Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The genetic basis for the distinctive capacity of influenza A/WSN/33 (H0N1) virus (WSN virus) to produce plaques on bovine kidney (MDBK) cells was found to be related to virus neuraminidase. Recombinant viruses that derived only the neuraminidase of WSN virus were capable of producing plaques, whereas recombinant viruses identical to WSN except for neuraminidase did not produce plaques. With viruses that do not contain WSN neuraminidase, infectivity of virus yields from MDBK cells was increased approximately 1,000-fold after in vitro treatment with trypsin. In contrast, no significant increase in infectivity was observed after trypsin treatment of viruses containing WSN neuraminidase. In addition, polyacrylamide gel analysis of proteins of WSN virus obtained after infection of MDBK cells demonstrated that hemagglutinin was present in the cleaved form (HA1 + HA2), whereas only uncleaved hemagglutinin was obtained with a recombinant virus that derived all of its genes from WSN virus except its neuraminidase. These data are in accord with the hypothesis that neuraminidase may facilitate production of infectious particles by removing sialic acid residues and exposing appropriate cleavage sites on hemagglutinin.
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PMID:Virulence factors of influenza A viruses: WSN virus neuraminidase required for plaque production in MDBK cells. 56 60

Myxoviruses (ortho- and paramyxoviruses) possess on their surface two virus-specific glycoproteins, the functions of which are largely understood; These glycoproteins are synthesized on the rought endoplasmic reticulum, and during their transport to the plasma membrane on smooth intracellular membranes, they undergo modification through proteolytic cleavage. In this way, the orthomyxovirus hemagllutinin is converted from a high-molecular weight form (HA) into two smaller cleavage products (HA1 and HA2). With the paramyxoviruses, the glycoprotein F, which is responsible for fusion and hemolysis, is derived from proteolytic cleavage of a precursor, F0. Furthermore, with a few strains of avirulent NDV, a precursor of the hemagglutinin-neuraminidase complex, (HN0), has been identified which again, as a result of proteolysis, undergoes cleavage to HN. Whether cleavage takes place is as much dependent on the structure of the glycoprotein as on the host cell type. Proteolytic cleavage is indeed not necessary for virus particle production but is required for infectivity. Virus particles which possess the uncleaved glycoproteins may be activated by in vitro treatment with trypsin. As evidenced by experiments with orthomyxovirus recombinants, the glycoproteins alone do not determine the pathogenicity of the viruses. With paramyxoviruses, the pathogenic and apathogenic strains show clear differences in their host range spectrum which is directly related to the sensitivity of their glycoproteins twoard proteases. These observations provide an initial sketch for the molecular basis of infectivity and pathogenicity with myxoviruses.
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PMID:[Correlation between structure and pathogenicity of myxoviruses (author's transl)]. 57 26

The low pH-induced fusion of influenza virus with intact erythrocyte plasma membranes is preceded by a delay time following pH reduction, that is itself pH- and temperature dependent. At 37 degrees C/pH 4.8, lipid mixing between virus and target membranes begins < 2 s after pH reduction, whereas at 4 degrees C/pH 4.8, fusion does not commence until > 10 min after pH reduction. We have found that within this time period at 4 degrees C, a population of virus acquires the capacity to subsequently undergo fusion with high efficiency at elevated temperatures and pH 7.4. Both the kinetics and the extent of this pH 7.4 fusion depend upon the time of pre-incubation at pH 4.8/4 degrees C. Incubation at pH 7.4/4 degrees C, following this pre-incubation does not result in fusion, but the capacity to fuse at pH 7.4/37 degrees C is retained for a time period exceeding 1 h. The longevity of this fusion committed state makes it amenable to biochemical and immunological analysis. We have shown that it is insensitive to dithiothreitol, neuraminidase and trypsin, but is incapacitated by thermolysin or protease K. We conclude that only the HA2 sub-unit of influenza haemagglutinin is a necessary protein component of later stages of the fusion pathway.
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PMID:A long-lived state for influenza virus-erythrocyte complexes committed to fusion at neutral pH. 139 18

Microtubules have been implicated in the transport of vesicles carrying newly synthesized proteins from the trans-Golgi network (TGN) to the cell surface. We have established a quantitative in vitro binding assay to investigate the putative interaction between these exocytic carrier vesicles and the microtubules at the molecular level. TGN-derived exocytic carrier vesicles, labeled with C6NBD-ceramide metabolites or viral glycoproteins, were obtained from polarized filter-grown MDCK II cells by perforation of the apical membrane with a nitrocellulose filter. These exocytic vesicles were incubated with taxol-polymerized tubulin and cytosol, layered on top of a 30% sucrose cushion and subjected to centrifugation. Quantitation of vesicles co-sedimenting with microtubules was done by measuring NBD-fluorescence of viral glycoproteins in the pellet and supernatant fractions. About 25% of the label sedimented through the cushion in the presence of microtubules and cytosol. Both apically and basolaterally targetted carrier vesicles containing influenza virus HA2 or vesicular stomatitis virus G protein, respectively, associated with the microtubules. Only 2-5% NBD-fluorescence was obtained in the pellet when no cytosol or microtubules were added to the vesicles. Negative-stain electron microscopy of resuspended pellets showed distinct microtubule-vesicle complexes. Heat inactivation or treatment of cytosol with N-ethylmaleimide (NEM), or trypsinization of vesicles inhibited the binding of vesicles to microtubules. Furthermore, coating of microtubules with brain microtubule-associated proteins abolished binding. These data suggest that NEM-sensitive cytosolic proteins are required for microtubule-vesicle association, and that the vesicles are bound via trypsin-sensitive receptor proteins on their surface.
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PMID:Binding of exocytic vesicles from MDCK cells to microtubules in vitro. 238 28

Mouse-pathogenic influenza A/Aichi 2/68 (H3N2) grown in cell culture and having uncleaved hemagglutinin HA after treatment with trypsin underwent proteolytic shearing of HA (m.w. 75 kD) into two fragments: NAL (60 kD) and HA2 (15 kD); its lethal effect on mice inoculated intranasally increased more than 200-fold. The virus treated with chemotrypsin underwent similar shearing of HA into HAL and HA2; however, its lethal effect on mice was weak, analogous to that of intact virus with uncleaved HA.
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PMID:[The trypsin cleavage of hemagglutinin enhances the infectivity of the influenza virus in mice]. 238 64

To study the immune response of the chicken to specific influenza proteins, we have constructed a recombinant vaccinia virus containing the hemagglutinin gene of influenza A/Turkey/Ireland/1378/83 (H5N8). In mammalian cell culture the hemagglutinin expressed by this recombinant virus was full-length, cleaved into HA1 and HA2 in the absence of trypsin, and transported to the cell surface, confirming that other virus products are not required for cleavage activation. Chickens inoculated through the wing web with the live recombinant virus produced extremely low levels of hemagglutination-inhibiting or infectivity-neutralizing antibody. However, they were protected from lethal H5 influenza virus challenge. Protection extended to the antigenically distinct virulent H5 viruses, Chicken/Pennsylvania/1370/83 and Chicken/Scotland/59. Chemically bursectomized vaccinated chickens were not protected, whereas normal chickens with very low antibody levels (less than 10) obtained by passive transfer were protected in a dose-dependent fashion. This indicates that despite the low antibody titers induced by vaccination, protection was mediated by antibody.
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PMID:Protection of chickens from lethal influenza infection by vaccinia-expressed hemagglutinin. 326 29

One of the unresolved questions concerning the acquisition of virulence by the A/Chicken/Pennsylvania/83 (H5N2) influenza virus is which gene segments other than the hemagglutinin (HA) showed changes that were relevant. To answer this question, reassortants were made possessing the hemagglutinin gene of the virulent virus and the seven other genes from the avirulent parent. Since both the virulent and avirulent H5N2 strains are antigenically almost indistinguishable, it was necessary to transfer the genes of interest to a "carrier" virus before the appropriate reassortment could be selected. The gene compositions of the reassortants was established by a combination of sequence analysis and migration on polyacrylamide gels. These analyses established that the avirulent influenza virus present in April 1983 possessed seven of the eight gene segments necessary for virulence; mutation(s) in the HA gene were required for acquisition of virulence. Other viruses such as A/Seal/Mass/1/80 (H7N7) could provide the other genes necessary for virulence. Two changes in the HA have been associated with the acquisition of virulence; these are at amino acid residues 23 and 78 (H3 numbering) (Y. Kawaoka and R.G. Webster, Virology, 146, 130-137 (1985]. Isolation of an amantadine-resistant avirulent revertant virus provided the opportunity to determine which of the two amino acid changes in HA is critical. Sequence analysis of the revertant virus revealed amino acid changes at residues 23 in HA1 and 40 in HA2 (H3 numbering). The change at residue 23 of HA1 is probably associated with reversion to avirulence, of cleavability of HA, and inability to plaque in tissue culture without trypsin; while the change at residue 40 of HA2 may be associated with the amantadine-resistant phenotype. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/Pennsylvania/1/83 (H5N2) was probably all that was required to produce the highly virulent Chicken/Pennsylvania virus; the avirulent virus already possessed the other genes necessary for virulence.
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PMID:Molecular changes in A/Chicken/Pennsylvania/83 (H5N2) influenza virus associated with acquisition of virulence. 394 82

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).
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PMID:An efficient method for introducing defined lipids into the plasma membrane of mammalian cells. 631 96

At calcium-specific ionophore A23187 concentrations of approximately 0.25 microM [which still allow assembly and release of fowl plague virus (FPV) particles] post-translational proteolytic cleavage of the viral hemagglutinin precursor HA into the fragments HA1 and HA2 is inhibited. The resulting virus particles with uncleaved hemagglutinin, that cannot be obtained under normal conditions, provide a suitable substrate for in vitro assays of the protease sensitivity of the FPV hemagglutinin. Proteolytic activation is accomplished with trypsin. Treatment with cathepsin B at low pH yields aberrant cleavage products suggesting that the cellular cleavage enzyme is not of lysosomal origin. A protease that cleaves the FPV hemagglutinin in the correct place can be detected in lysates of MDBK cells. This enzyme is calcium dependent and has a neutral pH optimum.
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PMID:Inhibition of proteolytic cleavage of the hemagglutinin of influenza virus by the calcium-specific ionophore A23187. 644 5

The A/Chick/Penn/83 (H5N2) influenza virus that appeared in chickens in Pennsylvania in April 1983 and subsequently became virulent in October 1983, was examined for plaque-forming ability and cleavability of the hemagglutinin (HA) molecule. The avirulent virus produced plaques and cleaved the HA only in the presence of trypsin. In contrast, the virulent virus produced plaques and cleaved the HA precursor into HA1 and HA2 in the presence or absence of trypsin. The apparent molecular weight of the HA1 from the avirulent virus was higher than that from the virulent virus, but when the viruses were grown in the presence of tunicamycin, the molecular weights of HA were indistinguishable. Two of nine monoclonal antibodies to the HA of the avirulent virus indicate that there is at least one epitope on the HA that is different between the virulent and avirulent viruses. The amino acid sequences of the HAs from the two viruses were compared by sequencing their respective HA gene. The nucleotide sequence coding for the processed HA polypeptide contained 1641 nucleotides specifying a protein of 547 amino acids. The amino acid sequences of the virulent and avirulent viruses were indistinguishable through the connecting peptide region, indicating that the difference in cleavability of the H5 HA is not directly attributed to the amino acid sequence of the connecting peptide. Four of seven nucleotide changes resulted in amino acid changes at residues 13, 69, and 123 of HA1 and at residue 501 of the HA2 polypeptide. Since there were no deletions or insertions in the amino acid sequence of the virulent or avirulent viruses, the possibility exists that the difference in molecular weight is due to loss of a carbohydrate side chain in the virulent strain. The amino acid change in the virulent strain at residue 13 is the only mutation that could affect a glycosylation site and this is in the vicinity of the connecting peptide. It is postulated that the loss of this carbohydrate may permit access of an enzyme that recognizes the basic amino acid sequences and results in cleavage activation of the HA in the virulent virus.
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PMID:Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? 651 14


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