Of the inhibiting ligands examined, only the sCD4 neutralization-to-binding ratio correlated with sensitivity to cold. efficiency with which the envelope glycoproteins interact with CD4, is usually shown for comparison because some of the correlation analyses utilized this parameter. (B) Cell-surface immunoprecipitation of envelope glycoproteins. COS-1 cells were transfected with the indicated envelope glycoproteins and labeled with 35S-cysteine/methionine. Two days after transfection, cells were incubated with the 2G12 antibody (1 g/ml) in binding buffer (PBS made up of 3% BSA) for 30 min at room heat. Cells were then washed three times with binding buffer and lysed using NP-40 buffer (0.5 M NaCl, 10 mM Tris, pH 7.5 and 0.5% [vol/vol] NP-40). Envelope glycoprotein-2G12 complexes were precipitated with Protein A-Sepharose beads and analyzed by SDS-PAGE. The envelope glycoprotein Amyloid b-Peptide (1-43) (human) bands around the gel were detected by PhosphorImager. (C) Virion incorporation of envelope glycoproteins. Viruses made up of the indicated Amyloid b-Peptide (1-43) (human) envelope glycoprotein variants were generated and purified by ultracentrifugation through a 20% sucrose cushion. The control viruses produced in the pcDNA-transfected cells do not contain envelope glycoproteins. Pellets were then resuspended in PBS and virion content determined by reverse transcriptase (RT) activity. Samples were analyzed by SDS-PAGE (gel loading normalized by RT activity) and Western blotted using pooled serum obtained from HIV-1 infected individuals. The positions of the p24 capsid protein and gp120 envelope glycoprotein are indicated. Results are representative of those obtained Rabbit Polyclonal to STAT5A/B in two impartial experiments.(TIF) ppat.1002101.s001.tif (2.2M) GUID:?873B1D72-CDC9-4801-BA51-3A1267C93995 Figure S2: Envelope Glycoprotein binding and neutralization by the trimer-specific antibody PG16. (A) Binding of PG16 (0.2 g/ml) to COS-1 cells expressing the indicated envelope glycoproteins at 4C in the absence and presence of sCD4 (20 g/ml). Data are offered as mean percentage of PG16 binding ( SEM) relative to the binding of the 2G12 antibody (2 g/ml) to each envelope glycoprotein variant; the data were derived from duplicate experiments. (B) Neutralization of viruses made up of the indicated envelope glycoprotein variants by the PG16 antibody. Residual contamination represents the percentage of contamination measured after incubation with the indicated concentration of the PG16 antibody relative to that observed in the absence of antibody. The envelope glycoproteins are color coded according to their relative CD4-impartial infectivity (observe left column in Physique 4A). Data symbolize the means derived from three replicate samples.(TIF) ppat.1002101.s002.tif (712K) GUID:?62C56C72-FBD8-4C2B-B0B6-AAE1772F01C4 Physique S3: Contamination of CD4?CCR5+ and CD4+CCR5+ cells by viruses that contain a deletion of the gp120 V1/V2 loops (V) and/or the gp41 changes. The mean luciferase activity ( SEM) from an experiment performed with three replicate samples is usually offered (37,500 RT models added per well). In the bottom panel, contamination of CD4?CCR5+ cells is usually expressed as the percentage of infection of CD4+CCR5+ cells.(TIF) ppat.1002101.s003.tif (752K) GUID:?5A26889E-5AFB-488A-BF1C-A72CCCB88C33 Figure S4: Exposure of the gp41 HR1 coiled coil around the HIV-1 envelope glycoproteins. (A) Effect of envelope glycoprotein cytoplasmic tail deletion on contamination of CD4?CCR5+ and CD4+CCR5+ cells. Viruses that express the luciferase gene and contain the indicated full-length or cytoplasmic tail-deleted (ct) envelope glycoproteins were generated and RT activity measured. Virus preparations were incubated with CD4?CCR5+ or CD4+CCR5+ cells (10,000 RT models per well). The mean luciferase activity ( SEM) from an experiment performed with three replicate samples is usually presented. (B) Effect of heat on binding of C34-Ig to cell surface-expressed envelope glycoproteins. COS-1 cells transiently expressing the indicated envelope glycoproteins were incubated with sCD4 (40 g/ml) or buffer for 3 min at 37C. Samples were subsequently washed three Amyloid b-Peptide (1-43) (human) times to remove extra sCD4 and then incubated with C34-Ig (40 g/ml) for 30 min at the indicated heat. Binding of C34-Ig to the envelope glycoproteins is usually shown in the top panel. Binding of the CD4-Ig probe (0.6 g/ml) after incubation for 30 min at the indicated temperature is shown in the bottom panel. Data symbolize the means ( SEM) derived from duplicate samples. (C) Binding of C34-Ig to V1/V2 loop-deleted envelope glycoproteins in the absence and presence of sCD4. COS-1 cells that transiently express the indicated cytoplasmic tail-deleted envelope glycoproteins were incubated with C34-Ig (30 g/ml) at 26C for 30 min in the absence or presence of sCD4 (20 g/ml). Data symbolize imply ( SEM) binding values after subtraction of background values of C34-Ig binding to mock-transfected cells. In the bottom panel, C34-Ig binding in the absence of sCD4 is usually expressed as the percentage of C34-Ig binding in the presence of sCD4. Amyloid b-Peptide (1-43) (human) (D) Binding of C34-Ig (40 g/ml) to COS-1 cells expressing the indicated cytoplasmic tail-deleted envelope glycoprotein variants at 26C, in the absence or presence of sCD4 (20 g/ml). Binding of.