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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.
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PMID:Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport. 173 2

Besides the rapid diagnostic tests based on influenza A and B antigens nucleoproteins detection, which are routinely used, the isolation of influenza strains is still required to obtain recent variant isolates for full antigenic characterization, in order to up-date the influenza vaccine composition. To increase the rapidity and the efficacy of the virus growth, we implemented a culture test in 24-well plates by centrifugation of samples on to LLCMK2 cells in the presence of trypsin. This test was routinely applied to 331 nasopharyngeal swabs collected during the influenza A outbreak in the winters 1988-1989 and to 962 in 1989-1990. The centrifugation culture assay has been compared with the direct detection of NP antigens in the clinical samples by immunofluorescence and capture ELISA tests and with the conventional virus isolation by inoculation of the samples to embryonated eggs and to LLCMK2 cell cultures. Compared with the NP antigen detection tests, the centrifugation culture assay closely correlated (r = 0.95) and the sensitivity and specificity were also excellent, 93.4% and 99.6%, respectively. Compared with the conventional culture assays, the centrifugation culture markedly increased the performance (five times) and rapidity (2 days) of influenza virus isolation and identification.
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PMID:Comparison between three rapid methods for direct diagnosis of influenza and the conventional isolation procedure. 179 40

We have studied the kinetics of low-pH-induced fusion between erythrocyte membranes and membranes containing influenza virus hemagglutinin by using assays based on the fluorescence dequenching of the lipophilic dye octadecylrhodamine. Stopped-flow mixing and fast data acquisition have been used to monitor the early stages of influenza virus fusion. We have compared this with the kinetics observed for fusion of an NIH 3T3 cell line, transformed with bovine papillomavirus, which constitutively expresses influenza virus hemagglutinin (GP4f cells). Virus and GP4f cells both display a pH-dependent time lag before the onset of fluorescence dequenching, but of an order of magnitude difference, ca. 2 s versus ca. 20 s. We have adopted two strategies to investigate whether the difference in lag time reflects the surface density of acid-activated hemagglutinin, able to undergo productive conformational change. (i) Hemagglutinin expressed on the cell surface requires proteolytic cleavage with trypsin from an inactive HAO form; we have limited the extent of proteolysis. (ii) We have used infection of CV-1 cells with a recombinant simian virus 40 bearing the influenza virus hemagglutinin gene. The surface expression of hemagglutinin is a function of time postinfection. For low-pH-induced fusion of both types of cell with erythrocytes, the lag time decreases with increasing hemagglutinin densities. Our results do not indicate a cooperative phenomenon at the level of the principal rate-determining step. We also show in the instance of virus fusion, that the magnitude of the delay time is a function of the target membrane transbilayer lipid distribution. We conclude that for a given amount of pH-activated hemagglutinin per unit area of membrane, the kinetics of fusion is determined by nonspecific physical properties of the membranes involved.
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PMID:Delay time for influenza virus hemagglutinin-induced membrane fusion depends on hemagglutinin surface density. 185 19

In the polarized kidney cell line MDCK, the influenza virus hemagglutinin (HA) has been well characterized as a model for apically sorted membrane glycoproteins. Previous work from our laboratory has shown that a single amino acid change in the cytoplasmic sequence of HA converts it from a protein that is excluded from coated pits to one that is efficiently internalized. Using trypsin or antibodies to mark protein on the surface, we have shown in MDCK cells that HA containing this mutation is no longer transported to the apical surface but instead is delivered directly to the basolateral plasma membrane. We propose that a cytoplasmic feature similar to an endocytosis signal can cause exclusive basolateral delivery.
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PMID:A single amino acid change in the cytoplasmic domain alters the polarized delivery of influenza virus hemagglutinin. 186 Aug 78

The virulent avian influenza virus A/Ty/Ont/7732/66 (H5N9) (Ty/Ont) causes severe destruction of the lymphoid cells in infected birds. Previous studies have suggested that viral infection of macrophages may be involved. However, Ty/Ont failed to replicate productively in primary cultures of chicken macrophages. Therefore, in an effort to develop an in vitro system for our studies, we examined the susceptibility of an avian macrophage cell line, HD11, to Ty/Ont. We found that Ty/Ont replicated in the HD11 cells to high titres, as measured by haemagglutination (HA) assays and infectivity yields. To determine whether this property was unique to Ty/Ont, we also examined the replication of influenza viruses representative of all 13 HA subtypes and an attenuated variant of Ty/Ont. All of the tested viruses replicated in HD11 cells; the avirulent strains required the presence of trypsin in the culture medium whereas virulent viruses and the attenuated variant of Ty/Ont did not. These results suggest that the HD11 cells can support the replication of a wide variety of influenza viruses and that this continuous avian cell line may prove useful for in vitro studies on these viruses.
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PMID:Replication of influenza A viruses in an avian macrophage cell line. 187 96

The surface glycoproteins of influenza A viruses are the viral components first recognized by the immune system of the infected host, and they are the viral proteins first to contact the infecting cell. Cleavage of the hemagglutinin (HA) is the presupposition for the uptake and fusion between viral and endosomal membranes at a relatively low pH. If this cleavage does not occur during synthesis and migration within the cell, an external trypsin-like protease has to activate the virus with a non-cleaved HA. This latter property is presumably the reason, why such a large reservoir of non-pathogenic influenza A viruses could be built up in water fowl. Especially feral ducks can disseminate influenza viruses along their flight routes all over the world. The role of the neuraminidase (NA) in the infectious process is not so clear. Its main task in the natural infection seems to be removal of mucoids at the site of entry and in this way to start the primary infection. The synthesis of the viral proteins is a highly regulated process. There is not only a transcriptional but also a translational control. The viral glycoproteins belong to the late proteins. Specifically their synthesis can be inhibited by compounds acting in completely different ways like a specific methylase inhibitor (3DA-Ado), a protein phosphokinase C inhibitor (H7), or a lipid solvent (DMSO). It remains to be determined whether the underlining mechanism is in all these cases the same, namely posttranscriptional modification of viral mRNA. All these viral components do not act separately but they cooperate in their functions and sometimes interfere with each other.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Synthesis and function of influenza A virus glycoproteins. 193 Jan 3

Influenza B/Beijing/1/87 neuraminidase heads were isolated from virus via trypsin digestion and characterized by PAGE, N-terminal sequencing, electron microscopy, and enzyme activity. The heads were crystallized into two crystal forms; tetragonal plates, like other neuraminidase crystals described before, that diffract to medium resolution (3 A) and a new form consisting of trigonal prisms or needles that diffract to high resolution (at least 2 A). The gene segment coding for neuraminidase was sequenced and compared with the neuraminidase sequence of B/Lee/40. The deduced amino acid sequences for neuraminidase showed only a 7% difference, whereas those for the NB proteins differed by 20%.
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PMID:Sequence and crystallization of influenza virus B/Beijing/1/87 neuraminidase. 198 52

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion.
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PMID:Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion. 198 76

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


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