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)

Glycoprotein D (gD) of herpes simplex virus contains three utilized sites (Asn-X-Ser/Thr) for addition of asparagine-linked carbohydrates (N-CHO). Previously, we used oligonucleotide-directed mutagenesis to alter serine or threonine residues to alanine at each N-CHO addition site. Studies with monoclonal antibodies showed that a mutant protein lacking all three sites (now designated AAA) was structurally altered because of the amino acid change at residue 96 as well as the absence of the N-CHO. In this study, we constructed additional single mutations at site 1 (residues 94 and 96) and found that in most cases, the amino acid change itself adversely affected the conformation of gD. However, changing asparagine 94 to glutamine (Q) at site 1 had the least effect on gD. We constructed a second triple mutant, QAA, which lacked all three N-CHO signals. The antigenic conformation of QAA was similar to that of gD produced in the presence of tunicamycin (TM-gD). However, binding of MAbs to the AAA protein or to single mutants altered at site 1 was reduced compared with TM-gD. Wild-type gD and QAA proteins were equally susceptible to digestion by trypsin or Staphylococcus aureus V8 protease. In contrast, the AAA protein was more sensitive to trypsin but less sensitive to V8, again suggesting conformational alterations of the AAA protein. Despite what appeared to be large changes in structure, each mutant complemented the infectivity of a virus lacking gD (F-gD beta). We conclude that the N-CHO and amino acids at N-CHO site 1 play an important role in forming and/or maintaining gD structure, but none of the N-CHO are required for gD to function in the complementation assay.
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PMID:Absence of asparagine-linked oligosaccharides from glycoprotein D of herpes simplex virus type 1 results in a structurally altered but biologically active protein. 164 38

Work done in our laboratories, using a murine model, indicates that suppression of host immune responses might be due to secretion of soluble factors by tumor cells. The H238 cells (BALB/c embryonic fibroblasts transformed by UV-inactivated herpes simplex virus Type 2) exhibit progressive tumor growth with subsequent decrease in lymphoproliferation. To further study the suppressive effects of a tumor, H238 conditioned medium (CM) was tested for its ability to block murine and human mitogenic and allogeneic lymphocyte responses. PHA, Con A and LPS were used as mitogens. Lymphoproliferation, in the presence of increasing amounts of H238 CM, resulted in a greater degree of suppression of [3H]thymidine ([3H]Tdr) uptake, in both human and mouse systems. The kinetics of proliferation in the presence of concentrated H238 CM (cCM) showed that depression was evident regardless of the time of cCM addition, thereby affecting it at any stage of the cell cycle. Treatment of H238 cCM using acid (pH 2.3), base (pH 9.6), trypsin (100 micrograms/ml), heat (56 degrees C, 100 degrees C) and freeze-thawing, restored PHA-stimulated lymphoproliferation. Dialysis of H238 cCM showed that the molecular weight of the suppressor lies between 15 and 25 kDa. Northern blot analysis demonstrated the presence of a TGF-beta transcript in H238 cells. Neutralization of the H238 cCM with monoclonal antibody to TGF-beta resulted in complete abrogation of suppressive activity in spleen cell lymphoblastogenesis. These results suggest that TGF-beta appears to be the main inhibitor of immune responses found in this HSV-2-induced murine tumor cell line. Such tumor-induced modulations may contribute to the outcome of immunotherapy in the tumor-bearing host.
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PMID:Suppression of immune responses by herpes virus type 2-transformed murine tumor cells. 166 30

Direct virus inactivation of tachyplesin I and related isopeptides, which are antimicrobial peptides isolated from the hemocytes of the horseshoe crab (Tachypleus tridentatus and Limulus polyphemus), was examined against several viruses. Vesicular stomatitis virus (VSV) was inactivated by incubation with tachyplesin I and its isopeptides. Influenza A (H1N1) virus was slightly inactivated by tachyplesin I, whereas herpes simplex virus 1 and 2, adenovirus 1, reovirus 2 and poliovirus 1 were resistant to inactivation. The inactivation of VSV by tachyplesin I depended on the concentration, the time and the temperature of incubation. Pretreatment of tachyplesin I with trypsin or lipopolysaccharide of gram-negative bacteria entirely abolished the antiviral activity. Electron microscopy of VSV treated with tachyplesin I showed naked and damaged virions. These data suggest that tachyplesin I directly inactivates the VSV by destroying its envelope subunits.
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PMID:Direct virus inactivation of tachyplesin I and its isopeptides from horseshoe crab hemocytes. 166 45

Translation of in vitro-synthesized herpes simplex virus type 2 (HSV-2) gG-2 mRNA in a reticulocyte lysate system was used to study the processing of HSV-2 gG-2. In the presence of canine pancreatic microsomal membranes, a single species that is protected from trypsin digestion was detected. This product comigrates with the 104,000-Mr (104K) high mannose intermediate seen in HSV-2-infected-cell lysates. Endo-beta-N-acetylglucosaminidase H treatment of the in vitro-synthesized 104K protein yielded a single product migrating at 100 K. The 72K and 31K cleavage products of gG-2 were not observed in the in vitro system. These data show that the molecular weight of the nonglycosylated form of the gG-2 protein is 100,000 and that the cotranslational processing of this protein in the endoplasmic reticulum yields the 104K high-mannose intermediate.
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PMID:In vitro synthesis and processing of herpes simplex virus type 2 gG-2, using cell-free transcription and translation systems. 215 14

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.
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PMID:Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8). 215 71

Although the mortality rate after herpes simplex virus type 2 inoculation was not significantly different between pregnant mice and nonpregnant mice, systemic interferon production was very high during late pregnancy compared with that in nonpregnant mice. Antiviral activity was detected in placentas from all noninfected pregnant mice (80 to 320 U/ml in 20% suspension). The antiviral activity had a broad spectrum and was also effective in the cells of other species; an antiviral effect was shown even if the cells were treated after challenge with a virus. In addition, this activity was not inactivated by antimouse interferon-neutralizing antisera. The molecular weight of this placental antiviral substance was estimated to be 200,000 to 450,000 daltons by gel filtration, and it was inactivated by heat, acid, and trypsin. Noninterferon antiviral activity (40 to 80 U/ml) was also detected in more than half the sera (61.5%) of noninfected mice in late pregnancy.
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PMID:Study of interferon production during pregnancy in mice and antiviral activity in the placenta. 241 60

Herpes simplex virus type 1 was purified by density gradient centrifugation, and the virion-associated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By Western blot (immunoblot) analysis with an anti-ICP4 monospecific serum, the results indicated that ICP4, one of the five immediate-early proteins of herpes simplex virus type 1, was associated with the purified virions. To define the location of ICP4 within the virion, trypsin digestion experiments were performed. Purified virions were treated with trypsin in the presence or absence of detergent. The virus envelope appeared to protect ICP4 from the trypsin, since virus-associated ICP4 was sensitive to digestion only after detergent treatment. In addition, ICP4 remained associated with the virus particle when the virion-specific glycoproteins were removed after detergent treatment. Finally, ICP4 was not detected in purified preparations of type A and B capsids isolated from the nuclear fraction of virus-infected cells. The above-mentioned data suggest that detectable amounts of ICP4 are present within the tegument region of the virion.
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PMID:A major transcriptional regulatory protein (ICP4) of herpes simplex virus type 1 is associated with purified virions. 254 9

A method of in situ hybridization with biotinylated probes is described for specific detection of varicella-zoster virus (VZV) DNA in infected cell cultures and in human biopsied or autopsied materials. From molecularly cloned EcoRI-fragments of VZV DNA, we selected as probes for in situ hybridization three fragments (B, H, and K) which had any detectable homology neither with human cell DNA nor with DNAs of VZV-related viruses (herpes simplex virus type 1, type 2 and human cytomegalovirus). The procedure of in situ hybridization was based on that of Brigati et al. (Virology 126, 32-50, 1983), but following modifications were made. 1) The concentration of probe DNA was lowered to 100 ng per ml. 2) Streptavidin-biotin system was used for detection of hybridization. 3) Acid phosphatase was used to generate color development discernible from melanin present in skin. 4) Before hybridization, deparaffinized sections were treated with trypsin by the procedure routinely employed for detection of viral antigens in paraffin-embedded sections.
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PMID:Detection of varicella-zoster virus DNA in cultured cells and tissue specimens by in situ hybridization using biotin-labeled probes. 283 43

An antiserum was produced to the oligomeric form of glycoprotein B (gB) induced by herpes simplex virus type 1 (HSV-1) strain 17. This antiserum gave a single common precipitin line in agar gel immunodiffusion with HSV-1, HSV-2, bovine mammillitis virus (BMV) and equine herpesvirus type 1 (EHV-1). It also neutralized HSV-1, HSV-2 and BMV but not EHV-1. Absorption of the antiserum with excess HSV-2 or BMV antigen resulted in an HSV-1-specific neutralizing antiserum. In immunoprecipitation, two proteins, gB and pgB, were precipitated from HSV-1- and HSV-2-infected cells and at least three from BMV- and EHV-1-infected cells. Glycoprotein B and pgB of three HSV-1 and three HSV-2 strains and the corresponding antigenically related glycoproteins of BMV- and EHV-1-infected cells were labelled with 125I, digested with trypsin and the resulting peptides separated by two-dimensional thin-layer chromatography or high-pressure liquid chromatography. The resulting profiles were found to be almost identical, suggesting considerable structural conservation of the peptide backbone of the antigenically related glycoproteins of these four viruses.
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PMID:Antigenic and biochemical analysis of gB of herpes simplex virus type 1 and type 2 and of cross-reacting glycoproteins induced by bovine mammillitis virus and equine herpesvirus type 1. 298 66

Herpes simplex virus type 1 (HSV-1) infection induces the appearance of viral analogues of human Fc IgG and C3 receptors on the surface of human cells. The virally induced C3 receptor(s) has been broadly defined as a C3b receptor, but its ligand binding characteristics have not been rigorously defined. In this study, human epidermal cells, A431 cells, and human umbilical vein endothelial cells infected with HSV-1 demonstrated rosetting with sheep erythrocytes (E) coated with IgG (E-IgG) or the complement components C3b (EAC3b) or iC3b (EAC3bi), but not with E-IgM, C4 (EAC14), C3d (EAC3d), or E alone. Rosetting was markedly enhanced by pretreatment of HSV-1-infected cells with neuraminidase. Unlike human C3 receptors, the HSV-1-induced C3 receptor was found to be trypsin resistant. To determine whether HSV-1 induced CR1-like receptors or CR3-like receptors, infected cells were pretreated with EDTA, which is known to inhibit native CR3 function. EDTA failed to prevent rosetting with EAC3bi. Furthermore, blocking studies using monoclonal antibodies against CR1 and CR3 revealed that the anti-CR1 antibody 5C11 consistently blocked EAC3b and EAC3bi rosetting with HSV-1-infected cells in a dose dependent manner, but monoclonal antibodies against CR3 did not. This study indicates that the HSV-1-induced C3 receptor is an analogue of CR1.
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PMID:Characterization of the C3 receptor induced by herpes simplex virus type 1 infection of human epidermal, endothelial, and A431 cells. 302 70


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