The conserved membrane-proximal external region (MPER) of HIV-1 envelope is a target for the rare broadly neutralizing 2F5, Z13, and 4E10 monoclonal antibodies (mAbs). Enough time during which virus is sensitive to 2F5 mAb-mediated neutralization is approximately 3-fold longer when the mutation is present. These data suggest that a major contribution to the L669S mutant virus phenotype of enhanced susceptibility to MPER mAbs is prolonged exposure of the MPER neutralizing epitope during viral entry. and and and and and and and and and Clones. Mutant TND_669S, wild-type TND_669L, 7534.2, 7534.11, and QZ4734 (previously described in ref. 21) were generated using bulk PCR from plasma from clade B HIV-1+ infected subjects. For mutant TND_669S, subsequent single-genome amplification of the plasma indicated that the L669S mutation was likely not present in vivo; therefore, it could CCT137690 be the result of the cloning process. Alignment of 1 1,963 complete HIV-1 sequences at http://HIV-1.lanl.gov revealed only 1 1 sequence (0.05%) containing this L669S mutation. Molecular Cloning of Full-Length Envelopes, Production of Pseudotyped Viruses, and Neutralization Assay. Cloning strategy of full-length gp160 has been described previously (40, 41). Production and titration of the env-pseudotyped viruses were conducted following procedures modified from methods previously described (40). The 50% tissue culture infectious dose (TCID50) of each virus preparation was determined (42). Neutralization assays with pseudotyped viruses were performed on TZM-bl cells on 96-well flat-bottom plates as previously described (40). The IC50 was determined as the concentration of Ab able to inhibit virus infection by 50% compared to the virus control (41). Time Course of CCT137690 2F5 Neutralization Assay. The time course of neutralization of 2F5 mAb or T20 peptide was determined in a synchronized postattachment HIV-1 pseudotyped virus neutralization assay as described earlier (22). TZM-bl cells (104/well) were plated and allowed to adhere overnight. Each of the plates was then cooled and incubated on ice for 2 h following addition of cold Env pseudotyped viruses. To CCT137690 remove unbound viruses, plates were washed with fresh, cold medium. Warm medium (150 L/well) was added to each well followed by 100 L of inhibitory concentrations of either 2F5 mAb (5 or 20 g/mL) or T20 peptide (20 g/mL) at different time intervals (0, 10, 20, 40, 60, 120, 180, and 240 min) after contamination. A32 mAb and scrambled T20 peptide were used as controls. Infectivity was measured by relative light units (RLUs) as described above for the standard neutralization assay. SPR Assays. All SPR binding assays were performed on a BIAcore 3000 instrument at 25 C and data analyses were performed using the BIAevaluation 4.1 software (BIAcore) as previously described (35). Kinetics and Affinity of 2F5 and 4E10 mAb Binding to Peptide Epitopes. Biotinylated versions of peptides MPER657C671 Rabbit polyclonal to WBP11.NPWBP (Npw38-binding protein), also known as WW domain-binding protein 11 and SH3domain-binding protein SNP70, is a 641 amino acid protein that contains two proline-rich regionsthat bind to the WW domain of PQBP-1, a transcription repressor that associates withpolyglutamine tract-containing transcription regulators. Highly expressed in kidney, pancreas, brain,placenta, heart and skeletal muscle, NPWBP is predominantly located within the nucleus withgranular heterogenous distribution. However, during mitosis NPWBP is distributed in thecytoplasm. In the nucleus, NPWBP co-localizes with two mRNA splicing factors, SC35 and U2snRNP B, which suggests that it plays a role in pre-mRNA processing. (EQELLELDKWASLWN) and MPER657C671/L669S (EQELLELDKWASSWN), MPER656C683 and MPER656C683/L669S, and control peptides with scrambled sequences were individually anchored on a BIAcore SA sensor chip as described previously (35, 43). Each peptide was injected until 100C150 response units (RU) of binding to streptavidin were observed. Specific binding responses of mAb binding were obtained following subtraction of nonspecific binding around the scrambled peptide surfaces. Rate constants were measured using the bivalent analyte model (to account for the avidity of bivalent Ig molecules) and global curve fitting to binding curves obtained from 2F5 mAb titrations, which ranged from 0.01 to119 nM. 2F5 mAb was injected at 30 L/min for 2C6 min and glycine-HCl (pH 2.0) and surfactant P20 (0.01%) were used as the regeneration buffer. Kinetics and Affinity of 2F5 mAb and 4E10 mAb Binding to PeptideCLiposome Conjugates. 2F5 and 4E10 mAb binding CCT137690 to peptideCliposome conjugates was examined using a BIAcore L1 sensor chip as described previously (35). PeptideCliposome conjugates were made following an extrusion method as described earlier, using phospholipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-sn-glycero-3-phosphate, and cholesterol at a molar ratio of 45:25:20:1.33 and a peptide to lipid.