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

The M2 protein of influenza A H1N1 strains PR8, WS, and WSN is present in homooligomeric forms in virions grown in the allantoic cavity of embryonated eggs. The bulk of the virion M2 is detected as tetramers and dimers. The oligomeric forms of PR8 virions differ from those of WS and WSN not only in apparent molecular weight (MW) but also in that they seem to be composed of two types of monomers differing in MW by approximately 1.5 kDa. Evidence from monoclonal antibody binding (or lack of it) and from in vitro trypsin digestion suggests that, in ovo, the external NH2 region of some PR8 M2 monomers is proteolytically trimmed, resulting in heterogeneous oligomers composed of cleaved and uncleaved monomers in different proportions.
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PMID:Oligomeric organization and strain-specific proteolytic modification of the virion M2 protein of influenza A H1N1 viruses. 172 10

The aims of these studies are (1) to determine whether, and by what mechanism(s), underexpression of M1 and/or NS1 protein restricts replication and cytopathogenicity in mouse brain cells of human influenza viruses which are closely related to the neurovirulent WSN variant but not selected for the neurovirulent phenotype; (2) to learn, ultimately, whether similarly restricted replication in natural infections might be enough to cause direct or indirect, immunologically mediated, neuropathology. On the basis of immunostaining, we have suggested that, in "aged" mouse embryo brain (MEB) cell cultures infected with A/PR/8/34 (PR8) or A/WS/33 (WS), M1 protein expression is restricted mainly in mature astrocytes (the dominant cell type in such cultures), but not in mature oligodendrocytes or neurons. Here we show that amounts of radiolabeled M1 protein in lysates of MEB cultures infected with PR8, WS, or WSN differ in proportion to previously reported single-cycle yields of trypsin-activated infectious virions. Low or undetectable cell-associated M1 does not reflect accelerated degradation, but tends to be accompanied by increased M2 protein (a product of spliced mRNA7). Radiolabeled NS1 is reduced, NS2 relatively increased, in "aged" MEB cultures infected at low m.o.i. with PR8, at high m.o.i. with WS as well, but not with WSN. In contrast, actively dividing and differentiating astrocyte-enriched or "young" MEB cultures tend to produce greatly increased amounts of NS2 even though NS1 may be at "normal" levels, both relative to those in similarly infected CEF cultures. We show, in extension of comparative studies by others on permissive and abortive FPV-infected cell systems, that virus strain-, cell type-, and perhaps differentiation-dependent variations in efficiency of mRNA 7 and 8 transcription and/or splicing are primary factors controlling variable expression of M and NS proteins in mouse brain cell cultures.
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PMID:Replication of H1N1 influenza viruses in cultured mouse embryo brain cells: virus strain and cell differentiation affect synthesis of proteins encoded in RNA segments 7 and 8 and efficiency of mRNA splicing. 214 Jun 29

A trypsin inhibitor, 6-amidino-2-naphthyl p-guanidinobenzoate (FUTHAN) reduced both the number and size of plaques of influenza virus A/WSN/33 (H1N1) that can grow without trypsin treatment in MDCK cells. The resulting virus particles with uncleaved hemagglutinin (HA) in the presence of FUTHAN was activated to produce infectious virions by trypsin treatment. Uncleaved HA of WSN virus grown in the presence of FUTHAN was found to be accumulated by protein analysis of WSN virus labeled biosynthetically with [35S]-methionine. It was strongly suggested that FUTHAN inhibited viral replication by preventing proteolytic cleavage of HA.
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PMID:Inhibition of influenza virus A/WSN replication by a trypsin inhibitor, 6-amidino-2-naphthyl p-guanidinobenzoate. 235 Mar 38

The mandatory step in reproduction of myxoviruses (influenza viruses and paramyxoviruses) is proteolytic shearing of viral glycoproteins activating the infectivity of virions. Such activation of myxoviruses is realized by trypsin-like proteases of the host. This study demonstrated that proteolytic activation of virions could be inhibited by a physiological inhibitor of proteases, aprotinine. A single injection of aprotinine (preparations Gordox or Contrycal) into chick embryos infected with various influenza viruses (WSN/34, Udorn/72) and paramyxoviruses (Sendai/960, NDV/La Sota, NDV/Queensland) blocked shearing of viral glycoproteins, HA, FO, HNO as a result of which noninfectious virions with unsheared glycoproteins were predominantly synthesized. In aprotinine-treated embryos, multicycle virus infection was markedly decreased which led to the 10(4)-fold or greater reduction of the virus yield. The antiviral effect of protease inhibitors and possibilities of their practical use in viral diseases are discussed.
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PMID:[Suppression of the proteolytic activation of myxoviruses in infected chick embryos by using aprotinin]. 240 86

The combined presence of WSN gene segments 6 (neuraminidase), 7 (M1 and M2), and 8 (NS1 and NS2) in reassortants of WSN with A/Aichi/2/68 (H3N2) has been found by others to be necessary for full expression of neurovirulence in mice. We are examining the expression of the analogous three gene segments in brains of mice after intracerebral infection with non-neuroadapted strains A/WS/33 (WS) (from which WSN was derived) and A/PR/8/34 (PR8). Our aim is to determine possible mechanisms by which one or more of the five gene products may restrict replication of these strains in mouse brain cells to a single cycle, yielding noninfectious hemagglutinating particles (incomplete growth cycle). We found that minority subsets of such particles did produce plaques, provided they were activated by trypsin (analogous to other abortive systems producing virions with uncleaved HA), a step obviated for some WSN virions by indirect promotion of hemagglutinin cleavage by the neuraminidase of that strain. The percentage of such potentially infectious virions, relative to total hemagglutinating particles, was significantly lower in WS- or PR8-infected than in WSN-infected brains, suggesting possible defects in synthesis or function of M1 protein in the former. Cells in immunostained sections and appropriate bands in Western blots (immunoblots) of viral proteins electrophoretically separated from lysates of PR8-infected brains reacted with antibody to nucleoprotein but not to M1 protein. Either method revealed the presence of both proteins in WSN-infected brains. In contrast, Western blot analyses of particles concentrated from PR8-, WS-, or WSN-infected brains by hemadsorption, elution, and pelleting did reveal NP and M1 bands with comparable relative peroxidase-antiperoxidase staining intensities. The findings suggest that availability of M1 protein is a factor influencing the extent or rate of assembly of potentially infectious (i.e., trypsin-activated) progeny virions in mouse brains and that in this respect the two non-neurovirulent strains differ from WSN quantitatively rather than qualitatively.
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PMID:Role of hemagglutinin cleavage and expression of M1 protein in replication of A/WS/33, A/PR/8/34, and WSN influenza viruses in mouse brain. 264 24

The responses of mouse embryo brain (MEB) cell cultures and of Madin-Darby canine kidney cells and chicken embryo fibroblasts to infection with A/PR/8/34 (PR8), A/WS/33 (WS), or the neurovirulent WSN variant were compared in terms of (i) single-cycle yields of hemagglutinating and associated neuraminidase (NA) activities and plaque-forming particles, the latter with or without trypsin activation [PFU(TR++) or PFU(TR--), respectively], and (ii) expression of nucleoprotein (NP), M1, and NS1 protein, determined for specific cell types by immunostaining, for whole culture lysates by Western blot analysis of NP and M1. Primary MEB cultures grown in serum-enriched medium were infected after 6 days (young), when none of the cells reacted specifically and exclusively with any of the nerve cell marker antibodies used, or after greater than or equal to 21 days (aged), when astrocytes (the predominant cell type), neurons, and oligodendrocytes were morphologically and immunologically mature. Secondary astrocyte-enriched cultures were used when they contained 90 to 99% of their cells as astrocytes at an early stage of differentiation. By all criteria, young MEB cultures were only marginally less permissive for each of the three viruses than were chicken embryo fibroblasts or Madin-Darby canine kidney cells. Aged MEB cultures, by comparison, produced undiminished NP, hemagglutinin, and neuraminidase, but yields of PFU(TR++) and expression of M1 protein (relative to NP) were reduced for all three viruses, most for PR8 and least for WSN; relative reduction of NS1 protein was demonstrable only in PR8-infected aged cultures. Immunostaining revealed low levels of M1 and NS1 expression only in astrocytes, not in oligodendrocytes and neurons. In PR8-infected mature astrocytes, NP accumulated in the nucleus; it persisted in some cells for at least 8 weeks after infection. The presence of NP did not seem to interfere with cell division. Secondary MEB cultures containing 90 to 99% immature astrocytes were less restricted than were aged primary cultures. Thus, it appears that reduced permissivity of nerve cell cultures, as measured in this study, is most closely correlated with advancing differentiation and maturity of astroglial cells. Assembled virions, including those that score as PFU(TR++) in restricted cultures (e.g., PR8-infected aged MEB), may be mainly products of mature oligodendrocytes and neurons.
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PMID:Effects of cell differentiation on replication of A/WS/33, WSN, and A/PR/8/34 influenza viruses in mouse brain cell cultures: biological and immunological characterization of products. 264 25

Adsorption and penetration into chick fibroblast cells of WSN virus with completely cleaved hemagglutinin precursor (virus grown in chick embryos), partially cleaved hemagglutinin precursor (virus grown in chick fibroblasts), and virus treated with trypsin added to infected cells or virions were studied. Treatment with trypsin in both instances resulted in complete cleavage of hemagglutinin precursor and in more than 100-fold increase of the infectious activity. Chick embryo-grown virus adsorbed in the same manner as the culture-grown virus not treated with trypsin, but penetrated into the cells 2--3 times more effectively. The culture-grown virus after treatment with trypsin adsorbed better but penetrated in the same way as the original culture-grown virus. Treatment of the infected cells with trypsin led to more effective penetration with unchanged adsorption. The experimental results suggest that cleavage of hemagglutinin precursor is essential both for adsorption and penetration. Depending on the method of treatment, the effectiveness of one or the other process is increased.
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PMID:[The role of hemagglutinin cleavage in influenza virus adsorption and penetration into the cell]. 738 84

MDCK cells (a continuous line of dog kidney) were infected with 3H-uridine-labeled influenza virus, the WSN strain, and localization of parental structure in the cells was determined at various intervals after inoculation using the cell autoradiography method. At 15 and 30 min postinfection radioactive granules were found in nuclei and some of them concentrated around nucleoli. One hour after infection the granules were found both in nuclei and in cytoplasm. Two hours after infection the bulk of the granules was found in the cytoplasm localized in a limited poorly strained area of the cytoplasm contiguous to the nucleus. The parental structures recovered from the cytoplasm and the nucleus were identified by centrifugation in sucrose density and cesium chloride density gradients as viral ribonucleoproteins. The amount of the granules in the nuclei at early stages of infection increased proportionately to hemagglutinin precursor cleavage in the original virus. The cleavage was achieved by treating the virus or virus-producing cells with trypsin or allantoic fluid from noninfected chick embryos. The experimental results indicate that parental ribonucleo-proteins immediately after infection penetrate into the nucleus and then are transported into the cytoplasm.
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PMID:[Localization and transport of influenza virus parental ribonucleoproteins in infected cells]. 743 32

When influenza virus A/WSN/33 (H1N1) was grown in MDBK or CV-1 cells in serum-free medium, progeny virus released from these cells contained only uncleaved hemagglutinin (HA). This virus was unable to infect CV-1 cells unless subjected to cleavage activation by trypsin, but it was infectious for MDBK cells without such treatment. Temperature shift experiments demonstrated that HA of radiolabeled input virus was cleaved within 30 min after inoculation of MDBK cells. As indicated by its resistance to trypsin cleavage, the virus was already internalized at this stage. Cleavage was not observed after inoculation of CV-1 cells. The serine-protease inhibitor leupeptin blocked virus growth when added to MDBK cells during the initial phase of the replication cycle. The inhibitor did not show this effect, when the input virus contained already cleaved HA. These results demonstrate that activation of HA of the WSN strain can occur in MDBK cells, but not in CV-1 cells, at the stage of virus entry, presumably by an endosomal protease.
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PMID:Cell tropism of influenza virus mediated by hemagglutinin activation at the stage of virus entry. 805 55


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