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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Ribonucleoprotein (RNP) cores extracted from virions of wild-type (Edmonston strain) measles virus (MV) or obtained from MV-infected cells (cRNP) were shown to be capable of transcribing RNA in vitro but at relatively low efficiency. The tightly bound matrix (M) protein could be effectively removed from virion RNP (vRNP) and from cRNP by exposure to buffers of high ionic strength (0.5 to 1.0 M KCl) but only at pH 8.0 or higher. The vRNP and cRNP cores complexed with M protein exhibited markedly reduced transcriptional activity at increasing concentrations, whereas vRNP and cRNP cores free of M protein exhibited linear and substantially higher transcriptional activity; these data suggest that M protein is the endogenous inhibitor of MV RNP transcription. M-gene cDNA clones derived from three strains of wild-type (wt) MV and 10 clones from mRNAs isolated from the brain tissue of patients who had died from subacute sclerosing panencephalitis (SSPE) and from measles inclusion body encephalitis (MIBE) were recloned in the pTM-1 expression vector driven by the bacteriophage T7 RNA polymerase expressed by a coinfecting vaccinia virus recombinant. All 10 mutant SSPE and MIBE clones expressed in vitro and in vivo M proteins that reacted with monospecific anti-M polyclonal antibody and migrated on polyacrylamide gels to positions identical to or only slightly different from those of the M proteins expressed by wt MV clones. When reconstituted with cRNP cores, the three expressed wt M proteins and 6 of the 10 mutant-expressed M proteins showed equivalent capacity to down-regulate MV transcription. Three of the M proteins from SSPE clones and one from the MIBE clone showed little or no capacity to down-regulate transcription when reconstituted with cRNP cores. The only plausible explanations for loss of transcription inhibition activity by the four SSPE/MIBE M proteins were exceedingly high degrees of hypermutations leading to U-->C transitions and cloning-corrected mutations in the initiator codon (ATG-->ACG) of the four M genes. However, only the hypermutated M protein expressed by the MIBE cDNA clone exhibited virtually no capacity to bind cRNP cores in a reconstitution assay. These experiments provide some preliminary data to support the hypothesis that MV encephalitis may result from certain selective mutations in the M gene.
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PMID:Transcription inhibition and other properties of matrix proteins expressed by M genes cloned from measles viruses and diseased human brain tissue. 810 16

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

A system has been established allowing the rescue of replicating measles viruses (MVs) from cloned DNA. On one hand, plasmids were constructed from which MV antigenomic RNAs with the correct termini are transcribed by phage T7 RNA polymerase. On the other hand, helper cells derived from the human embryonic kidney 293 cell line were generated constitutively expressing T7 RNA polymerase together with MV nucleocapsid protein and phosphoprotein. Simultaneous transfection of the helper cells with the MV antigenomic plasmid and with a plasmid encoding the MV polymerase under direction of a T7 promoter led to formation of syncytia from which MVs were easily recovered. A genetic tag comprising three nucleotide changes was present in the progeny virus. As a first application of reverse genetics, a segment of 504 nucleotides from the 5' non-coding region of the fusion gene was deleted, leading to an MV variant whose replication behaviour in Vero cells was indistinguishable from that of the laboratory Edmonston B strain. Since no helper virus is involved, this system, in principle, should be applicable to the rescue of any member of the large virus order Mononegavirales, i.e. viruses with a nonsegmented negative-strand RNA genome.
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PMID:Rescue of measles viruses from cloned DNA. 884 71

Rinderpest virus is a morbillivirus and is the causative agent of a widespread and important disease of cattle. The viral genome is a single strand of RNA in the negative sense. We have constructed plasmids containing cDNA copies of the 5' and 3' termini of the virus separated by a reporter gene and have shown that antigenome-sense RNA transcripts of these model genomes can be replicated, transcribed, and packaged by helper virus, both rinderpest virus and the related measles virus. Further, these genome analogs can be replicated and transcribed by viral proteins expressed from cDNA clones by using a recombinant vaccinia virus expressing T7 RNA polymerase (MVA-T7). Using this latter system, we have rescued live rinderpest virus from a full-length cDNA copy of the genome of the RBOK vaccine strain. The recombinant virus appears to grow in tissue culture identically to the original virus.
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PMID:Rescue of rinderpest virus from cloned cDNA. 899 50

A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids. The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs. Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation. The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus. Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L. Furthermore, it is useful for investigating later steps in the MV life cycle.
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PMID:Rescue of measles virus using a replication-deficient vaccinia-T7 vector. 902 30

Measles virus (MV) isolated in B95a cells, a marmoset B-cell line, retains full pathogenicity for cynomolgus monkeys, while its derivative obtained by adaptation to the growth in Vero cells, a monkey kidney cell line, loses the pathogenic potential (F. Kobune, H. Sakata, and A. Sugiura, J. Virol. 64:700-705, 1990). Here, we show with a pair of strains, a fresh isolate (9301B) in B95a cells and its Vero cell-adapted form (9301V), that the in vivo attenuation parallels the decrease of replication and syncytium-inducing capabilities in the original B95a cells and that these in vitro phenotypes are attributable to impediment of transcription, which is already obvious at the level of primary transcription catalyzed by the virion-associated RNA polymerase. On the other hand, cell fusion assays detected no functional difference between the glycoproteins of the two viruses. Essentially the same transcriptional impediment with reduced syncytium induction following Vero cell adaptation was found with two other pairs of strains that had been similarly prepared. Nucleotide sequence comparison between the 9301B and 9301V viruses revealed that a few (at most five) amino acid changes, which sporadically took place in the polymerase (L and P proteins) and/or accessory V and C proteins, were responsible for the in vitro and in vivo attenuation through adaptation to growth in Vero cells.
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PMID:Measles virus attenuation associated with transcriptional impediment and a few amino acid changes in the polymerase and accessory proteins. 976 10

Measles virus encodes three proteins required for the encapsidation, transcription and replication of viral genomes. The genes for these proteins have been inserted into the vaccinia virus genome together with the gene for the bacteriophage T7 RNA polymerase. Cells infected with this recombinant virus were able to encapsidate, transcribe and replicate a CAT gene positioned in the negative polarity behind a T7 promoter and flanked by measles virus genomic termini. Inhibition of the accumulation of the nucleocapsid proteins by actinomycin D led to an increase in CAT expression. Thus the measles virus polymerase activity, encoded by the vaccinia genome, was regulated by the level of measles proteins just as the authentic polymerase. The recombinant vaccinia described in this study could be useful for the production of measles virus-like particles encoding foreign genes and employed in vaccination or gene therapy strategies.
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PMID:A functional measles virus replication and transcription machinery encoded by the vaccinia virus genome. 1032 36

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

Molecular determinants of neuropathogenesis have been shown to be present in the hemagglutinin (H) protein of measles virus (MV). An H gene insertion vector has been generated from the Edmonston B vaccine full-length infectious clone of MV. Using this vector, it is possible to insert complete H open reading frames into the parental (Edtag) background. The H gene from a rodent brain-adapted MV strain (CAM/RB) was inserted into this vector, and a recombinant virus (EdtagCAMH) was rescued by using a modified vaccinia virus which expresses T7 RNA polymerase (MVA-T7). The recombinant virus grew at an equivalent rate and to similar titers as the CAM/RB and Edtag parental viruses. Neurovirulence was assayed in a mouse model for MV encephalitis. Viruses were injected intracerebrally into the right cortex of C57/BL/6 suckling mice. After infection mice inoculated with the CAM/RB strain developed hind limb paralysis and ataxia. Clinical symptoms were never observed with an equivalent dose of Edtag virus or in sham infections. Immunohistochemistry (IHC) was used to detect viral antigen in formalin-fixed brain sections. Measles antigen was observed in neurons and neuronal processes of the hippocampus, frontal, temporal, and olfactory cortices and neostriatum on both sides of symmetrical structures. Viral antigen was not detected in mice infected with Edtag virus. Mice infected with the recombinant virus, EdtagCAMH, became clinically ill, and virus was detected by IHC in regions of the brain similar to those in which it was detected in animals infected with CAM/RB. The EdtagCAMH infection had, however, progressed much less than the CAM/RB virus at 4 days postinfection. It therefore appears that additional determinants are encoded in other regions of the MV genome which are required for full neurovirulence equivalent to CAM/RB. Nevertheless, replacement of the H gene alone is sufficient to cause neuropathology.
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PMID:The H gene of rodent brain-adapted measles virus confers neurovirulence to the Edmonston vaccine strain. 1040 Jul 89

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein. When aligned with amino acid sequences of L proteins from other negative-sense RNA viruses, the Sendai L protein contains six regions of good conservation, designated domains I-VI, which have been postulated to be important for the various enzymatic activities of the polymerase. To directly address the roles of domains IV and VI, 14 site-directed mutations were constructed either by changing clustered charged amino acids to ala or by substituting selected Sendai L amino acids with the corresponding sequence from measles virus L. Each mutant L protein was tested for its ability to transcribe and replicate the Sendai genome. The series of mutations created a spectrum of phenotypes, from those with significant, near wild-type, activity to those being completely defective for all RNA synthesis. The inactive L proteins, however, were still able to bind P protein and form a polymerase capable of binding the nucleocapsid template. The remainder of the mutations reduced, but did not abolish, enzymatic activity and included one mutant with a specific defect in the synthesis of the leader RNA compared with mRNA, and three mutants that replicated genome RNA much more efficiently in vivo than in vitro. Together, these data suggest that even within a domain, the function of the Sendai L protein is likely to be very complex. In addition, SS3 and SS10 L in domain IV and SS13 L in domain VI were shown to be temperature-sensitive. Both SS3 and SS10 gave significant, although not wild-type, activity at 32 degrees C; however, each was completely inactivated for all RNA synthesis at 37 and 39.6 degrees C. SS13 was completely inactive only when synthesized at the higher temperature. Each polymerase synthesized at 32 degrees C could only be partially heat inactivated in vitro at 39.6 degrees C, suggesting that inactivation involves both thermal lability of the protein and temperature sensitivity for its synthesis.
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PMID:Mutations in conserved domains IV and VI of the large (L) subunit of the sendai virus RNA polymerase give a spectrum of defective RNA synthesis phenotypes. 1075 21


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