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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

1. The hemagglutinating capacity, enzymic activity, and infectivity of several influenza viruses were destroyed by repeated freezing and thawing of dialyzed allantoic fluids containing them. 2. Influenza virus degraded by freezing and thawing, by treatment with 5 M urea, or by heating at 65 degrees C. still combined with homologous antibody and was demonstrable by blocking of the hemagglutination-inhibition and virus neutralization reactions. 3. After 50 cycles of freezing and thawing, much of the blocking antigen activity was not sedimented by centrifugation at 120,000 g for 2 hours, and electron microscopy showed complete disruption of the virus particles. So called soluble blocking antigen was obtained from four strains of influenza A, the Lee strain of influenza B, mumps, and Newcastle disease viruses. 4. Soluble blocking antigens from influenza A viruses were highly strain-specific; gave little or no reaction in complement-fixation tests; stimulated but little antibody production in rabbits and did not induce immunity in mice; caused reactivation of infective virus in neutral mixtures of homologous virus and immune serum. 5. Repeatedly frozen and thawed influenza virus preparations did not interfere with the propagation of infective virus in the allantoic sac. The blocking antigen activity they contained was precipitated by half saturated ammonium sulfate, destroyed by trypsin, chymotrypsin, or heating at 56 degrees C. for 30 minutes, but was unaffected by desoxyribonuclease or ribonuclease. 6. These findings are in accord with the view that soluble blocking antigen obtained from influenza virus particles on disruption by repeated freezing and thawing is protein in nature and represents the essential antigenic material of the intact virus.
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PMID:Disruption of influenza virus; properties of degradation products of the virus particle. 1315 79

Influenza A virus replicates in the respiratory epithelium and induces an inflammatory infiltrate comprised of mononuclear cells and neutrophils. To understand the development of the cell-mediated immune response to influenza and how leukocyte trafficking to sites of inflammation is regulated, we examined the chemokine expression pattern in lung tissue from A/PR/8/34-infected C57BL/6 mice using an RNase protection assay. Monocyte chemoattractant protein 1, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, MIP-3alpha, regulated on activation, normal T expressed and secreted (RANTES), MIP-2, and interferon-inducible protein 10 (IP-10) mRNA expression was up-regulated between days 5 and 15 after infection, consistent with a role for these chemokines in leukocyte recruitment to the lung. Low levels of expression were detected for the CC chemokine receptors (CCR)2 and CCR5, whereas CXC chemokine receptor (CXCR)3 was significantly up-regulated by day 10 after infection, coinciding with peak inflammatory cell infiltration in the airways. As RANTES, IP-10, and their receptors were up-regulated during influenza virus infection, we investigated leukocyte recruitment and viral clearance in mice deficient in RANTES or CXCR3, the receptor for IP-10. Leukocyte recruitment and viral replication in influenza-infected RANTES knockout(-/-) mice were similar to that in control mice, showing that RANTES is not essential for the immune response to influenza infection. Similarly, leukocyte recruitment and viral replication in CXCR3-/- mice were identical to control mice, except at day 8 postinfection, where fewer lymphocytes, neutrophils, and eosinophils were detected in the bronchoalveolar lavage of CXCR3-/- mice. These studies suggest that although the chemokines detected may play a role in regulating leukocyte trafficking to the lung during influenza infection, some may be functionally redundant.
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PMID:Chemokine expression during the development and resolution of a pulmonary leukocyte response to influenza A virus infection in mice. 1524 Jul 57

Cleavage of phosphodiester bonds by small ribonuclease mimics within different bulge-loops of RNA was investigated. Bulge-loops of different size (1-7 nt) and sequence composition were formed in a 3' terminal fragment of influenza virus M2 RNA (96 nt) by hybridization of complementary oligodeoxynucleotides. Small bulges (up to 4 nt) were readily formed upon oligonucleotide hybridization, whereas hybridization of the RNA to the oligonucleotides designed to produce larger bulges resulted in formation of several alternative structures. A synthetic ribonuclease mimic displaying Pyr-Pu cleavage specificity cleaved CpA motifs located within bulges faster than similar motifs within the rest of the RNA. In the presence of 10 mM MgCl2, 75% of the cleavage products resulted from the attack of this motif. Thus, selective RNA cleavage at a single target phosphodiester bond was achieved by using bulge forming oligonucleotides and a small ribonuclease A mimic.
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PMID:Enhanced RNA cleavage within bulge-loops by an artificial ribonuclease. 1573 40

Conjugates of pancreatic RNase and ligand-free human serum albumin (LFHSA) have been obtained. The number of hydrophobic binding sites both for initial HSA and LFHSA has been determined by the polarised luminescence method. Interaction between RNase and HSA involves additional electrovalent linkage. Unlike initial enzyme, conjugates exhibit activity toward double-strand RNA. After intravenous injection, transferase activity of unmodified enzyme remains in the blood during 20 min., whereas 30-40% of this activity is detected at the fourth day after administration of RNase conjugates. A single dose administration of LFHSA-RNAse conjugates exhibited high antiviral activity in mice, infected with influenza A and influenza B viruses.
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PMID:[Conjugates of pancreatic ribonuclease and ligand-free human serum albumin]. 1611 87

Nucleocapsids released from influenza virions by sodium deoxycholate sedimented heterogeneously in sucrose gradients. Highly infectious virus (complete) preparations yielded nucleocapsids with peak distributions at 64 and 56S; von Magnus type virus (incomplete) lacked 64S nucleocapsids. Treatment of influenza virus nucleocapsids with pancreatic ribonuclease rendered the associated viral ribonucleic acid (RNA) molecules acid-soluble, indicating that capsid proteins do not completely surround the viral RNA's. However, the capsid proteins remained associated after enzymatic hydrolysis of the RNA, as judged by persistently high sedimentation rates. Sedimentation rates of viral nucleocapsids reflected the sedimentation rates of the associated RNA's: 64S nucleocapsids contained 18S RNA, whereas 56S nucleocapsids contained 15S RNA, although in both cases RNA's sedimenting at 4 to 13S were also recovered. Furthermore, just as incomplete virions lacked 64S nucleocapsids, they also lacked 18S RNA. These findings support the hypothesis that the influenza virus genome is divided among several distinct pieces of RNA.
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PMID:Some properties of influenza virus nucleocapsids. 1678 96

Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.
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PMID:An antigenic epitope of influenza virus nucleoprotein (NP) associated with polymeric forms of NP. 1831 50


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