions with immunoglobulin G derived from sera of monkeys hyperimmunized against measles specifically removes activity sedimenting in the density region of measles virions. The requirements of the reaction, which is RNase sensitive, are similar to those reported for other paramyxovirus-associated activities, including detergent, divalent cation, ribonucleoside triphosphates, and a reducing agent. The size classes of RNA synthesized correspond to those found in measles-infected cells, including 50, 35, and 16 to 20S. The product RNA of the Edmonston B virus-stimulated reaction was rendered RNase resistant by annealing with RNA extracted from purified Edmonston B virions. RNA from uninfected Vero cells was ineffective in the annealing reaction.
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PMID:Characterization of an RNA-dependent RNA polymerase activity associated with measles virus. 64 73

The nucleotide sequence of the L gene of Marburg virus, strain Musoke, has been determined. The L gene has a single long open reading frame encoding a polypeptide of 2330 amino acids (MW 267,175) that represents the viral RNA-dependent RNA polymerase. The putative transcription start signal (3'CUACCUAUAAUU 5') and the termination signal (3' UAAUUCUUUUU 5') of the gene could be identified. Computer-assisted comparison of the L protein with L proteins of other nonsegmented negative-stranded RNA viruses (Paramyxoviridae: Sendai virus, Newcastle disease virus, human parainfluenza 3 virus, measles virus, human respiratory syncytial virus; Rhabdoviridae: vesicular stomatitis virus, rabies virus) revealed significant homologies primarily in the N-terminal half of the proteins. We have identified three common conserved boxes (A, B, and C) among filo-, paramyxo-, and rhabdovirus L proteins, which are probably involved in the polymerase function. The L proteins can be divided into an N-terminal half, which seems to accommodate the common enzymatic sites, and a C-terminal half carrying virus specific peculiarities. The data presented here suggest a common evolutionary history for all nonsegmented negative-stranded RNA viruses and show that filoviruses are more closely related to paramyxo- than to rhabdoviruses.
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PMID:The nucleotide sequence of the L gene of Marburg virus, a filovirus: homologies with paramyxoviruses and rhabdoviruses. 154 52

Synthesis of virus-specific RNAs in human HEP-2 and L-41 cells chronically infected with measles virus was studied in comparison with synthesis of viral RNA in acutely infected L-41 cells. The RNA, a component of RNP isolated from chronically infected cells, was shown to be represented mainly by "minus" chains and to contain 23-25% "plus"-RNA. It was demonstrated by blotting hybridization that 1 species of genomic RNA with a molecular weight of 5 megadaltons was synthesized in acute infection whereas in chronically infected cells a small amount of subgenomic RNAs was additionally detected in RNP. The level of virus genome transcription in chronically infected cells was 7-8 fold lower than that in acute infection. The RNA-transcriptase activity of RNP isolated from chronically infected HEP-2 and L-41 cells was also lower than RNP activity from acutely infected L-41 cells. The observed features of virus-specific RNA synthesis in chronically infected cells seem to be likely to play a role in the maintenance of virus persistence.
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PMID:[RNA analysis of the measles virus in a human cell culture of the chronic infection]. 242 50

The complete sequence of the gene encoding the matrix protein (M) of human parainfluenza virus type 3 (PIV-3) was determined from cDNA clones and from primer extension dideoxy sequencing of the viral genome. The M mRNA is 1150 nucleotides in length, exclusive of polyadenylate, and codes for a protein of 353 amino acids, having a calculated molecular weight of 39,480. The M protein of PIV-3 was found to have a high degree of sequence homology with that of a closely related paramyxovirus, Sendai virus, and to a lesser extent it contained sequence homology with two more distant paramyxoviruses, measles virus and canine distemper virus. We also determined the sequences of the intergenic junctions for the first four genes of PIV-3: NP, P, M, and F. Comparison of these sequences yielded a consensus mRNA start sequence of 5'-AGGANNAAAGA-3', an mRNA end sequence of 5'-UAAGAAAAA-3', and an intergenic sequence of 5'-CUU-3'. The end sequence of the M gene is unusual in that it contains an eight base insertion prior to the A5 tract found in the consensus sequence. This disruption appears to cause a high frequency of readthrough by the viral transcriptase at this junction.
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PMID:Complete nucleotide sequence of the matrix protein mRNA and three intergenic junctions of human parainfluenza virus type 3. 302 66

Persistent measles virus infection of human HEp-2 or L-41 cells was accompanied by pronounced structural and functional changes of isolated intracellular viral nucleocapsids (NCs). The bulk of persistent NCs possessed altered conformation and a "string-of-beads" appearance, contained substantial amounts of subgenomic size RNAs, exhibited reduced transcriptase activity in vitro and lacked infectivity on transfection of susceptible cells. Immunogold staining revealed negligible binding of anti-P protein monoclonal antibodies to the "string-of-beads" type NCs, thus suggesting their non-functional state.
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PMID:Non-infectious morphologically altered nucleocapsids of measles virus from persistently infected cells. 359 84

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

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

Transcription by nonsegmented negative-strand RNA viruses is mediated by the viral RNA-dependent RNA polymerase and transcriptional cofactor P. The P protein is activated by phosphorylation, an event initiated by cellular kinases. The kinase used differs among this group of RNA viruses; vesicular stomatitis virus and respiratory syncytial virus utilize casein kinase II (CKII), whereas human parainfluenza virus type 3 utilizes PKC isoform zeta (PKC-zeta) for activation of its P protein. To identify the cellular kinase(s) involved in the phosphorylation of the canine distemper virus (CDV) P protein, we used recombinant CDV P in phosphorylation assays with native kinase activities present in CV1 cell extracts or purified CKII and PKC isoforms. Here, we demonstrate that the CDV P protein is phosphorylated by two cellular kinases, where PKC-zeta has the major and CKII the minor activities. In contrast, the P protein of another member of the morbillivirus genus, measles virus, is phosphorylated predominantly by CKII, whereas PKC-zeta has only minor activity. Selective inhibition of PKC-zeta activity within CV1 cells eliminated permissiveness to CDV replication, indicating an in vivo role for PKC-zeta in the virus replication cycle. The broad tissue expression of PKC-zeta parallels the pantropic nature of CDV infections, suggesting that PKC-zeta activity is a determinant of cellular permissiveness to CDV replication.
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PMID:Phosphorylation of canine distemper virus P protein by protein kinase C-zeta and casein kinase II. 918 3

A reverse transcription-PCR and hybridization-enzyme immunoassay (RT-PCR-EIA) has been developed to identify the major agents of bronchiolitis in infants: respiratory syncytial viruses A and B (RSVA and RSVB) and parainfluenzavirus 3 (PIV3). Two primer sets (P1-P2 and P1-P3) were selected in a conserved region of the polymerase L gene. In infected cell cultures, this method detected RSVA (n = 14), RSVB (n = 13), and PIV3 (n = 13), with the exclusion of PIV1 (n = 4), PIV2 (n = 3), measles virus (n = 6), mumps virus (n = 4), influenza A virus (n = 11), and influenza B virus (n = 4). The differentiation of the amplicons by restriction fragment length polymorphism (RFLP) showed a PvuII site for PIV3 strains and an AvaII site for RSV strains, with RSVA distinguished from RSVB by BglII. The hybridization-EIA, using three internal probes specific for each virus, correlated with the immunofluorescence assay (IFA) and RFLP results. Clinical aspirates from 261 infants hospitalized with bronchiolitis were tested by IFA, viral isolation technique (VIT), and RT-PCR-EIA. RT-PCR-EIA detected RSV sequences in 103 samples (39.4%), and IFA-VIT detected RSV sequences in 109 cases (41.7%). A few samples (2.6%) were IFA-VIT positive but PCR negative, and one sample was RT-PCR-EIA positive only. RT-PCR-EIA detected PIV3 sequences in 14 of the 15 IFA-VIT-positive isolates. The two methods showed very good correlation (96.9%), but RT-PCR-EIA was clearly more efficient in typing, leaving 5% non-A, non-B isolates, while IFA failed to resolve 23% of the isolates. The two methods contradicted each other for <5% of the isolates.
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PMID:Detection of respiratory syncytial virus A and B and parainfluenzavirus 3 sequences in respiratory tracts of infants by a single PCR with primers targeted to the L-polymerase gene and differential hybridization. 950 15

Reverse transcriptase (RT) activity has been detected recently in all chicken cell-derived measles and mumps vaccines. A study of a vaccine manufactured in Europe indicated that the RT is associated with particles containing endogenous avian retrovirus (EAV-0) RNA and originates from the chicken embryonic fibroblasts (CEF) used as a substrate for propagation of the vaccine. We investigated the origin of RT in measles and mumps vaccines from a U.S. manufacturer and confirm the presence of RT and EAV RNA. Additionally, we provide new evidence for the presence of avian leukosis virus (ALV) in both CEF supernatants and vaccines. ALV pol sequences were first identified in particle-associated RNA by amplification with degenerate retroviral pol primers. ALV RNA sequences from both the gag and env regions were also detected. Analysis of hypervariable region 2 of env revealed a subgroup E sequence, an endogenous-type ALV. Both CEF- and vaccine-derived RT activity could be blocked by antibodies to ALV RT. Release of ALV-like virus particles from uninoculated CEF was also documented by electron microscopy. Nonetheless, infectivity studies on susceptible 15B1 chicken cells gave no evidence of infectious ALV, which is consistent with the phenotypes of the ev loci identified in the CEF. PCR analysis of ALV and EAV proviral sequences in peripheral blood mononuclear cells from 33 children after measles and mumps vaccination yielded negative results. Our data indicate that the sources of RT activity in all RT-positive measles and mumps vaccines may not be similar and depend on the particular endogenous retroviral loci present in the chicken cell substrate used. The present data do not support transmission of either ALV or EAV to recipients of the U.S.-made vaccine and provide reassurance for current immunization policies.
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PMID:Evidence of avian leukosis virus subgroup E and endogenous avian virus in measles and mumps vaccines derived from chicken cells: investigation of transmission to vaccine recipients. 1036 36


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