Infection in vitro, this protein was tested for clinical efficacy in HIV-1-infected individuals; however, no effect on plasma viral loads was observed [13]. Further examination revealed that doses of sCD4 that were significantly higher than those achieved in the clinical trial were required to neutralize primary clinical isolates of HIV-1, in contrast to the relatively sensitive, laboratory-adapte
Infection in vitro, this protein was tested for clinical efficacy in HIV-1-infected individuals; however, no effect on plasma viral loads was observed [13]. Further examination revealed that doses of sCD4 that were significantly higher than those achieved in the clinical trial were required to neutralize primary clinical isolates of HIV-1, in contrast to the relatively sensitive, laboratory-adapte
Recombinant HIV-1 viruses that express the luciferase reporter gene were generated by transfection of 293T cells using the calcium phosphate transfection method (Promega). Cells were seeded in 100-mm tissue culture dishes (approximately 46106 cells per dish) and transfected the next day with 10 mg of the HIV-1 packaging construct pCMVDP1DenvpA [50], 10 mg of the firefly luciferase-expressing const
N the exposure of the coreceptor-binding site on gp120 and the HR1 coiled coil on gp41 after pulse activation by sCD4. To measure the progression of conformational changes that immediately follow HIV-1 envelope glycoprotein activation, we developed a novel enzyme-linked immunosorbent assay (ELISA)based system that utilizes live cells as a platform for expression of membrane-bound trimeric envelope
Amic changes in conformation and function of the HIV-1 envelope glycoproteins, immediately after engagement of the activating molecules. Using these tools, we found that SCMs inactivate envelope glycoprotein function by an activation-triggered inhibition process, through induction of a metastable activated state.Materials and Methods Reagents and AntibodiesFour-domain sCD4 (molecular weight 50 kDa
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GCl2, 25 mM Tris, pH 7.5 and 140 mM NaCl). For pulse activation experiments, the COS-1 cells were incubated with sCD4 (40 mg/ml, 0.8 mM) or 191 (360 mM) suspended in blocking buffer for three minutes, washed three times with blocking buffer and incubated for different time periods until the C34-Ig or 48d antibodies were added (at 40 mg/ml or 1 mg/ml, respectively, for 30 minutes). To study the tem
Washing buffer (140 mM NaCl, 1.8 mM CaCl2, 1 mM MgCl2 and 20 mM Tris, pH 7.5). A horseradish peroxidase-conjugated antibody specific for the Fc region of human IgG was then incubated with the samples for 45 minutes at room temperature. Cells were washed 5 times with blocking buffer and 5 times with washing buffer. HRP enzyme activity was determined after the addition of 33 ml per well of a 1:1 mix