Comparative proteomic analysis of HIV-1 particles reveals a role for Ezrin and EHD4 in the Nef-dependent increase of virus infectivity

Abstract

Nef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ∼30 and ∼70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.

Fig 1

Optimization of HIV-1_NL4-3 particles purification. Mock or pNL4-3 (WT)-transfected cells were incubated 24 h in complete medium (A and B), DMEM, CD293, or Free style 239 medium (C) as indicated. Supernatants were then harvested, cleared by low-speed centrifugation and filtered prior to subsequent purification steps. (A) Supernatants were layered on top of a 20% sucrose cushion and ultracentrifuged. (B and C) Supernatants were layered on top of a discontinuous sucrose gradient composed of 20 and 60% sucrose cushions. After ultracentrifugation, the 20 to 60% interphase was recovered, diluted in PBS, and subjected to an additional ultracentrifugation step. Pellets were resuspended in DIGE buffer, resolved by 12% acrylamide SDS-PAGE, and visualized by silver staining. About 75% of virions released by cells grown in a T75 flask were loaded in each lane. (D) Material from 200 ml (∼20 T75 flasks) of mock-, pNL4-3 (WT)-, or pNL4-3 Xho (nef-deleted)-transfected 293T cell supernatants were pelleted as described in panel C. Pellets were resuspended in 200 μl of DIGE buffer, 13 μl of which was mixed with reducing loading buffer and resolved by 12% acrylamide SDS-PAGE without boiling. The proteins were then visualized by silver staining. Nine samples prepared independently are resolved on this gel.

Fig 2

Differential gel electrophoresis of WT and nef-deleted NL4-3 particles. WT and nef-deleted viral particles were purified as described for Fig. 1 and processed for DIGE. (A) Portions (50 μg) of proteins of each WT, nef-deleted and IS sample labeled with Cy5, Cy3, and Cy2, respectively, were resolved by 2-D electrophoresis, and the gel was scanned at the wavelength corresponding to Cy5 or Cy3 in order to visualize proteins from WT or nef-deleted samples (left and middle panels, respectively). The Cy2 acquisition colored in blue was overlaid with that of Cy3 and Cy5 colored in red and yellow, respectively (right panel). Proteins enriched in WT or nef-deleted viruses appear as purple (red + blue, red arrows) or green (yellow + blue, green arrows) spots, respectively, while proteins equally abundant in both samples appear as white spots. Differential protein enrichment was determined by DeCyder, and candidates with statistically significant differences (labeled spots) were extracted for MS analysis and protein identification. (B) 3-D density plot of proteins significantly enriched in WT or nef-deleted virions. For each spot, the protein identity is shown, together with the enrichment factor, and the statistical significance was calculated from three gels prepared as described in Materials and Methods. (C) One WT and one nef-deleted sample were resolved separately by 2-D gel electrophoresis, followed by Western blotting and immunodetection with a Nef-specific or a p24-specific antibody as indicated. Immunodetections of Nef and p24 from SDS-PAGE of WT NL4-3 are shown on the side.

Fig 3

Manipulation of Ezrin or CD81 expression levels in virus-producing cells. HXBc2 Env or VSV-G pseudotyped GFP-reporter viruses were made in the presence or in the absence of Nef (Nef and Nef Xho, respectively) from cells transfected with control, CD81, or Ezrin-specific siRNA (A and B) or pCDNA3.1-, CD81-, or Ezrin-encoding plasmids (C and D). (A and C) Portions (15 μg) of virus-producing cell lysate protein were resolved by SDS-PAGE, followed by Western blotting and immunodetection with the indicated antibodies. (B and D) HeLa-CD4 cells were incubated with the indicated viruses (∼50 or 10 ng of p24 of HXBc2 Env or VSV-G-pseudotyped viruses, respectively), and virus infectivity was calculated by normalizing the percentage of GFP-positive cells to that measured when cells were incubated with viruses produced from cells transfected with control siRNA or plasmid in the absence of Nef. Representative results of experiments performed three times in duplicate are shown in panels A and C. Panels B and D present averages ± the standard deviations (SD) calculated from three independent experiments. Asterisks indicate a WT/nef-deleted virus infectivity ratio significantly different from that obtained in mock conditions (Student t test; *, P < 0.05).

Fig 4

Manipulation of EHD4 or ALG-2 expression levels in virus-producing cells. Virions were produced as in Fig. 3 from cells transfected with control or EHD4- or ALG-2-specific siRNA (A and B) or pCDNA3.1-, EHD4-, or ALG-2-encoding plasmids (C and D). (A and C) Cell lysates were analyzed as described in Fig. 3A and C with EHD4- and ALG-2-specific antibodies. (B and D) Virus infectivity was measured as in Fig. 3B and D. Unlike endogenous EHD4 detection, which required enhanced chemiluminescence (ECL) plus reagents (A, upper panel), overexpressed EHD4 was readily detected with ECL reagents; however, under these conditions, endogenous EHD4 appeared as a faint band (C, upper panel). Representative results of experiments performed three times in duplicate are shown in panels A and C. Panels B and D present averages ± the SD calculated from three independent experiments. Asterisks indicate a WT/nef-deleted virus infectivity ratio significantly different from that obtained in mock conditions (Student t test; *, P < 0.05).

Fig 5

Simultaneous extinction of Ezrin and EHD4 in virus-producing cells. (A) HXBc2 Env-pseudotyped GFP reporter viruses produced in 293T cells were purified as in Fig. 1, and pelleted material was analyzed by SDS-PAGE, followed by immunodetection to visualize p24, Ezrin, EHD4, and HA-tagged Nef. (B to D) Virions were produced from 293T cells transfected with control or EHD4 and Ezrin siRNA. Cell lysates and pelleted virions were analyzed by Western blotting, followed by immunodetection with EHD4, Ezrin, and p24-specific antibodies as indicated (B). The virus concentration was measured in the supernatants of transfected cells and normalized to that of virions produced from control siRNA-treated cells in the absence of Nef (D). HeLa-CD4 cells were incubated with increasing amounts of each virus, and the percentage of GFP-positive cells was measured by fluorescence-activated cell sorting (FACS) at 60 h postinfection (D, left panels). Virus infectivity was normalized to that of virus produced from cells transfected with control siRNA in the absence of Nef as in Fig. 3B (D, right panel). Representative results of experiments performed twice (A) or three times (B and D, left panel) are shown. Panels C and D present the averages ± the SD calculated from three independent experiments. Asterisks indicate a WT/nef-deleted virus infectivity ratio significantly different from that obtained in mock conditions (Student t test; **, P < 0.005).

Fig 6

Contribution of Ezrin and EHD4 to WT and nef-deleted HIV-1 biogenesis in SupT1 cells. (A) SupT1 cells transduced with vectors expressing Ezrin or EHD4-specific shRNAs, either separately or simultaneously were solubilized and ∼15 μg of cells lysate proteins were resolved by SDS-PAGE, followed by Western blotting and immunodetection with the indicated antibodies. The parental SupT1 cell line was analyzed in similar conditions. (B) WT and nef-deleted viruses encoding GFP were produced from the SupT1 cell lines described in panel A and incubated with HeLa cells. Virus infectivity was measured and Nef potency was calculated and normalized to that of virus produced from parental SupT1 cells as described in Table 2. (C) SupT1 cell lines were infected with replication-competent WT or nef-deleted viruses encoding GFP. At the indicated time points, cells were analyzed by FACS, and the percentages of GFP-positive cells was plotted as a function of time. Representative results of experiments performed three times are shown in panel C. Panel B presents the averages ± the SD calculated from three independent experiments. Asterisks indicate values significantly different from that obtained in mock conditions (Student t test; *, P < 0.05).