Real-time DNA binding measurements of the ETS1 recombinant oncoproteins reveal significant kinetic differences between the p42 and p51 isoforms.

The sequence-specific DNA binding of recombinant p42 and p51 ETS1 oncoprotein was examined quantitatively to determine whether the loss of the Exon VII phosphorylation domain in p42 ETS1 or the phosphorylation of expressed Exon VII in p51 ETS1 had an effect on DNA binding activity. The kinetics of sequence-specific DNA binding was measured using real-time changes in surface plasmon resonance with BIAcore (registered trademark, Pharmacia Biosensor) technology. The real-time binding of p42 and p51 ETS1 displayed significant differences in kinetic behavior. p51 ETS1 is characterized by a fast initial binding and conversion to a stable complex, whereas p42 ETS1 exhibits a slow initial binding and conversion to a stable complex. All of the p51 ETS1 DNA binding states are characterized by rapid turnover, whereas the p42 ETS1 DNA binding states are 4-20 times more stable. A model describing these kinetic steps is presented. Stoichiometric titrations of either p42 or p51 ETS1 with specific oligonucleotides show 1:1 complex formation. The DNA sequence specificity of the p42 and p51 ETS1 as determined by mutational analysis was similar. The in vitro phosphorylation of p51 ETS1 by CAM kinase II obliterates its binding to specific DNA, suggesting that the regulation of p51 ETS1 sequence-specific DNA binding occurs through phosphorylation by a calcium-dependent second messenger. The p42 ETS1 lacks this regulatory domain (Exon VII), and binding to its specific DNA sequence is not sensitive to calcium signaling.

Heterogeneity of Nef proteins in cells infected with human immunodeficiency virus type 1.

Human T-lymphocytic cell line H9 infected with the HTLV-IIIB isolate of human immunodeficiency virus type 1 (HIV-1) synthesizes two forms of the Nef protein (p25 and p27) that differ both in molecular weight and charge. Different subpopulations of viruses were isolated from the HTLV-IIIB stock which induce expression of only p25 or p27. Cells infected with HIV-1 derived from the HXB3 clone of the HTLV-IIIB isolate made only the p25 species, whereas the 8E5/LAV cell line which harbors a single defective LAV provirus produces only the p27 species. These findings are consistent with the notion that the HTLV-IIIB isolate consists of at least two distinct variants with different nef genes, one specifying p25 and the other encoding p27. After a considerable number of passages in culture, H9 cells chronically infected with the HTLV-IIIB isolate produced high levels of p25 and lower levels of p27. Passages in culture appear to select for a subpopulation of virus variants that specify high levels of p25 Nef expression.

Comparative analysis of gp41 antigens by enzyme-linked immunosorbent assays for detecting antibodies to human immunodeficiency virus type 1.

We have compared the antigenic qualities of human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein with a synthetic oligopeptide (peptide R21S) and a bacterially synthesized protein (protein 566), which are homologous with the N-terminal region of gp41, in enzyme-linked immunosorbent assays (ELISA) for detecting antibodies to HIV-1 in sera of patients with the acquired immunodeficiency syndrome (AIDS) or the aids-related complex (ARC). Although the use of all three types of antigens readily allowed the detection of antibodies in human sera, ELISA employing purified gp41 glycoprotein and the protein 566 were more specific and sensitive than the peptide R21S ELISA.

Partial purification of native HIV transmembrane protein gp41: generation of polyclonal and monoclonal antibodies.

We partially purified the human immunodeficiency virus (HIV) glycoprotein gp41 from infected H9 cells by immunoaffinity chromatography using a column containing the M25 monoclonal antibody (diMarzo-Veronese et al., 1985). A pH 11.5 buffer worked best for eluting the glycoprotein from this column. The eluted gp41 was used in a sensitive slot blot immunoassay to detect antibodies to HIV in human sera and to prepare rabbit polyclonal antibodies and the 41-1S mouse monoclonal antibody. These antibodies reacted with gp41 in immunoprecipitation and in Western blot assays, but did not neutralize HIV in a syncytium-forming microassay. A pH 2.5 buffer was found to be the most effective solution for eluting gp41 from a 41-1S monoclonal antibody column.

Enzyme-linked immunosorbent assay for determination of antibodies against herpes simplex virus types 1 and 2 in human sera.

A rapid and reproducible enzyme-linked immunosorbent assay (ELISA) is described for determining antibodies in human sera against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2). The sera were absorbed for 30 min with heterologous virus-infected-cell extracts to remove cross-reacting antibodies and then were applied to ELISA plates containing the target antigens, immunoaffinity-purified HSV-1 glycoproteins gC and gD and HSV-2 glycoproteins gD and gF. The absorbance index, defined as the ratio of A414 generated by a serum sample absorbed with a heterologous virus-infected-cell extract versus the A414 of a serum sample absorbed with an uninfected-cell extract, was used to determine the presence or absence of antibodies to HSV-1 and HSV-2. Results of the ELISA for detecting antibodies against HSV-2, when compared with results obtained for the same sera by the microneutralization test, showed an index of overall agreement of 91%. Results of the ELISA for detecting antibodies against HSV-1, when compared with microneutralization test results for sera negative for HSV-2 antibodies but positive for HSV antibodies by ELISA, showed an index of agreement of 99%.

Detection in antisera of antibodies that cross-react with herpes simplex virus type 1 glycoprotein gC.

Herpes simplex virus type 1 (HSV-1) glycoprotein gC was purified by affinity chromatography with an immunosorbent column containing monoclonal antibody to HSV-1 gC, and its reactivity with rabbit antisera was measured by enzyme-linked immunosorbent assay and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of radioimmunoprecipitates. Positive reactions were detected between HSV-1 gC and rabbit hyperimmune antisera to both HSV-1 and HSV-2. Electrophoretic analysis also revealed reactivity between the rabbit antisera and peptides of HSV-1 gC generated by partial digestion with trypsin. These findings indicate that HSV-1 gC has one or more cross-reactive or type-common determinants that are readily detected, and therefore, the presence of antibodies reacting with HSV-1 gC in sera may not necessarily be indicative of an earlier infection with HSV-1.

Herpes simplex virus type 2 glycoprotein gF and type 1 glycoprotein gC have related antigenic determinants.

The 104-S monoclonal antibody immunoprecipitated from herpes simplex virus type 2 (HSV-2)-infected cell extracts the 75,000-molecular-weight glycoprotein gF and its 65,000-molecular-weight precursor (pgF). The precursor pgF was sensitive to endoglycosidase H digestion, indicating the presence of high mannose-type oligosaccharides, whereas the stable gF product was sensitive to neuraminidase digestion, indicating the presence of sialic acid residues. The 104-S antibody also weakly precipitated the 130,000-molecular-weight herpes simplex virus type 1 (HSV-1) glycoprotein gC from both infected cell extracts and purified preparations obtained through the use of monoclonal antibody-containing immunoadsorbent columns. Immunofluorescence tests demonstrated that the 104-S antibody reacted with antigen present in cells infected with HSV-2 strain 333 and HSV-1 strain 14012 but not with antigen present in cells infected with HSV-1 strain MP, a strain deficient in HSV-1 gC production. These findings indicate that HSV-1 gC and HSV-2 gF have antigenic determinants that are related.