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)

An alternative approach to structure-function analysis of vesicular stomatitis virus (VSV) gene products and their interactions with one another during each phase of the viral life cycle is described. We showed previously by using the vaccinia virus-T7 RNA polymerase expression system that when cells expressing the nucleocapsid protein (N), the phosphoprotein (NS), and the large polymerase protein (L) of VSV were superinfected with defective interfering (DI) particles, rapid and efficient replication and amplification of (DI) particle RNA occurred. Here, we demonstrate that all five VSV proteins can be expressed simultaneously when cells are contransfected with plasmids containing the matrix protein (M) gene and the glycoprotein (G) gene of VSV in addition to plasmids containing the genes for the N, NS, and L proteins. When cells coexpressing all five VSV proteins were superinfected with DI particles, which because of their defectiveness are unable to express any viral proteins or to replicate, DI particle replication, assembly, and budding were observed and infectious DI particles were released into the culture fluids. Omission of either the M or G protein expression resulted in no DI particle budding. The vector-supported DI particles were similar in size and morphology to the authentic DI particles generated from cells coinfected with DI particles and helper VSV and their infectivity could be blocked by anti-VSV or anti-G antiserum. The successful replication, assembly, and budding of DI particles from cells expressing all five VSV proteins from cloned cDNAs provide a powerful approach for detailed structure-function analysis of the VSV gene products in each step of the replicative cycle of the virus.
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PMID:Cells that express all five proteins of vesicular stomatitis virus from cloned cDNAs support replication, assembly, and budding of defective interfering particles. 184 19

Interferons alpha and beta induce an efficient antiviral state against influenza virus in mouse cells that possess the Mx gene, but not in mouse cells that lack this gene. In Mx-containing cells treated with interferon the amount of viral mRNA synthesized as a result of primary transcription is drastically reduced. Only two viral mRNAs could be detected by Northern analysis and by translating the poly(A)+ RNA from infected cells in wheat germ extracts: a reduced amount of the mRNA for nonstructural protein 1 and an even lower amount of the mRNA for the matrix protein. The other viral mRNAs were not made in detectable amounts. In addition, the rate of viral mRNA synthesis catalyzed by the inoculum transcriptase, measured by in vitro RNA synthesis catalyzed by permeabilized cells, was severely inhibited. In contrast, interferon treatment of cells lacking the Mx gene had little or no effect on either the steady-state level or the rate of synthesis of viral mRNAs made by the inoculum transcriptase. These results indicate that the interferon-induced Mx gene product, a 75,000-molecular-weight protein that accumulates in the nucleus, inhibits influenza viral mRNA synthesis which occurs in the nucleus. No Mx-specific effect acting directly on viral protein synthesis in the cytoplasm was observed.
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PMID:Inhibition of influenza viral mRNA synthesis in cells expressing the interferon-induced Mx gene product. 241 49

The matrix protein (M1) of influenza A virus, which has a critical role in viral assembly and can inhibit the viral transcriptase complex, has the ability to bind RNA. The RNA-binding property of M1 is specific for single-stranded RNA, like that of influenza nucleoprotein (NP) and shows similar sensitivity to pH and to salt concentration. M1:RNA complexes are stable, once formed, to competition from excess single-stranded RNA. The possible location of the RNA-binding regions in the M1 protein is discussed.
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PMID:RNA-binding properties of influenza A virus matrix protein M1. 247 6

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

Sendai virions, disrupted in 2% Triton X-100 in 1 M KCl, were separated into nucleocapsids and envelope proteins by centrifugation. The nucleocapsids, representing 46% of the virion proteins, had a buoyant density of 1.29 gm/cm(3) in D(2)O sucrose. RNA-dependent transcriptase activity associated with them had a ninefold greater specific activity than transcriptase assayed in unfractionated detergent-disrupted virions. These enzyme-active nucleocapsids contained only two polypeptides, the largest virion polypeptide (molecular weight 75,000) and the nucleocapsid structure unit (molecular weight 60,000). Virion envelope proteins, either glycoproteins or nonglycosylated matrix protein, inhibited nucleocapsid-associated polymerase activity; brief heat denaturation abolished their inhibitory activity. Yeast RNA stimulated nucleocapsid-associated enzyme, suggesting that stimulatory polyanions act at the enzyme-template level.
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PMID:Sendai virion transcriptase complex: polyeptide composition and inhibition by virion envelope proteins. 435 18

One of the major structural proteins of vesicular stomatitis virus is a small, nonglycosylated, matrix protein which associates with the nucleocapsid core during final stages of morphogenesis and budding. Biochemical and genetic studies suggested that the matrix protein regulates RNA synthesis both in vitro and in vivo. We have purified biologically active matrix protein from the virus and have directly shown that it significantly inhibits RNA synthesis in vitro mediated by the virion-associated RNA polymerase at low ionic strength (0.02 M). The inhibition was greater than 80% when the ratio of matrix protein to the major nucleocapsid protein in the transcribing complex was 2:1 (wt/wt). The inhibition was found to be at the level of RNA chain elongation and not at the initiation step. Electron microscopic studies revealed that inhibition of transcription by matrix protein was accompanied by a profound structural change of the transcribing nucleocapsid from an extended structure to a highly compact form. At higher ionic strength (0.12 M), the matrix protein failed to interact with the nucleocapsid. The matrix protein appears to be involved in condensing the nucleocapsid and blocking transcription during maturation of the virus particle.
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PMID:Purified matrix protein of vesicular stomatitis virus blocks viral transcription in vitro. 629 18

Influenza viruses are spherical, about 1000 A in diameter, and consist of an as yet undefined central structure containing the eight negative-sense RNA molecules of the genome (1) in association with the transcriptase required for mRNA synthesis, an abundant nucleoprotein, and an equally abundant matrix protein. This core is surrounded by a membrane derived from the cell surface in a budding process by which newly formed viruses are released from the infected cell. During infection cell membranes are modified by the incorporation of newly synthesized virus membrane proteins, and the finally released viruses contain exclusively two different types of virus-specified glycoprotein, hemagglutinin and neuraminidase, and a proton channel protein, M2. All three of these molecules have been studied extensively, particularly the glycoproteins, and in this paper information on their structures and functions will be summarized and related to modifications in cellular membranes that occur during virus infection.
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PMID:Influenza viruses and cell membranes. 755 5

The orientation of the TATA box is thought to direct downstream transcription of eukaryotic genes by RNA polymerase II. However, the putative TATA box in the promoter of the bone sialoprotein (BSP) gene, which codes for a tissue-specific and developmentally regulated bone matrix protein, is inverted (5'-TTTATA-3') relative to the consensus TATA box sequence (5'-TATAAA-3') and is overlapped by a vitamin D3-response element. Here we show that the inverted TATA sequence in the rat BSP gene binds to recombinant TATA-box-binding protein (TBP) with an affinity similar to that observed with the consensus TATA box, and site-directed point mutations in the inverted TATA sequence (mutating TTTATA into TCTCTA) abrogate both TBP binding and BSP promoter activity. However, when the inverted TATA sequence is changed to a canonical TATAAA, the TBP- and vitamin D3 receptor-binding properties together with the BSP promoter activity are retained. In addition, we found that the TBP is required to reconstitute in vitro transcription driven by the BSP promoter. These studies, which have revealed a naturally occurring inverted TATA box that can bind TBP and direct downstream transcription, demonstrate that the orientation of the TATA box does not determine the direction of transcription in higher eukaryotic genes. Consequently, the inverted TATA box that is conserved in the human, rat and mouse BSP gene promoters will provide an excellent in vivo model to investigate the polarity of the transcription factor IID-DNA complex and its relation to downstream transcription.
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PMID:An inverted TATA box directs downstream transcription of the bone sialoprotein gene. 764 64

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

A Sendai virus expression vector in the form of a transcribing copy-back defective interfering RNA was constructed and shown to efficiently express a tagged matrix protein in the only context of a Sendai virus infection. In an attempt to identify relevant M protein domains involved in viral assembly and budding, a series of deletion mutants were tested for their ability to bind to cellular membrane fractions. The deletion of a region spanning amino acids 105-137 significantly decreased this binding when the protein was expressed in a system driven by the T7 RNA polymerase away from any other viral proteins. Plus or minus charges were introduced in the hydrophobic portion of a predicted amphiphilic helix in this region, and M proteins with altered membrane binding properties were produced. The genes encoding these mutant M proteins were then inserted in the Sendai virus vector and shown to be expressed at levels similar to that of the endogenous wild-type M protein. The presence of a negative charge in the hydrophobic region of the putative amphiphilic helix prevented the incorporation of the mutant protein into virus particles and appeared to decrease the efficiency of virus particle budding. In contrast, the introduction of a positive charge appeared to increase the M mutant uptake into virions. The use a Sendai virus vector has therefore been shown instrumental in the identification of mutant M proteins interfering with the viral assembly-budding process.
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PMID:A Sendai virus vector leading to the efficient expression of mutant M proteins interfering with virus particle budding. 866 24


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