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:3.1.27.1 (RNase)
16,360 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The transcription and replication of influenza virus RNA (vRNA) were reconstituted in vivo. The experimental approach involved the transfection of plasmids encoding the viral subunits of the polymerase and the nucleoprotein into cells infected with a vaccinia virus recombinant virus expressing the T7 RNA polymerase. As templates, one of two model RNAs was transfected: vNSZ or cNSZ RNA. The RNAs were 240 nucleotides in length, contained the terminal sequences of the NS viral segment, and were of negative or positive polarity, respectively. The accumulation of cRNA and mRNA in cells transfected with vNSZ RNA and the accumulation of vRNA and mRNA in cells transfected with cNSZ RNA were determined by RNase protection assays with labeled vNSZ-L or cNSZ-L probes. The patterns of protected bands obtained indicated that both cRNA replication intermediate and mRNA accumulated when the system was reconstituted with vNSZ RNA. Likewise, both vRNA and mRNA accumulated after reconstitution with cNSZ RNA. The reconstitution of incomplete systems in which any of the subunits of the polymerase or the model RNA were omitted was completely negative for the accumulation of cRNA or vRNA, indicating that the presence of the PB2 subunit in the polymerase is required for replication of vRNA.
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PMID:The influenza A virus PB2 polymerase subunit is required for the replication of viral RNA. 899 63

When MDCK cells in a semiconfluent monolayer were infected with 5 p.f.u. per cell of influenza virus A/PR/8/34 (H1N1), a majority of the cells continued to grow stably upon subsequent cultivation with a growth medium containing 50% foetal calf serum. While growing, the cells spontaneously excreted virus, the amount of which declined gradually as the passage number of the cells increased. The extent of virus shedding was significantly increased when the cells were subsequently maintained in a medium containing 0.2% bovine serum albumin. Within the cells, viral messenger RNAs for all eight genes of A/PR/8 were demonstrated by PCR indicating that endogenous viral genes were constitutively transcribed. However, viral proteins as well as viral genes were not demonstrable by radioimmunoprecipitation or ribonuclease protection assays, respectively.
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PMID:Spontaneous excretion of virus from MDCK cells persistently infected with influenza virus A/PR/8/34. 904 5

Glycoproteins generally exist as populations of glycosylated variants (glycoforms) of a single polypeptide. Although the same glycosylation machinery is available to all proteins that enter the secretory pathway in a given cell, most glycoproteins emerge with characteristic glycosylation patterns and heterogeneous populations of glycans at each glycosylation site. The factors that control the composition of the glycoform populations and the role that heterogeneity plays in the function of glycoproteins are important questions for glycobiology. A full understanding of the implications of glycosylation for the structure and function of a protein can only be reached when a glycoprotein is viewed as a single entity. Individual glycoproteins, by virtue of their unique structures, can selectively control their own glycosylation by modulating interactions with the glycosylating enzymes in the cell. Examples include protein-specific glycosylation within the immunoglobulins and immunoglobulin superfamily and site-specific processing in ribonuclease, Thy-1, IgG, tissue plasminogen activator, and influenza A hemagglutinin. General roles for the range of sugars on glycoproteins such as the leukocyte antigens include orientating the molecules on the cell surface. A major role for specific sugars is in recognition by lectins, including chaperones involved in protein folding. In addition, the recognition of identical motifs in different glycans allows a heterogeneous population of glycoforms to participate in specific biological interactions.
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PMID:Glycosylation: heterogeneity and the 3D structure of proteins. 906 19

The interaction of influenza virus NS1 protein with other viral products in the infected cell was analysed by co-immunoprecipitation studies. The three subunits of the polymerase and the nucleoprotein, but not M1 protein, were co-immunoprecipitated by NS1-specific serum but not when control serum was used. Such co-immunoprecipitation was not sensitive to RNase treatment of the immunoprecipitates. Co-immunoprecipitation was also obtained when the viral transcription-replication system was reconstituted in vivo by transfection of cDNAs and model vRNA template into vaccinia virus-T7-infected cells. Analysis of the RNA pulled-down in the NS1-specific precipitates indicated the presence of both vRNA and mRNA. These results are discussed in the context of the phenotype of virus temperature-sensitive mutants affected in the NS1 gene.
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PMID:Influenza virus NS1 protein interacts with viral transcription-replication complexes in vivo. 934 63

The M1 protein of influenza virus inhibits the in vitro transcriptase activity of ribonucleoprotein cores from virions. This inhibitory activity is thought to be relevant in vivo because accumulation of M1 at the late stages of viral replication may be the cue to halt viral mRNA production. A model influenza reporter genome was used to explore the effect of M1 on the activity of the influenza virus transcriptase complex within cultured cells. Expression of M1 in cells bearing the model influenza virus reporter genome was accompanied by a reduction of CAT gene expression to 12% of control levels. Quantification of RNA by ribonuclease protection assay revealed that the influenza reporter genome mRNA levels in M1-expressing cells were reduced by approximately 74% compared with those of cells expressing a control protein. These findings are consistent with the proposed model in which M1 is responsible for limiting viral transcription during late stages of infection. By expressing truncated forms of M1, the inhibitory activity was found to reside within the amino-terminal half of the M1 protein. Two independent inhibitory domains were identified in this region: one between amino acid residues 1-90 and the other spanning residues 91-127.
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PMID:The matrix 1 protein of influenza A virus inhibits the transcriptase activity of a model influenza reporter genome in vivo. 974 Jul 76

The viral factor responsible for triggering the acute phase response, or 'flu' syndrome, associated with many acute viral infections is not defined. One candidate viral factor is double-stranded RNA (dsRNA) generated during viral replication. In this report we demonstrate by reverse-transcriptase polymerase-chain reaction that nuclease-stable viral RNA was released from influenza-infected MDCK epithelial cells at the time of cell lysis. Removal of virion-associated RNA by ultracentrifugation left equal amounts of positive- and negative-strand viral RNA in the medium that resisted degradation by endogenous RNase in the medium and by exogenous RNase added prior to phenol extraction. These data are the first demonstration that viral RNA with characteristics of dsRNA is spontaneously released from dying influenza virus-infected cells, and thus is available to amplify cytokine induction and contribute to systemic disease.
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PMID:Spontaneous release of stable viral double-stranded RNA into the extracellular medium by influenza virus-infected MDCK epithelial cells: implications for the viral acute phase response. 993 Jan 93

Influenza virus infection is known to shut off the expression of host genes. To study the mechanism, we examined the effects of influenza A/Udorn/72 virus infection on the heat induction of a major heat shock protein, HSP70, in Madin-Darby canine kidney cells. The induction of HSP70 protein synthesis was progressively suppressed with postinfection time when heat shock was applied. Northern hybridization analysis revealed the appearance of longer, heterogeneous HSP70 transcripts in the range of 2.7 to 30 kb with a concomitant decrease in the amount of the mature 2.7-kb mRNAs in the nucleus of the infected cells. Such longer beta-actin transcripts were also observed but with much less intensity. The longer HSP70 transcripts contained the downstream sequence of the polyadenylation site, as demonstrated by RNase protection with an antisense RNA probe containing the sequence through the polyadenylation sites. This clearly proved that influenza virus infection inhibits the polyadenylation-site cleavage of the pre-mRNAs by the host cleavage and polyadenylation machinery. One temperature-sensitive mutant virus carrying a temperature-sensitive mutation on the NS1 gene failed to inhibit the cleavage at the nonpermissive temperature, indicating that the NS1 protein is involved in the inhibition of the pre-mRNA cleavage. This is the first report of the down-regulation of cellular mRNA maturation at the point of polyadenylation-site cleavage by virus infection and identifies a new mechanism by which the influenza virus shuts off host gene expression.
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PMID:Influenza virus inhibits cleavage of the HSP70 pre-mRNAs at the polyadenylation site. 998 87

Influenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome, including the unique cap-I-dependent RNase activity. To map the important domains for RNA polymerization, cap-I-dependent RNase, and cap-I-binding activity, we generated site-specific antibodies against overlapping 150-amino-acid peptides that cover each entire subunit. Monospecific antibodies against each subunit inhibited RNA synthesis in vitro. Those against PB1 and PB2 inhibited the cap-I-dependent RNase activity, but those against PB2 alone slightly inhibited the cap-I-binding activity. Antibodies against the N-terminal amino acids 1-159 of PB2 that overlap the PB1-binding site on PB2 and the C-terminal amino acids 501-617 of PA that overlap the putative nucleotide-binding site and PB1-binding site on PA inhibited RNA polymerizing activity as well as monospecific antibodies. Those against the N-terminal (amino acids 1-159); the central region (amino acids 305-559) of PB2, where a part of the cap-binding domain predicted previously is localized; the N-terminal (amino acids 1-222) of PB1; and amino acids 301-517 and 601-716 of PA inhibited the cap-I-dependent RNase activity. The cap-binding domain on PB2 could be mapped in amino acids 402-559, where one of the cap-binding domains mapped previously overlapped.
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PMID:Molecular mapping of influenza virus RNA polymerase by site-specific antibodies. 1008 33

To characterize the sites and nature of binding of influenza A virus matrix protein (M1) to ribonucleoprotein (RNP), M1 of A/WSN/33 was altered by deletion or site-directed mutagenesis, expressed in vitro, and allowed to attach to RNP under a variety of conditions. Approximately 70% of the wild-type (Wt) M1 bound to RNP at pH 7.0, but less than 5% of M1 associated with RNP at pH 5.0. Increasing the concentration of NaCl reduced M1 binding, but even at a high salt concentration (0.6 M NaCl), approximately 20% of the input M1 was capable of binding to RNP. Mutations altering potential M1 RNA-binding regions (basic amino acids 101RKLKR105 and the zinc finger motif at amino acids 148 to 162) had varied effect: mutations of amino acids 101 to 105 reduced RNP binding compared to the Wt M1, but mutations of zinc finger motif did not. Treatment of RNP with RNase reduced M1 binding by approximately half, but even M1 mutants lacking RNA-binding regions had residual binding to RNase-treated RNP provided that the N-terminal 76 amino acids of M1 (containing two hydrophobic domains) were intact. Addition of detergent to the reaction mixture further reduced binding related to the N-terminal 76 amino acids and showed the greatest effect for mutations affecting the RNA-binding regions of basic amino acids. The data suggest that M1 interacts with both the RNA and protein components of RNP in assembly and disassembly of influenza A viruses.
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PMID:Association of influenza virus matrix protein with ribonucleoproteins. 1043 36

Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein.
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PMID:Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E. 1275 27


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