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)

Rabies virion-associated transcriptase activity was investigated in vitro and compared with that of the New Jersey serotype of vesicular stomatitis virus. The concentration of detergent that affected [3H]GMP incoporation into acid-insoluble material was significantly different for both viruses. Vesicular stomatitis virus New Jersey required 0.05 to 0.1% nonionic detergent, whereas rabies virion could not be fully activated unless 4 to 5% detergent was used. Other optimal conditions were as follows: 40 mM NaCl, 5 mM Mg2+, 40 mM Tris-hydrochloride (pH 7.4), 5 mM dithiothreitol, and 30 degrees C. The reaction required four nucleoside triphosphates. The initial rate of RNA synthesis by rabies virion enzyme was 140 pmol of GMP incorporated/mg of viral protein per h and linearly increased until about 8 h, with a slight initial lag phase. The enzyme activity that correlated with the content of L protein was highest when rabies virions were grown at 33 degrees C. The product was single-stranded RNA, which was complementary in base sequences to rabies viral RNA. Most of the RNA synthesized sedimented at 6-16S.
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PMID:Transcriptase activity associated with rabies virion. 2 66

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

Phosphorylation of rabies virus proteins was followed in vivo and in vitro. The N and M1 proteins were both found to be phosphorylated. The M1 protein was present in the virion in two phosphorylated states, but only the hypophosphorylated form of M1 was found in infected cells. The hypothesis that some of the M1 molecules become hyperphosphorylated during the maturation process by a membrane-bound kinase was examined. The phosphorylation of the viral proteins by the kinase present in purified rabies virions was studied using an in vitro transcriptase assay: under the conditions of the assay, additional phosphate groups were rapidly attached to the N protein. The M1 protein was similarly hyperphosphorylated although more slowly. Whether the hyperphosphorylation of the N protein is responsible for the poor efficiency of the in vitro transcriptase reaction is not clear. No detectable change in the phosphorylation of cellular proteins was observed in the course of rabies virus infection.
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PMID:Phosphorylation of the N and M1 proteins of rabies virus. 299 64

We have now completed the rabies genome structure by the cloning and the sequencing of the entire L gene and the 5' untranscribed region. The L gene encodes a single open reading frame 2142 amino acids in length (244,206 Da) that corresponds to the viral RNA-dependent RNA polymerase. In contrast with other isofunctional proteins, the rabies polymerase exhibits a high degree of homology with the vesicular stomatitis virus polymerase, and a lesser degree, although significant, with those of Sendai virus and Newcastle disease virus, which suggests a differential evolution of the different cistrons. We have observed several strongly conserved stretches which may designate the independent functional domains of this multifunctional protein. In addition to the conservation of related transcription signals (N. Tordo et al. (1986) Proc. Natl. Acad. Sci. USA 83, 3914-3918.), this highlights the striking selective pressure on elements involved in transcription and replication mechanisms, and provides further evidence for a common ancestry of Rhabdoviridae and Paramyxoviridae families. The terminal complementarity observed in the rabies genome suggests the conservation of important genomic signals.
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PMID:Completion of the rabies virus genome sequence determination: highly conserved domains among the L (polymerase) proteins of unsegmented negative-strand RNA viruses. 340 52

We analyzed cell extracts from BHK(21) cells infected with vesicular stomatitis virus (VSV) and rabies virus for in vitro RNA polymerase activity. Cells infected with VSV B virions exhibited several complexes with in vitro RNA polymerase activity in sucrose gradients. These complexes synthesize VSV transcriptase product (4 to 18S) polyadenylated in RNA complementary to virion RNA. Cells infected with a high multiplicity of B virions and T particles show only one RNA polymerase complex active in vitro. This complex sediments at 110S and makes only small (2S) RNA. Carrier BHK(21) cells persistently noncytopathically infected with VSV contain several complexes active in RNA polymerase, but both exhibit very low activity. Cytoplasms of cells noncytopathically infected with rabies virus also show very low levels of a complex containing RNA polymerase activity. No transcriptase nor any other in vitro polymerase activity could be found associated with purified rabies virions, although they do carry out primary transcription in cells treated with actinomycin D and cycloheximide before infection.
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PMID:Transcribing complexes in cells infected by vesicular stomatitis virus and rabies virus. 436 91

Infectious B virions of vesicular stomatitis virus were 100% lethal to BHK(21) (baby hamster kidney) cells when infecting alone, and persistent noncytocidal infection could not be achieved with cloned B virions alone. However, a mixture of B virions and homologous, short, defective, interfering particles (T particles) of a temperature-sensitive mutant of the virus regularly established persistently infected, noncytocidal carrier cultures. A long T particle was generated during establishment of the carrier culture; we show that this long T particle can establish and maintain persistent noncytocidal infection even when it infects cells along with virulent wild-type B virions. This long T particle causes the production of wild-type B virions with greatly reduced virion transcriptase (EC 2.7.7.6; RNA nucleotidyltransferase) levels when coinfecting the same cells, so it appears to prevent cytopathology by regulating virus transcription. The implications of these findings for rabies and other slowly progressing noncytocidal infections are discussed.
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PMID:Persistent noncytocidal vesicular stomatitis virus infections mediated by defective T particles that suppress virion transcriptase. 437 Feb 55

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

Rabies virus M protein was expressed in Escherichia coli in the form of a fusion protein with maltose binding protein (MBP) and purified by amylose affinity column chromatography after extraction. In order to investigate the possible regulatory role of M protein in viral transcription, an assay system for rabies virion-associated transcriptase activity was established by using the ribonucleoprotein (RNP) cores prepared from purified virions. Analysis of the products of the transcription assay system showed that the products are sensitive to RNase and are positive-strand RNA. Addition of the fusion protein to the system after cleavage with a proteinase Factor Xa (FXa), which cleaves the fusion protein into the M protein and MBP, resulted in an efficient and dose-dependent inhibition of the transcription. Furthermore, addition to the system of anti-M protein monoclonal antibody significantly restored the transcription. Control experiments with the same transcription assaying system using rabies virus nucleoprotein expressed as a fusion protein with MBP and cleaved with FXa did not result in an inhibition of the transcription. These results suggest that the M protein of rabies virus has the property to down-regulate virion-associated transcription.
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PMID:Rabies virus M protein expressed in Escherichia coli and its regulatory role in virion-associated transcriptase activity. 864 3

Rhabdoviruses such as rabies virus (RV) encode only five multifunctional proteins accomplishing viral gene expression and virus formation. The viral phosphoprotein, P, is a structural component of the viral ribonucleoprotein (RNP) complex and an essential cofactor for the viral RNA-dependent RNA polymerase. We show here that RV P fused to enhanced green fluorescent protein (eGFP) can substitute for P throughout the viral life cycle, allowing fluorescence labeling and tracking of RV RNPs under live cell conditions. To first assess the functions of P fusion constructs, a recombinant RV lacking the P gene, SAD DeltaP, was complemented in cell lines constitutively expressing eGFP-P or P-eGFP fusion proteins. P-eGFP supported the rapid accumulation of viral mRNAs but led to low infectious-virus titers, suggesting impairment of virus formation. In contrast, complementation with eGFP-P resulted in slower accumulation of mRNAs but similar infectious titers, suggesting interference with polymerase activity rather than with virus formation. Fluorescence microscopy allowed the detection of eGFP-P-labeled extracellular virus particles and tracking of cell binding and temperature-dependent internalization into intracellular vesicles. Recombinant RVs expressing eGFP-P or an eGFP-P mutant lacking the binding site for dynein light chain 1 (DLC1) instead of P were used to track interaction with cellular proteins. In cells expressing a DsRed-labeled DLC1, colocalization of DLC1 with eGFP-P but not with the mutant P was observed. Fluorescent labeling of RV RNPs will allow further dissection of virus entry, replication, and egress under live-cell conditions as well as cell interactions.
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PMID:Tracking fluorescence-labeled rabies virus: enhanced green fluorescent protein-tagged phosphoprotein P supports virus gene expression and formation of infectious particles. 1550 20

Foreign viral proteins expressed by rabies virus (RV) have been shown to induce potent humoral and cellular immune responses in immunized animals. In addition, highly attenuated and, therefore, very safe RV-based vectors have been constructed. Here, an RV-based vaccine vehicle was utilized as a novel vaccine against severe acute respiratory syndrome coronavirus (SARS-CoV). For this approach, the SARS-CoV nucleocapsid protein (N) or envelope spike protein (S) genes were cloned between the RV glycoprotein G and polymerase L genes. Recombinant vectors expressing SARS-CoV N or S protein were recovered and their immunogenicity was studied in mice. A single inoculation with the RV-based vaccine expressing SARS-CoV S protein induced a strong SARS-CoV-neutralizing antibody response. The ability of the RV-SARS-CoV S vector to confer immunity after a single inoculation makes this live vaccine a promising candidate for eradication of SARS-CoV in animal reservoirs, thereby reducing the risk of transmitting the infection to humans.
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PMID:A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies. 1583 55


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