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 large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) was identified in sucrose gradient-purified HSV-2 virions by immunoprecipitation/immunoblotting with antibody specific for the protein kinase (PK) domain. Immunoblotting of individual gradient fractions indicated that ICP10 cosediments with the major capsid protein and the highest virus titers. ICP10 was not labeled by iodination of purified virions, indicating that it is not located on the virion surface. After envelope glycoproteins were removed by detergent treatment, ICP10 was associated with capsid-tegument particles and became sensitive to trypsin digestion. The capsid-tegument-associated ICP10 was phosphorylated and had PK activity in vitro and on Immobilon membranes. A mutant ICP10 protein deleted in the PK domain (p95) was also associated with purified virions (ICP10deltaPK virus) but it lacked PK activity. The data indicate that ICP10 is contained within the tegument component where it retains intrinsic PK activity.
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PMID:The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is associated with the virion tegument and has PK activity. 926 54

The proteases encoded by herpesviruses including herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV) are attractive targets for antiviral drug development because of their important roles in viral replication. We randomly screened a chemical compound library for inhibitory activity against HSV-1 protease. 1,4-Dihydroxynaphthalene and three naphthoquinones were found to be potent inhibitors of HSV-1 protease with IC50 values of 6.4 to 16.9 microM. Inhibitory mode analysis of the compounds against HSV-1 protease suggested that, in spite of structural similarities, only 1,4-dihydroxynaphthalene was a competitive inhibitor, whereas the three naphthoquinones were noncompetitive inhibitors. Among all assayed dihydroxynaphthalene derivatives in the chemical compound library, 1,4-dihydroxynaphthalene proved to be the most potent inhibitor of HSV-1 protease. Therefore, the two hydroxyl groups located at positions 1 and 4 on the naphthalene structure seemed essential for exertion of a potent inhibitory activity against HSV-1 protease. In addition, we have found that these compounds are also potent inhibitors of HCMV protease with extremely low micromolar IC50 values. This differed from the results of inhibitory mode analysis of HSV-1 protease, 1,4-dihydroxynaphthalene was a noncompetitive inhibitor of HCMV protease, and three naphthoquinones were competitive inhibitors. These compounds showed no effective inhibitory activity against several mammalian serine proteases (trypsin, chymotrypsin, kallikrein, plasmin, thrombin and Factor Xa) at 100 microM. These results suggest that 1,4-dihydroxynaphthalene and three naphthoquinones may be useful in the development of nonpeptidic antiherpesvirus agents.
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PMID:Selective nonpeptidic inhibitors of herpes simplex virus type 1 and human cytomegalovirus proteases. 1125 77

Although various cell lines have been used for virus isolation, few study of virus isolation using MRC-5 cell, a human embryonic lung fibroblasts, have been reported in Japan. MRC-5 and other cell lines (Caco-2, Vero, RD-18s, LLC-MK2, HeLa, MDCK, FL, B95a and HMV-II), and suckling mouse were compared for isolation of viruses from clinical specimens. A total of 3,284 specimens, collected from clinics and hospitals in Saitama Prefecture between July 1997 and August 2001, were inoculated in these cells. A total of 1,252 viral strains were isolated and 1,190 viral strains of these were identified. MRC-5 detected 209 of specimens positive for various viruses. As for adenovirus, a total of 132 viral strains were isolated using cell lines described above, and 100 of 132 viral strains were isolated in MRC-5. MRC-5 showed the highest sensitivity for isolation of adenovirus 3 and 7 (79.1% and 100%) of all other cells. The sensitivity in isolation of these viruses in HeLa was 58.1% and 50.0%, respectively. It showed that MRC-5 is able to isolate enterovirus, especially coxsackie virus A16 and enterovirus 71 with a high sensitivity (85.7% and 73.7%). RD-18s detected 35.7% and 26.3% of coxsackie virus A16 and enterovirus 71 isolates, LLC-MK2 detected 60.7% and 47.4%, and Vero detected 48.6% and 52.6%, respectively. Coxsackie virus B group was not isolated, except for a few coxsackie virus B 5 strains. Enteroviruses except coxsackie virus A16 and enterovirus 71 were isolated more frequently in Caco-2 and RD-18s. Seven hundred thirteen strains of influenza viruses were isolated in MDCK and Caco-2, but none was isolated in MRC-5. It was probably due to the maintenance medium without trypsin. The isolation rate of herpes simplex virus in Vero was 88.9% and MRC-5 showed 77.8%, it was high secondary to Vero by MRC-5. However, the CPE was detected in a few days in MRC-5, it was earlier than in Vero. The MRC-5 is possible to be maintained without changing the maintenance medium and passaged for 2 weeks, and clear CPE was observed. On the other hand, the disadvantages in using the MRC-5 were that the passage was limited and that the split ratio was only 1:2. However, the MRC-5 was used successfully for virus isolation, especially coxsackie virus A16, enterovirus 71 and adenoviruses, from clinical specimens.
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PMID:[The usefulness of human lung embryonal fibroblast cells (MRC-5) for isolation of enteroviruses and adenoviruses]. 1213 51

Herpes simplex virus (HSV)-1 has been discovered in placental tissue from spontaneous miscarriages, but reports of transplacental transmission and fetal infection are extremely rare. Previously, we demonstrated that the villous syncytiotrophoblast, which forms a continuous layer between the maternal and fetal circulation, is resistant to HSV entry. Here, we tested our hypothesis that the villous syncytiotrophoblast prevents transplacental transmission of HSV secondary to decreased expression of HSV entry mediators (HveA, HveB, and HveC). In addition, we investigated the ability of HSV to infect extravillous trophoblast cells, which mediate placental attachment to the uterine wall, and the expression of HSV receptors in these cells. We performed fluorescence-activated cell sorting (FACS) analyses and immunostaining to demonstrate that HveA, HveB, and HveC were not expressed in third-trimester villous trophoblast cells. Consequently, villous explants obtained from third-trimester placentas were resistant to infection by a recombinant HSV-1 vector, HSV-1 KOS, but approximately 20% of mesenchymal cells within the villous core were infected when villous explants were pretreated with trypsin to disrupt the villous trophoblast layer. Conversely, FACS analysis and immunostaining demonstrated that extravillous trophoblast cells expressed HveA, HveB, and HveC, and these cells were efficiently infected by HSV vectors. Infection of extravillous trophoblast cells by HSV-1 was not reduced when the cells were pretreated with an antibody against HveA but was partially reduced when the cells were pretreated with antibodies directed against HveB and HveC. Thus, the decreased expression of herpesvirus entry mediators in villous syncytiotrophoblast prevents placental villous infection, thereby limiting maternal-fetal transmission of HSV.
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PMID:Syncytiotrophoblast is a barrier to maternal-fetal transmission of herpes simplex virus. 1239 Aug 90

Herpes simplex virus-1 (HSV-1) virions are large, complex enveloped particles containing a proteinaceous tegument layer connected to an icosahedral capsid. The major capsid protein, VP5 (149 kDa), makes up both types of capsomere, pentons and hexons. Limited trypsin digestion of VP5 identified a single stable 65 kDa fragment which represents a proposed protein folding nucleus. We report the 2.9 A crystal structure of this fragment and its modeling into an 8.5 A resolution electron cryomicroscopy map of the HSV-1 capsid. The structure, the first for any capsid protein from Herpesviridae, revealed a novel fold, placing herpesviruses outside any of the structurally linked viral groupings. Alterations in the geometrical arrangements of the VP5 subunits in the capsomeres exposes different residues, resulting in the differential association of the tegument and VP26 with the pentons and hexons, respectively. The rearrangements of VP5 subunits required to form both pentavalent and hexavalent capsomeres result in structures that exhibit very different electrostatic properties. These differences may mediate the binding and release of other structural proteins during capsid maturation.
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PMID:Structure of the herpesvirus major capsid protein. 1257 12

Chemical modification of the proteins bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin and chicken lysozyme by 3-hydroxyphthalic anhydride (3-HP) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. Of the three enveloped viruses tested, human herpes simplex virus type 1 (HSV-1), bovine parainfluenza virus type 3 and porcine respiratory corona virus, only HSV-1 proved sensitive to the 3-HP-proteins. All of the chemically modified proteins presented antiviral activity against HSV-1 when assayed before, during or after infection. However, to achieve HSV-1 inhibition, significantly higher concentrations of the modified proteins were required if present before infection as compared to during or after infection. Our results suggest that multiple mechanisms are involved in the inhibition of HSV-1 infection. Proteolytical digestion of albumin, alpha-lactalbumin, beta-lactoglobulin and lysozyme by trypsin, chymotrypsin and pepsin yielded several peptide fragments with antiherpetic activity. Chemical modification of these peptide fragments by 3-HP generated peptides with antiviral activity, however, this was almost always combined with a cytotoxic effect on the Vero cells. Overall, our results suggest that targeted chemical modification of some natural products might provide compounds effective against HSV-1 infection.
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PMID:The antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification. 1283 57

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.
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PMID:Proteins of purified Epstein-Barr virus. 1553 16

Enterococcus mundtii ST4V, isolated from soya beans, produces a 3950Da antibacterial peptide active against Gram-positive and Gram-negative bacteria, including Enterococcus faecalis, Streptococcus spp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus. The peptide also inactivated the herpes simplex viruses HSV-1 (strain F) and HSV-2 (strain G), a polio virus (PV3, strain Sabin) and a measles virus (strain MV/BRAZIL/001/91, an attenuated strain of MV). MV, HSV-1 and HSV-2 were 95.5%-99.9% inactivated by peptide ST4V at 400 microg/ml. Monkey kidney Vero cells were not inactivated, even at four times the level peptide ST4V displayed antiviral activity, indicating that the effect was not due to cytotoxicity. Complete inactivation or significant reduction in antimicrobial activity was observed after treatment of peptide ST4V with Proteinase K, pronase, pepsin and trypsin. No change in antimicrobial activity was recorded after treatment with alpha-amylase, suggesting that peptide ST4V was not glycosylated. This is the first description of an antibacterial and antiviral peptide with such broad-spectrum of activity, produced by a lactic acid bacterium.
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PMID:An antibacterial and antiviral peptide produced by Enterococcus mundtii ST4V isolated from soya beans. 1586 68

UL25 is one of seven herpes simplex virus-encoded proteins involved specifically in DNA encapsidation. Its role appears to be to stabilize the capsid so that DNA is prevented from escaping once it has entered. To clarify the function of UL25, we have examined capsids with the goal of defining where it is located. Analysis of trypsin-treated capsids showed that UL25 is sensitive to cleavage like other proteins such as the major capsid and portal proteins that are exposed on the capsid surface. Internal proteins such as the scaffolding protein and protease were not affected under the same experimental conditions. Capsids were also examined by electron microscopy after staining with gold-labeled antibody specific for UL25. Images of stained capsids demonstrated that most labeled sites (71% in C capsids) were at capsid vertices, and most stained C capsids had label at more than one vertex. A quantitative immunoblotting method showed that the capsid contents of UL25 were 56, 20, and 75 copies per capsid in A, B, and C capsids, respectively. Finally, soluble UL25 protein was found to bind in vitro to purified capsids lacking it. The amount of bound UL25 corresponded to the amount present in B capsids, and bound UL25 was found by immunoelectron microscopy to be located predominantly at the capsid vertices. The results are interpreted to suggest that five UL25 molecules are found at or near each of the capsid vertices, where they are exposed on the capsid surface. Exposure on the surface is consistent with the view that UL25 is added to the capsid as DNA is packaged or during late stages of the packaging process.
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PMID:Herpes simplex virus capsid structure: DNA packaging protein UL25 is located on the external surface of the capsid near the vertices. 1677 16

Studies to localize the herpes simplex virus 1 portal protein encoded by UL6, the putative terminase components encoded by UL15, UL 28, and UL33, the minor capsid proteins encoded by UL17, and the major scaffold protein ICP35 were conducted. ICP35 in B capsids was more resistant to trypsin digestion of intact capsids than pUL6, pUL15, pUL17, pUL28, or pUL33. ICP35 required sectioning of otherwise intact embedded capsids for immunoreactivity, whereas embedding and/or sectioning decreased the immunoreactivities of pUL6, pUL17, pUL28, and pUL33. Epitopes of pUL15 were recognized roughly equally well in both sectioned and unsectioned capsids. These data indicate that pUL6, pUL17, pUL28, pUL33, and at least some portion of pUL15 are located at the external surface of the capsid.
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PMID:Herpes simplex virus 1 DNA packaging proteins encoded by UL6, UL15, UL17, UL28, and UL33 are located on the external surface of the viral capsid. 1692 Aug 25


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