Figure 1.
Intracellular staining of HIV-transfected HEK293T cells.
The ER and Golgi apparatus (green) were labelled with an anti-PDI and an anti-giantin antibody, respectively. Gp120 (red) was visualized with antibody 2G12. Overlay of gp120 and the ER or Golgi is shown as orange/yellow.
Figure 2.
Western blot analysis of glycosidase-treated cell lysates derived from wild-type (WT) and mutant N260Q gp160 HIV-transfected HEK293T cells.
Cell lysates were treated with either endoglycosidase H (EndoH) or peptide-N-glycosidase F (PNGaseF). The glycoprotein gp160 was detected with an anti-gp41 antibody (A) or an anti-gp120 antibody (B). GAPDH was used as an equal-sample loading control. M: MagicMark XP Western Protein Standard (Novex).
Figure 3.
Infectivity of WT and mutant virus strains.
Different concentrations of (WT and mutant) virus were added to C8166 cell cultures. Three to four days post infection, the viral infection was quantified by FACS analysis. Data (± SD) are the means of 2 to 3 independent experiments.
Figure 4.
Ribbon diagram representation of core gp120 with indication of Asn260 and Ser128.
In the shown orientation, the viral envelope would be on top of the picture and the cellular target membrane on the bottom of the picture. Asn260 is positioned in the C2 region. Ser128 can be found at the base of the V1/V2 loop region. Amino acid numbering is based on HIV-1 strain NL4.3, and corresponds with amino acids Asn262 and Ser128 in HIV-1 strain HXB2. Figure adopted from Kwong et al. 1998 [36].
Figure 5.
Western blot analysis of the incorporation levels of gp160, gp120 and gp41 in viral particles.
(A) Analysis of the level of gp160, gp120 and gp41 incorporation in virus particles, produced in the absence or presence of the lysosomal inhibitor chloroquine. P24 was quantified as an equal loading control. (B–C) Relative protein levels as quantified based on panel A using the ImageJ software, and normalized to equal p24 levels.
Figure 6.
Pulse-chase experiments to study signal peptide cleavage, oxidative folding and gp120 shedding.
HeLa cells transfected with WT or mutant gp160 were pulsed with Express 35S protein labelling mix during 10 min and chased with cold cysteine and methionine for the indicated time. Films in panel A were exposed for 7 days and in panel B and C for 14 days. (A) Reducing (R) SDS-PAGE to study signal peptide cleavage. (B) Non-reducing (NR) SDS-PAGE to study oxidative folding. (C) Detection of shed gp120 in culture medium. SP: signal peptide.
Figure 7.
Immunoprecipitation of WT and mutant gp160 with CD4-IgG2.
Aliquots of detergent lysates from Figure 6 were used for gp160 pull-down with CD4-IgG2, deglycosylation and subjection to SDS-PAGE. Films were exposed for 1 month.
Figure 8.
Western blot analysis of the expression levels of gp160, gp120 and gp41 in virus transfected HEK293T cells, in the absence or presence of the lysosomal inhibitor chloroquine.
WT, N260Q and N260Q/S128N virus transfected HEK293T cells were lysed and subjected to WB analysis (2 ng of p24 protein), to detect gp160, gp120 and gp41 in the cell lysates. GAPDH was used as an equal loading control.
Figure 9.
Flow cytometric analysis of gp120 expression on the surface of HIV-transfected HEK293T cells, in the absence or presence of chloroquine.
Two days after transfection in the absence or presence of chloroquine to produce WT, N260Q or N260Q/S128N virus, HEK293T cells were stained to detect gp120 on the cell surface with the primary antibody 2G12 and a secondary anti-human antibody labelled with Alexa Fluor 647. Data are the means (± SEM) of 5 independent experiments.
Figure 10.
Capacity of WT and mutant virus strains, produced in the absence or presence of chloroquine, to bind to sCD4.
45 ng p24 of virus was lysed using Triton-X100 and was brought into contact with ELISA strips coated with sCD4. The binding efficiency was calculated as relative to the binding of WT virus produced in the absence of chloroquine. Data are the means (± SEM) of 2–3 independent experiments.