Characterization of proteins induced by Herpesvirus saimiri: comparative immunoprecipitation and analysis of glycosylation.
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S Modrow
Publications and source records attributed to S Modrow.
Characterization of Herpesvirus saimiri and Herpesvirus ateles structural proteins.
The structural proteins of Herpesvirus saimiri strains 11 and 11 att and of Herpesvirus ateles strains 73 and 810 were characterized by electrophoresis in SDS-polyacrylamide gels. For H. saimiri 21 virus structural proteins could be identified with molecular weights ranging from 28,000 to 210,000 Da. For H. ateles 810 and H. ateles 73, 20 polypeptides were characterized. Using lactoperoxidase for iodination of surface proteins and immunoprecipitation, 5 polypeptides could be identified as envelope and 4 as capsid surface proteins.
Capsid↗
Herpesvirus saimiri-induced proteins in lytically infected cells. I. Time-ordered synthesis.
The addition of TPA (phorbol-12-myristate-13-acetate) to cultures during the lytic infection with herpesvirus saimiri led to an enhanced and accelerated production of polypeptides induced by H. saimiri and to a rapid shut-down of host cell protein synthesis and allowed a detailed analysis of the protein patterns. Analysis of sequential protein synthesis in owl monkey kidney cells lytically infected with H. saimiri 11 permitted the identification of 31 virus-induced polypeptides. The use of the amino acid analogues canavanine (for arginine) and azetidine (for proline) in parallel allowed experiments on the identification of proteins synthesized early and late during lytic infection.
Animals↗
Immunoprecipitation as a tool for studying humoral immunity of natural and experimental hosts of Herpesvirus saimiri.
Herpesvirus saimiri strain 11 and attenuated H. saimiri strain 11 proteins synthesized during the lytic cycle of virus replication were used for immunoprecipitation with various sera from natural (Saimiri sciureus) and experimental (Saguinus nigricollis, Saguinus fuscicollis, Aotus trivirgatus, New Zealand White rabbits) hosts. The analysis of the precipitates separated in sodium dodecyl sulfate:polyacrylamide gels revealed that in tumor-developing animals a specific set of viral polypeptides were precipitated, which were not precipitated by sera obtained from the natural host Saimiri sciureus. Using labeled proteins from H. saimiri 11 and its attenuated strain, respectively, a difference was shown after precipitation with a serum raised against infected cell proteins of H. saimiri 11.
Animals↗
Structural and immunological reactivity of the principal neutralizing determinant V3 of glycoprotein gp120 of HIV-1.
The variable domain V3 in the outer glycoprotein gp120 of HIV-1 is a highly important region with respect to immune response during the course of viral infection. Neutralizing antibodies are produced against this domain: in addition, it has been shown to be a functionally active epitope for T helper and cytotoxic T cells. The high degree of amino acid variability in individual HIV-isolates, however, limits the use of the V3-domain in approaches to vaccine development. In order to characterize the residues important for antibody interaction and binding to MHC class I proteins, we constructed a consensus sequence of the V3-domain with broad reactivity [1] and used synthetic peptides derived from this consensus sequence with individual residues altered to alanine. These peptides were used as antigens in ELISA tests to define the amino acids which are important for binding to human and rabbit/anti-peptide immunoglobulins. In addition, we used these alanine-derived peptides in interaction studies with human HLA-A2.1 and mouse H-2Dd by testing their capacity to stabilize the respective MHC class I protein complexes on the surface of mutant cell lines T2 and RMA-S transfected with Dd gene. The experimental tests allowed us to define individual residues involved in antibody and MHC-protein interaction, respectively. In a further approach, we used those results to design interaction models with HLA-A2.1 and H-2Dd. Therefore, a structural model for H-2Dd was built that exhibits an overall similar conformation to the parental crystal structure of HLA-A2.1. The resulting interaction models show V3-peptide bound in an extended beta-conformation with a bulge in its centre for both H-2Dd and HLA-A2.1 complexes. The N- and C-termini of V3 peptide reside in conserved pockets within both MHC-proteins. Anchoring residues could be determined that are crucial for the binding of the respective MHC class I haplotype. The cross-reactivity of V3-peptide in enhancing the expression of two different MHC class I molecules (H-2Dd and HLA-A2.1) is shown to be based on similar peptide binding that induces an almost identical peptide conformation.
AIDS Vaccines↗