Glycan deletion mutations cause neutralization resistance to subject BNAb antisera. To confirm the phenotype and assess the scale of neutralization resistance of glycan deletion mutants, each individual antiserum was titrated against each mutant that demonstrated significant neutralization resistance relative to its parental wild-type Env in the screening assay. The standard neutralization assay was used to measure neutralization resistance of the wild-type and mutant Envs in single experimental sets. The highest concentration of antiserum was about 2-fold higher than the IC₉₀ of a given antiserum for the parental wild-type Env, as determined in preliminary experiments. Neutralization was measured by determining the residual infectivity (%) after virus-antiserum incubation, normalized to that of the same virus without antibody incubation, which was set to 100%. The phenotype of significant neutralization resistance due to a glycan deletion mutation was indicated by a difference of 2 1:2 dilutions between the wild-type and mutant Envs for at least one point for the same level of RI%.

N234S and N241S mutations are specific for mapping IDGC BNAb epitopes. The N234S and N241S mutants and their parental wild-type HIV-1_YU2 gp160 were subjected to neutralization by NAbs that are known to bind epitopes outside the IDGC, as described in the legend to Fig. 3. The N362K mutant was also studied.

Neutralization of HIV-1 by subject antiserum is enhanced by enriching high-mannose glycans by use of kifunensine. Recombinant luciferase reporter viruses carrying HIV-1_YU2 gp160 were produced in the presence (50 μM or 200 μM) or absence of kifunensine in 293T cell culture medium. To determine if kifunensine treatment had an effect on neutralization of HIV-1_YU2 by subject antisera, the neutralization of each individual subject antiserum was titrated in the standard neutralization assay, using target viruses that were produced under kifunensine selection (YU2 + 50 μM Kif or YU2 + 200 μM Kif) or using the mock-treated parental virus (YU2). A significant effect on neutralization by treatment with kifunensine was defined by a difference of 2 1:2 dilutions at one or more points in the neutralization curve for the kifunensine-treated viruses compared to the mock-treated virus.

Glycan deletion mutants of HIV-1_YU2 and HIV-1_JR-FL gp160s. A panel of glycan deletion mutants was created by introducing point mutations into HIV-1_YU2 or HIV-1_JR-FL gp160 so as to eliminate the NXS/T N-linked glycosylation signals individually. The parental gp160 protein was expressed in the pSVIIIenv vector. The mutants were named as follows: original residue-position number-replacement amino acid. Position numbering follows the numbering convention of the analogous sequence of HIV-1_HXBc2 gp160 (27). This panel of mutants covers all N-linked glycosylation sites conserved in clade B HIV-1 strains. (A) HIV-1_YU2 gp160 was used as the primary model Env. (B) In cases where an HIV-1_YU2 gp160 mutant was functionally incapable of supporting virus entry, a replacement mutant of HIV-1_JR-FL gp160 was created to eliminate the N-linked glycosylation site at the same location. To assess the ability of the Env mutants to support virus entry, the wild-type and mutant Env proteins were pseudotyped in recombinant luciferase reporter viruses by transient transfection of 293T cells. Twenty microliters of each virus was incubated on the target cell line Cf2Th-CD4/CCR5 in a single-round entry assay. The level of Env function was determined by measuring the level of viral infection, reported as arbitrary luciferase units. *, glycan deletion mutant that could not support virus entry of both HIV-1_YU2 and HIV-1_JR-FL; #, glycan deletion mutant that could not support virus entry of HIV-1_YU2.

N332S and N386T mutations are specific for mapping ODGC BNAb epitopes. The N332S and N386T mutants and their parental wild-type HIV-1_JR-FL gp160 were subjected to neutralization by 2G12 as well as by selected NAbs of other known neutralizing epitopes, including the CD4BS (b12, VRC01, and F105), V3/CD4i (E51, 17b, and 48d), the V2 loop (PG9 and PG16), and MPER (2F5).

Effect of kifunensine treatment on entry function and neutralization sensitivity of HIV-1. HIV-1_YU2-pseudotyped viruses encoding firefly luciferase were produced in 293T cells in the presence or absence of various concentrations of the α-mannosidase inhibitor kifunensine. (A) Effect of kifunensine treatment on neutralization by MAb 2G12, an antibody specific for the ODGC, determined in a standard neutralization assay. (B) The ability of HIV-1_YU2-pseudotyped viruses produced in the presence of various concentrations of kifunensine to enter a target cell line was assessed by incubating 20 μl of each virus on CF2Th-CD4/CCR5 target cells in a single-round entry assay. (C) Effect of kifunensine treatment on neutralization by CD4 Ig, the natural ligand of HIV-1 Env, determined in a standard neutralization assay. (D) Effect of kifunensine treatment on neutralization by MAbs that target known epitopes. Epitope specificities for these MAbs are the CD4BS (b12, VRC01, and F105), V3/CD4i (E51, 17b, and 48d), the V2 loop (PG9 and PG16), and MPER (2F5).