Human immunodeficiency virus type 1 Nef associates with lipid rafts to downmodulate cell surface CD4 and class I major histocompatibility complex expression and to increase viral infectivity

Abstract

Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.

FIG. 1.

Subcellular targeting of Nef. (A) Membrane localization; 75 μg of pA-Nef or pA-Nef_G2A expression vector was transfected by electroporation into 1.5 × 10⁸ SupT1 cells. Cells were lysed after 48 h by freeze-thawing, and lysates were layered at the bottom of a sucrose gradient and spun overnight as described in Materials and Methods. Fractions were then solubilized and subjected to immunoprecipitation with a mouse anti-Nef antibody, followed by Western blot analysis with rabbit anti-Nef serum. For CD71 detection, fractions were analyzed by direct Western blot with a monoclonal anti-CD71 antibody. In both cases, development was performed with [¹²⁵I]protein A. The percentage of the total protein in each fraction was determined by dividing the phosphorimager units of the band in that fraction by the combined total value of that band in all fractions. (B and C) Raft localization; 10⁸ SupT1 cells were transfected with 50 μg of the pA-Nef expression vector as in panel A and harvested after 48 h. Cells were lysed in Triton X-100-containing buffer, and lysates were layered at the bottom of a sucrose gradient and spun overnight as described in Materials and Methods. The gradient was fractionated and analyzed for Nef by immunoprecipitation and Western blot or by direct Western blot for Lck and CD71. Panel B is a Western blot probed for the indicated protein and developed by ECL. Panel C shows the same gel quantitated with [¹²⁵I]protein A as described for panel A, and fractions were analyzed for Nef and CD71 as in panel A.

FIG. 2.

LAT-Nef and LAT_AA-Nef fusion proteins: construction and expression. (A) Schematic representation of LAT-Nef and LAT_AA-Nef and their expected subcellular destination. These constructs were built into vectors with expression driven by either the simian cytomegalovirus immediate-early promoter or the chicken actin promoter; 2 × 10⁷ SupT1 cells were transfected with 10 μg of the pA-based vectors expressing Nef, LAT-Nef, or LAT_AA-Nef. Lysates were then made and normalized for total protein content before being subjected to immunoprecipitation with a monoclonal anti-Nef antibody and Western blot analysis with a rabbit anti-Nef serum. The Western blot was developed by ECL (B) or [¹²⁵I]protein A (C). In panel C the amount of protein shown for LAT-Nef is a summation of both bands representing the partially and fully palmitoylated forms of the protein.

FIG. 3.

LAT-Nef and LAT_AA-Nef target to rafts and nonraft membrane, respectively; 10⁸ SupT1 cells were transfected with 40 μg of pA-Nef_G2A, pA-LAT-Nef, or pA-LAT_AANef expression vector. Lysates were made and subjected to membrane (A) and raft (B) isolation and gradient analysis as described for wild-type Nef in Fig. 1.

FIG. 4.

LAT-Nef downmodulates CD4 and class I MHC more efficiently than LAT_AA-Nef; 2 × 10⁷ SupT1 cells were transfected with Nef, Nef_G2A, LAT-Nef, or LAT_AA-Nef, expression vectors along with 2 μg of a GFP expression vector. In panels A and B, cells were transfected with 10 μg of each vector and incubated with PE-labeled antibody at the indicated times. In panels C and D, cells were transfected with the indicated amount of expression vector and incubated with PE-labeled antibody after 24 h. Cells were stained with either CD4-PE (A and C) or class I MHC A2.1-PE (B and D) antibodies and analyzed by flow cytometry. In each case, the data plotted are the CD4 or class I MHC mean fluorescence intensity of the GFP-positive gated population.

FIG. 5.

Functional differences between LAT-Nef and LAT_AA-Nef are highly reproducible. For CD4 and class I MHC downmodulation, 2 μg of GFP expression vector was cotransfected together with 10 μg of pA, pA-Nef, pA-LAT-Nef, or pA-LAT_AA-Nef expression vector into 2 × 10⁷ SupT1 cells. Cells were stained with antibodies against CD4 or class I MHC after 24 h. The LAT-Nef and LAT_AA-Nef CD4 and class I MHC activities of the GFP-positive populations were determined by flow cytometry. Results from nine measurements of CD4 downmodulation and 10 for class I MHC downmodulation are compiled.

FIG. 6.

Analysis of CD4 and class I MHC downmodulation by LAT-Nef and Nef containing mutations that affect these functions. (A and B) Vectors expressing the indicated proteins were transfected into SupT1 cells. Lysates were made and analyzed by Western blotting as described in Materials and Methods. The blots were probed with a rabbit polyclonal serum directed against Nef and developed by ECL. (C and D) SupT1 cells were transfected with Nef or LAT-Nef expression vector carrying the indicated mutation, together with 2 μg of a GFP expression vector. At 24 h posttransfection, cells were harvested and stained with either an anti CD4-PE antibody (C) or an anti-class I MHC A2.1-PE antibody (D). Samples were then analyzed by flow cytometry. In each case, the data plotted are the CD4 or class I MHC mean fluorescence intensities of the GFP-positive gated population. The sample marked mock underwent the transfection procedure with empty pA vector.

FIG. 7.

LAT-Nef enhances virion infectivity greater than LAT_AA-Nef. Virus vector was harvested at 3 days posttransfection from 5BD.1 cells transfected with pTR167 Nef(−) and 5, 10, or 15 μg of pCMVNef, pCMV LAT-Nef, or pCMVLAT_AA-Nef as indicated. For the control sample 5 μg of pCMV containing no insert was utilized. In all cases a vector that expressed Tat (pCMVTat) was also added. The supernatant was then used to infect HeLa CD4 cells, and hygromycin-resistant colonies were counted after 14 days. Vector titer was calculated as the number of hygromycin-resistant HeLa CD4 colonies formed per milliliter of supernatant. Each bar shows the data from independent duplicate experiments.

FIG. 8.

Incorporation of Nef proteins into virus particles produced in 293T cells. 293T cells were contransfected with the pNL4-3 proviral clone lacking Nef and vectors expressing either Nef, or Nef_G2A (10 μg) and LAT-Nef or LAT_AA-Nef (15 μg). (A) Cells were harvested at 36 h posttransfection, and lysates were made and analyzed by Western blotting as described in Materials and Methods. The blots were probed with a rabbit polyclonal serum directed against Nef and developed with [¹²⁵I]protein A. The amount of radioactivity in each band was then measured with a phosphorimager. The intensity of each band relative to the band from the sample transfected with the Nef expression vector is shown. (B) Virus particles were harvested, as described in Materials and Methods, at 72 h posttransfection from duplicate cultures that were transfected at the same time as those in part A. The particles were then lysed and subjected to Western blotting after normalizing for p24 content as measured by ELISA. The upper panel shows a portion of the Western blot that was probed with an anti-p24 monoclonal antibody capable of recognizing Pr55^gag. The blot was developed with ECL. The bottom panel shows a portion of the blot probed with a rabbit polyclonal serum directed against Nef. The blot was developed with [¹²⁵I]protein A. The intensity of each band relative to the band from the sample transfected with the Nef expression vector is shown after normalization for the slight differences in expression that were determined in part A.