Localization of the antigenic sites and intrinsic protein kinase domain within a 300 amino acid segment of the ribonucleotide reductase large subunit from herpes simplex virus type 2.
(1992)
Journal - Virology (UNITED STATES )
Abstract :
The 140-kDa ribonucleotide reductase (RR1) protein of herpes simplex virus type 2 (HSV-2) functions as the large subunit of virus-specified RR1 and exhibits an intrinsic protein kinase (PK) activity at its unique NH2-terminal region. The N-terminal half of RR1 contains the protein and DNA functions of the morphological transforming region III (mtrIII) of HSV-2. In the present study, we have expressed a number of truncated RR1 derivatives in a mammalian expression vector containing NH2-terminal RR1 gene fragments and amber mutants generated by site-specific mutagenesis. These derivatives, synthesized in transient expression assays, were used as test antigens to localize the epitopes of a panel of HSV-2 RR1-reactive monoclonal antibodies and to fine-map the PK catalytic domain. Our data show that the epitope for HSV-2-specific monoclonal antibody 6A-6 is located in a region of RR1 protein spanning aa 72-350. The epitopes for cross-reactive antibodies to HSV RR1, i.e., 48S and 51S, are formed predominantly by a stretch of amino acid residues specified by aa 350-376 of the RR1 molecule. The 6A-6 antibody utilized in conjunction with the RR1 amber mutants has allowed us to define a 278 aa domain within the NH2-terminal half of the 140-kDa RR1 (aa 72-350) that is sufficient for PK activity.
| ISSN : | 0042-6822 |
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| Mesh Heading : | Base Sequence Cloning, Molecular Epitopes Microscopy, Fluorescence Molecular Sequence Data Mutagenesis, Site-Directed Protein Kinases Restriction Mapping Ribonucleotide Reductases Simplexvirus genetics immunology genetics genetics enzymology genetics |
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| Mesh Heading Relevant : | immunology immunology immunology |
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Serologic specificity of antibodies to herpes simplex virus glycoprotein B present in human sera. Analysis by transient expression of glycoprotein B-derivatives.
(1990)
Journal - Archives of virology (AUSTRIA )
Abstract :
Glycoprotein B (gB) is an essential glycoprotein of herpes simplex virus (HSV) and a major target for cellular and humoral immune response in the infected host. In the present study, we have analyzed the pattern of reactivity of a panel of 23 HSV-seropositive patient sera using as test antigens gB derivatives made in COS cells in a transient expression assay. Our results show that nearly all the sera tested, reacted with wild-type gB or tgB (772) (that lacks 102 amino acids cytoplasmic domain). However, when tgB (477 amino acids) or gBdl (an inframe deletion between amino acids 477-772) were used as test antigens only 12 out of 23 sera tested positive. Further analysis using competition assays revealed that these sera can be classified into at least two groups: (i) that contain gB-reactive antibodies reactive to intact gB or tgB (772); (ii) that contain antibodies that recognize all forms of gB-derivatives tested. The results presented here underscore the potential limitations in using certain truncated forms of gB as antigens for subunit vaccine or in the serodiagnosis of HSV infection.
| ISSN : | 0304-8608 |
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| Mesh Heading : | Antibodies, Viral Antigens, Viral Cell Line Genes, Viral Herpes Simplex Humans Peptide Fragments Plasmids Radioimmunoprecipitation Assay Simplexvirus Transfection Viral Envelope Proteins immunology immunology immunology genetics |
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| Mesh Heading Relevant : | Antibody Specificity immunology immunology immunology |
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Oligomerization of herpes simplex virus glycoprotein B occurs in the endoplasmic reticulum and a 102 amino acid cytosolic domain is dispensable for dimer assembly.
(1990)
Journal - Virology (UNITED STATES )
Abstract :
Glycoprotein B (gB) is an essential protein specified by herpes simplex virus and a major envelope component of the virions. It is known to assemble into noncovalently associated dimers. The aim of this study was to investigate the role of topogenic domains of gB in dimer assembly and the intracellular location at which gB dimers are assembled. Therefore, dimer analyses were performed on intact gB and its three COOH-terminus-truncated gB derivatives encoding NH2-terminal 772, 586, and 477 amino acids (aa) of the mature gB, using SDS-polyacrylamide gel electrophoresis and sucrose gradient assays. Dimers were detected in gB and in tgB(772 aa), but were absent from tgB(586 aa) and from tgB(477 aa). These results showed that a 102 aa cytosolic domain (aa 773-874) is not required for the assembly of gB dimers. In addition, using endoglycosidase H treatment and dimer analysis of gB synthesized during 7 min pulse-labeling period, we have demonstrated that ER is the subcellular organelle at which gB monomers are assembled into dimeric forms.
| ISSN : | 0042-6822 |
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| Mesh Heading : | Amino Acids Cells, Cultured Cloning, Molecular Cytosol Endoplasmic Reticulum Gene Expression Humans Mutation Protein Conformation Simplexvirus Viral Envelope Proteins analysis chemistry genetics |
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| Mesh Heading Relevant : | metabolism analysis metabolism |
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