Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021400 (influenza)
 57,666 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Here we report the isolation of influenza virus A/turkey/Minnesota/833/80 (H4N2) with a mutation at the catalytic residue of the neuraminidase (NA) active site, rendering it resistant to the novel NA inhibitor 4-guanidino-Neu5Ac2en (GG167). The resistance of the mutant stems from replacement of one of three invariant arginines (Arg 292-->Lys) that are conserved among all viral and bacterial NAs and participate in the conformational change of sialic acid moiety necessary for substrate catalysis. The Lys292 mutant was selected in vitro after 15 passages at increasing concentrations of GG167 (from 0.1 to 1,000 microM), conditions that earlier gave rise to GG167-resistant mutants with a substitution at the framework residue Glu119. Both types of mutants showed similar degrees of resistance in plaque reduction assays, but the Lys292 mutant was more sensitive to the inhibitor in NA inhibition tests than were mutants bearing a substitution at framework residue 119 (Asp, Ala, or Gly). Cross-resistance to other NA inhibitors (4-amino-Neu5Ac2en and Neu5Ac2en) varied among mutants resistant to GG167, being lowest for Lys292 and highest for Asp119. All GG167-resistant mutants demonstrated markedly reduced NA activity, only 3 to 50% of the parental level, depending on the particular amino acid substitution. The catalytic mutant (Lys292) showed a significant change in pH optimum of NA activity, from 5.9 to 5.3. All of the mutant NAs were less stable than the parental enzyme at low pH. Despite their impaired NA activity, the GG167-resistant mutants grew as well as parental virus in Madin-Darby canine kidney cells or in embryonated chicken eggs. However, the infectivity in mice was 500-fold lower for Lys292 than for the parental virus. These findings demonstrate that amino acid substitution in the NA active site at the catalytic or framework residues, followed by multiple passages in vitro, in the presence of increasing concentrations of the NA inhibitor GG167, generates GG167-resistant viruses with reduced NA activity and decreased infectivity in animals.
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PMID:Catalytic and framework mutations in the neuraminidase active site of influenza viruses that are resistant to 4-guanidino-Neu5Ac2en. 909 7

Recent highly pathogenic (HP) field isolates of avian influenza (AI) virus from Mexico all possess an insertion of at least two basic amino acids (arg-lys) at the cleavage site of the hemagglutinin (HA) glycoprotein. One HP isolate has additional information which yields a 4 amino acid insert (arg-lys-arg-lys). We present here the nucleotide sequence of the HA gene of this unique isolate and compare it to recent H5N2 and other avian influenza isolates. The complete HA nucleotide sequence of the isolate and phylogenetic relationship suggest that it was derived in direct succession from a non-pathogenic strain isolated about 1 month earlier. The unique insertion sequence is a direct duplication of part of the purine-rich region preceding the arginine codon at the HA cleavage site. This evidence along with other data in this report provide compelling support for a proposed model explaining the mechanism of spontaneous, virulence-related insertions in type A influenza viruses.
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PMID:Virulence-associated sequence duplication at the hemagglutinin cleavage site of avian influenza viruses. 921 92

Based on molecular dynamics simulations, it is proposed that water within the binding groove of the human MHC class I molecule HLA-A2 plays a role in the formation of its complex with the influenza matrix protein (residues 58-66; GILGFVFTL) peptide. In these simulations, a loosely structured network of water molecules is present in the binding groove between the peptide and the MHC molecule, and may be important in completing the peptide-MHC interface. In two independent 400 ps simulations where groove-based water molecules were included, the peptide remained essentially in the conformation observed in the crystal structure. In contrast, in a 400 ps simulation in which no water molecules were placed between the peptide and the MHC molecule, the crystal structure conformation was rapidly lost. The basis for this behavior appears to be that the groove-based water molecules help to maintain the appropriate orientation of the Arg-97 side chain of HLA-A2 and, in turn, the conformation of the central part of the peptide.
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PMID:A model of water structure inside the HLA-A2 peptide binding groove. 931 Aug 37

The hemagglutinin (HA) protein of influenza B virus contains a single arginine residue at its cleavage site and the HA0 precursor is not cleaved to the HA1 and HA2 subunits by tissue culture cell-associated proteases. To investigate if an HA protein could be obtained that could be cleaved by an endogenous cellular protease, the cDNA for HA of influenza B/MD/59 virus was subjected to site-specific mutagenesis. Three HA mutant proteins were constructed, through substitution or insertion of arginine residues, that have 4, 5, or 6 basic residues at their cleavage sites. Chemical cross-linking studies indicated that all three HA cleavage site mutants could oligomerize to a trimeric species, like WT HA. The three HA cleavage site mutant proteins were efficiently transported to the cell surface and bound erythrocytes in hemadsorption assays. The mutants were cleaved at a low level to HA1 and HA2 by an endogenous host cell protease and cleavage could be increased somewhat by addition of exogenous trypsin. The fusogenic activities of the HA cleavage site mutants were assessed in comparison to the WT HA protein by determining their syncytium formation ability and by using an R18 lipid-mixing assay and a NBD-taurine aqueous-content mixing assay. While the fusion activity of the WT HA protein was dependent on exogenous trypsin to activate HA, the three HA cleavage site mutant proteins were able to induce fusion in the absence of trypsin when assayed with the R18 lipid-mixing and NBD-taurine aqueous-content mixing assays, but were unable to induce syncytium formation in either the presence or absence of exogenous trypsin. Our results suggest that while the presence of a subtilisin-like protease cleavage sequence at the influenza B virus HA1/HA2 boundary does enable some HA0 molecules to be cleaved intracellularly, it alone is not sufficient for efficient cleavage.
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PMID:Expression of influenza B virus hemagglutinin containing multibasic residue cleavage sites. 932 31

Infectivity and pathogenicity of influenza viruses are based on the interplay between the viral glycoprotein hemagglutinin (HA) and appropriate host proteases. HA receives its full biological activities by proteolytic cleavage of a precursor molecule at a definite cleavage site. Tryptase Clara, an arginine-specific protease secreted by the Clara cells in the bronchial epithelia, is a principal host protease responsible for the cleavage activation and pathogenicity of influenza viruses. Although influenza in normal individuals is usually confined to the upper respiratory tract, the infection often develops into fatal pneumonia in patients with chronic lung diseases, where bacterial infections often occur. Synergistic effects of bacterial infections on the pathogenesis of influenza viruses are described in regard to the cleavage activation of HA.
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PMID:[The role of cleavage activation of the hemagglutinin by host and bacterial proteases in the induction of the pathogenesis of influenza viruses]. 936 Mar 83

The solution NMR structure of the RNA-binding domain from influenza virus non-structural protein 1 exhibits a novel dimeric six-helical protein fold. Distributions of basic residues and conserved salt bridges of dimeric NS1(1-73) suggest that the face containing antiparallel helices 2 and 2' forms a novel arginine-rich nucleic acid binding motif.
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PMID:A novel RNA-binding motif in influenza A virus non-structural protein 1. 936 Jun 1

The influenza virus neuraminidase (NA)-specific inhibitor zanamivir (4-guanidino-Neu5Ac2en) is effective in humans when administered topically within the respiratory tract. The search for compounds with altered pharmacological properties has led to the identification of a novel series of influenza virus NA inhibitors in which the triol group of zanamivir has been replaced by a hydrophobic group linked by a carboxamide at the 6 position (6-carboxamide). NWS/G70C variants generated in vitro, with decreased sensitivity to 6-carboxamide, contained hemagglutinin (HA) and/or NA mutations. HA mutants bound with a decreased efficiency to the cellular receptor and were cross-resistant to all the NA inhibitors tested. The NA mutation, an Arg-to-Lys mutation, was in a previously conserved site, Arg292, which forms part of a triarginyl cluster in the catalytic site. In enzyme assays, the NA was equally resistant to zanamivir and 4-amino-Neu5Ac2en but showed greater resistance to 6-carboxamide and was most resistant to a new carbocyclic NA inhibitor, GS4071, which also has a hydrophobic side chain at the 6 position. Consistent with enzyme assays, the lowest resistance in cell culture was seen to zanamivir, more resistance was seen to 6-carboxamide, and the greatest resistance was seen to GS4071. Substrate binding and enzyme activity were also decreased in the mutant, and consequently, virus replication in both plaque assays and liquid culture was compromised. Altered binding of the hydrophobic side chain at the 6 position or the triol group could account for the decreased binding of both the NA inhibitors and substrate.
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PMID:Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors. 949 7

Influenza neuraminidase (NA) plays an important role in viral replication, and characterization of viruses resistant to NA inhibitors will help elucidate the role of active-site residues. This information will assist in designing better inhibitors targeted to essential active-site residues that cannot generate drug-resistant mutations. In the present study we used the benzoic acid-based inhibitor BCX-140 to select and characterize resistant viruses. BCX-140 binds to the NA active site in an orientation that is opposite that of a sialic acid-based compound, 4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid (GANA). Thus, the guanidino group of BCX-140 binds to Glu-276, whereas in GANA the guanidino group binds to Glu-119. We passaged influenza A/Singapore/1/57 (H2N2) in Madin-Darby canine kidney cells in the presence of BCX-140, and virus resistant to this inhibitor was selected after six passages. The NA of this mutant was still sensitive to inhibition by BCX-140. However, the mutant virus was resistant to BCX-140 in plaque and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Sequence analysis of hemagglutinin (HA) and NA genes revealed changes in both, although none were in the active site of the NA. Depending on the method of selection of the resistant virus, two types of changes associated with the sialic acid binding site were seen in the HA. One is a change in HA1 of Ala-133 to Thr, a residue close to the binding site, while the other change was Arg-132 of HA1 to Gln, which in HA1 of serotype H3 is a sialic acid contact (Asn-137). Binding studies revealed that both types of resistant viruses had reduced receptor binding affinity compared to that of the wild type. Thus, resistance to BCX-140 was generated by modifying the HA. NA active-site residue 276 may be essential for activity, and thus, it cannot be changed to generate resistance. However, drug-induced changes in the HA can result in a virus that is less dependent on NA activity for growth in cells and, hence, resistant to NA inhibitors.
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PMID:Generation and characterization of a mutant of influenza A virus selected with the neuraminidase inhibitor BCX-140. 955 86

The effectiveness and safety of known mutants of Escherichia coli heat-labile enterotoxin (LT) as an adjuvant for nasal influenza vaccine were examined. Six mutants, called LT7K (Arg to Lys), LT61F (Ser to Phe), LT112K (Glu to Lys), LT118E (Gly to Glu), LT146E (Arg to Glu) and LT192G (Arg to Gly) were constructed by the replacement of one amino acid at one position of the A1 subunit to another using site-directed mutagenesis. All mutants were confirmed to be less toxic than wild-type LT when analyzed using Y-1 adrenal cells in vitro. When influenza vaccine was administered intranasally with LT7K and LT192G, BALB/c mice developed high levels of serum and local antibodies to the HA molecules. The adjuvant activity of these mutant LTs corresponded to that of wild-type LT when 1 microgram of these mutant LTs (or wild-type LT) was coadministered with the vaccine. From the point of view of safety, LT7K was considered to be the most potent mucosal adjuvant and was examined in more detail. The adjuvant activity of the mutant was lowered more rapidly with a decrease in dose than was that of wild-type LT. The low level of adjuvant of a relatively small amount of LT7K was heightened by adding LTB to the mutant LT. These results suggest that LT7K supplemented with LTB can be used as a less toxic, effective adjuvant for nasal influenza vaccine.
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PMID:Mutants of Escherichia coli heat-labile enterotoxin as an adjuvant for nasal influenza vaccine. 960 38

Human histocompatibility leukocyte antigen B27 is highly associated with the rheumatic diseases termed spondyloarthropathies, but the mechanism is not known. B27 transgenic rats develop a spontaneous disease resembling the human spondyloarthropathies that includes arthritis and colitis. To investigate whether this disease requires the binding of specific peptides to B27, we made a minigene construct in which a peptide from influenza nucleoprotein, NP383-391 (SRYWAIRTR), which binds B27 with high affinity, is targeted directly to the ER by the signal peptide of the adenovirus E3/gp19 protein. Rats transgenic for this minigene, NP1, were made and bred with B27 rats. The production of the NP383-391 peptide in B27(+)NP1(+) rats was confirmed immunologically and by mass spectrometry. The NP1 product displaced approximately 90% of the 3H-Arg-labeled endogenous peptide fraction in B27(+)NP1(+) spleen cells. Male B27(+)NP1(+) rats had a significantly reduced prevalence of arthritis, compared with B27(+)NP- males or B27(+) males with a control construct, NP2, whereas colitis was not significantly affected by the NP1 transgene. These findings support the hypothesis that B27-related arthritis requires binding of a specific peptide or set of peptides to B27, and they demonstrate a method for efficient transgenic targeting of peptides to the ER.
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PMID:The specificity of peptides bound to human histocompatibility leukocyte antigen (HLA)-B27 influences the prevalence of arthritis in HLA-B27 transgenic rats. 1245 60


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