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. 2014 Jun 26;9(6):e101181.
doi: 10.1371/journal.pone.0101181. eCollection 2014.

Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity

Leen Mathys  1 , Katrien O François  1 , Matthias Quandte  2 , Ineke Braakman  2 , Jan Balzarini  1
Affiliations

Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity

Leen Mathys et al. PLoS One. .

Erratum in

  • PLoS One. 2014;9(10):e110202

Abstract

N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N) that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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
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
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
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 .
Figure 5
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
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
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
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
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
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.
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Grants and funding

This work was supported by grants from the KU Leuven Centers of Excellence (PF-10/018), the KU Leuven Geconcerteerde Onderzoeksacties (GOA) (no. 10/014), the Fonds voor Wetenschappelijk Onderzoek (FWO) (G.0485.08) and the CHAARM Network Project of the European Commission. Leen Mathys is supported by a fellowship from L’Oréal-UNESCO, in collaboration with the Belgian FWO (Aspirant). Matthias Quandte was supported by the European Union 7th framework program, ITN “Virus entry”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.