Fig 1.
Effects of SP mutations on the REJO Env expression and virus infectivity.
(A) Schematic representation of REJO WT and eight different SP mutations evaluated in this study. (B) Measurement of Env incorporation by Western blot. WT and mutant viruses were produced in transfected 293T cells, lysed, and analyzed by SDS-PAGE (4–20%) and Western blot. An anti-gp120 MAb cocktail (V3: 391/95-D, 694/98-D, 2219, 2558; C2: 847-D, 1006-30D; C5: 450-D, 670-D) and a p24 Gag MAb (91–5) were used to detect the relative levels of Env and Gag associated with virions. The ratios of Env/Gag were calculated. (C) The levels of Env incorporation into the mutant virions relative to that of WT were calculated based on their Env/Gag ratios (WT value was normalized to 100%). *, p< 0.01 (ANOVA). (D) Infectivity of WT vs. mutant viruses (K/R: top; H12: bottom) in CD4+ TZM.bl cells exposed to titrated viruses with equivalent p24 contents. (E) Correlation of virus infectivity in CD4+ TZM.bl cells with the incorporation of Env into virions by Spearman’s rank test. Infectivity was based on RLU produced upon infection with a fix amount of virus input (0.33 ng p24/ml).
Fig 2.
Neutralization of REJO WT and mutant viruses by different MAbs and CD4-IgG2.
Virus neutralization was measured using the TZM.bl target cells after the viruses were incubated with serially diluted MAbs at 37°C for 24 hours prior to addition of the target cells, except for PG9, PGT145, NIH45-46, and CD4-IgG2 which were pre-incubated with viruses for 1 hour. The irrelevant control MAb 1418 was included for negative control. Means and standard errors from two to three independent experiments are shown. *, p<0.05 from the two-way ANOVA as compared to neutralization of the WT virus. Data from two SP mutants (H12R and H12Q) are shown. Neutralization data of WT REJO produced in 293S GnTI- cells (WT GnTI-) are also presented for comparison.
Fig 3.
AUC values of REJO WT and SP mutant neutralization by MAbs targeting V2i, V3, V2q, and the CD4bs and by CD-IgG2.
Neutralization assays were performed for each of the MAb-virus pairs as described in Fig 2, and areas under the titration curves (AUCs) were calculated. AUCs that decreased or increased by >30% and had p<0.05 relative to the corresponding values for WT neutralization by the same MAbs or CD4-IgG2 are shown in red or blue. Neutralization data of REJO WT viruses produced in 293S GnTI- cells or in 293T cells in the presence of kifunensine are included for comparison.
Fig 4.
IC50 values of REJO SP mutants vs. WT by MAbs targeting V2i, V3, V2q, and the CD4bs and by CD-IgG2.
Neutralization assays were described in Fig 2. IC50 (μg/ml) values that increase or decrease by >3 fold and p<0.05 as compared to that of WT by the same MAbs or CD4-IgG2 are shown in red and blue. Data from REJO WT viruses produced in 293S GnTI- cells or in 293T cells in the presence of kifunensine are also presented.
Fig 5.
Effects of SP mutations on REJO virus capture and transmission by DC-SIGN.
(A) Parental Raji or Raji–DC-SIGN+ cells were incubated for 2 hours with WT or mutant viruses produced in 293T cells. Cells were washed extensively, and the amounts of p24 protein associated with the cells were measured by ELISA. (B) Parental Raji or Raji–DC-SIGN+ cells were incubated with WT and mutant viruses for 2 hours, washed to remove unbound viruses, and added to CD4+ TZM.bl cells. Viral transmission to the TZM-bl cells was determined by luciferase activity and calculated based on infection in TZM.bl cells without Raji cells as control (set to 100%). Background luciferase activity was determined in co-cultures without any virus. *, p< 0.05 as compared to WT (ANOVA). (C) Correlation of virus capture (top) and transmission (bottom) via DC-SIGN with the Env incorporation into the WT and mutant virions by Spearman’s rank test.
Fig 6.
Analysis of REJO Env sugar moieties by lectin-probed Western blotting.
The same amounts of Env from sucrose-pelleted virions were separated by SDS-PAGE (4–20%) under reducing conditions, blotted, and probed with an anti-gp120 MAb cocktail, an anti-gp41 MAb cocktail, and lectins (GNA, GRFT, and AAL). A) REJO WT virus produced in GnTI- cells or in 293T cells in the presence vs absence of kifunensine (25μM) known to alter Env glycan compositions. B) Quantification of total density of Env bands from REJO WT shown in A). C) REJO WT and mutant viruses produced in 293T cells. D) Density measurements of the upper and lower Env bands as recognized by anti-gp120 MAbs and different lectins. Density analysis was done by Image Lab software. The two Env species with distinct molecular masses are indicated by red and green arrows.
Fig 7.
HIV phenotypes with Env SP mutation relative to WT*.
Fig 8.
Proteomic and glycoproteomic analyses of virion proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Sucrose-pelleted virions were denatured with 8M in1 M ammonia bicarbonate buffer. The denatured proteins were than prepared for trypsin digestion at 37°C overnight. The samples containing peptides were acidified pH = 3 and desalted using C18 SPE column. The C18 elute was dried in the speed-vac and then resuspended in 0.2% formic acid. The samples (1 μg) were then subjected to LC-MS/MS. Relative abundance of different glycoforms found on 2 identified glycopeptides from SP mutants vs WT were calculated and shown as ratio of mutant to WT.
Fig 9.
Effects of SP mutations on JRFL Env expression, virus infectivity and reactivity to different MAbs.
(A) Schematic representation of JRFL WT and four different SP mutations evaluated in this study. (B) Measurement of Env incorporation by Western blot. JRFL WT and mutant viruses were produced in transfected 293T cells, lysed, and analyzed by SDS-PAGE (4–20%) and Western blot. An anti-gp120 MAb cocktail (V3: 391/95-D, 694/98-D, 2219, 2558; C2: 847-D, 1006-30D; C5: 450-D, 670-D) and a p24 Gag MAb (91–5) were used to detect the relative levels of Env and Gag associated with virions. The ratios of Env/Gag were calculated. (C) The levels of Env incorporation into JRFL mutant virions relative to that of WT were calculated based on their Env/Gag ratios (WT value was set to 100%). *, p< 0.01 (ANOVA). (D) Infectivity of JRFL WT vs. mutant viruses in CD4+ TZM.bl cells exposed to titrated viruses with equivalent p24 contents. (E) Correlation of virus infectivity in CD4+ TZM.bl cells with Env incorporation into the virions by Spearman’s rank test. Virus infectivity was based on RLU produced upon infection with a fix amount of virus input (0.9 ng p24/ml).
Fig 10.
Neutralization of JRFL WT and mutant viruses by different MAbs and CD4-IgG2.
Neutralization assays were performed for each of MAb-virus pairs as described in Fig 2. AUC values were calculated from titration curves. A) Titration curves of representative virus-MAb pairs. B) JRFL WT and mutant neutralization by MAbs targeting V2i, V3, V2q, and the CD4bs and by CD-IgG2. AUC values that decreased by >30% and had p<0.05 relative to WT are shown in red. Means and standard errors from two to three experiments are shown.
Fig 11.
Analysis of JRFL Env sugar moieties by lectin-probed Western blotting.
The same amounts of Env from JRFL WT and mutant viruses produced in 293T cells were separated by SDS-PAGE (10%) under reducing condition, blotted, and probed with an anti-gp120 MAb cocktail or lectins (GNA, GRFT, and AAL). A) Env reactivity with MAbs versus lectins. Two Env species with distinct molecular masses are indicated by red and green arrows. B) Density measurements of the upper and lower Env bands as recognized by anti-gp120 MAbs and different lectins. Density analysis was done by Image Lab software.