Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes

Abstract

Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles.

FIG.1.

Roles of viral proteins on HIV-1 envelope association with rafts. (A) Envelope expression and fusigenicity in the presence and absence of other viral proteins. 293T cells were cotransfected with pSVIIIenv (NL4.3 env) with either env⁻ pNL4.3 or pSV2tat72 and were immunostained for envelope. Cells were fixed and permeabilized with methanol-acetone (1:1) 48 h after transfection before immunostaining with anti-gp41 MAb Chessie 8 (top row of micrographs). Cell-cell fusion and syncytium induction were detected by cocultivation of 293T cells expressing NL4.3 envelopes in the presence and absence of env⁻ pNL4.3 with GHOST/CXCR4 cells. Cells were fixed and stained with 1% methylene blue and 0.25% basic fuchsin in methanol (bottom row of micrographs). Western blot of 293T cell lysate taken 48 h after transfection with pSVIIIenv (NL4.3 env) with either env⁻ pNL4.3 or pSV2tat72 is shown below the micrographs. (B) HIV-1 envelope association with DRMs in the presence and absence of viral proteins other than Tat and Rev. 293T cells were cotransfected with pSVIIIenv which expressed Rev and contained either NL4.3 and HXB2 envelopes together with env⁻ pNL4.3, which expresses all viral proteins except for envelope, or pSV2Tat72, which expresses Tat. Cells were lysed with cold 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (as described in Materials and Methods). Gradient fractions were Western blotted and probed for envelope (gp160 and gp41) and Gag (p55) with mouse MAbs. Note that both NL4.3 and HXB2 envelopes associate with DRM fractions in the presence of env⁻ pNL4.3 but were excluded from rafts when expressed in the absence of env⁻ pNL4.3 (bottom blots).

FIG. 2.

Effect of a matrix mutation (L30E) on envelope function. (A) 293T cells were transfected with pSVIIIenv (NL4.3 env) with either env⁻ pNL4.3 or env⁻ pNL4.3 (L30E) and assessed for envelope expression (left panels) and cell-cell fusion (right panels). (B) Impact of L30E mutation in p17^gag on virus infectivity tested on GHOST/CXCR4 cells and represented as infectivity (FFU) to particle (RT) ratio. (C) Envelope incorporation into virus particles was assessed by measuring the amounts of envelope in transfected 293T cell supernatants. Virus samples were precipitated and resuspended in PBS, and equivalent amounts of RT activity were then resolved on a sodium dodecyl sulfate-8% polyacrylamide gel, followed by Western blotting. Envelope and p24 were probed with Chessie 8 and 183-H12-5C monoclonal antibodies, respectively.

FIG. 3.

Effect of matrix mutation on envelope association with lipid rafts. 293T cells were cotransfected with pSVIIIenv (NL4.3 env) construct and either env⁻ pNL4.3 or env⁻ pNL4.3 (L30E). Cells were lysed with cold 0.5% Triton X-100 and homogenized, and postnuclear supernatants were subjected to sucrose density gradient centrifugation as described in Materials and Methods. Gradient fractions were subsequently probed for envelope (gp160 and gp41) and Gag (p55) with mouse MAbs by Western blotting. Note that envelope proteins coexpressed with the L30E Gag mutation failed to associate with DRM-L (bottom panel) compared with that with wild-type Gag (top panel).

FIG. 4.

Envelope association with lipid rafts in MOLT 4 T cells. MOLT 4 cells were infected with VSV-G⁺ pseudotype virions prepared with either pNL4.3 wt or pNL4.3 (L30E). After 48 h, cells were lysed with cold 0.5% Triton X-100 and homogenized, and postnuclear supernatants were subjected to sucrose density gradient centrifugation as described in Materials and Methods. Gradient fractions were subsequently probed for envelope (gp160 and gp41) and Gag (p55) with mouse MAbs by Western blotting. Note that envelope proteins coexpressed with the L30E Gag mutation failed to associate with DRM-L (bottom panel) compared with that with wild-type Gag (top panel).

FIG. 5.

Effect of β-methyl cyclodextrin on envelope and Gag association with lipid rafts. Untransfected 293T cells were treated with and without 10 mM β-methyl cyclodextrin (MβCD) and subsequently lysed with cold Triton X-100. Postnuclear supernatants were resolved by sucrose density centrifugation, and DRM and DSM fractions were analyzed for the presence of caveolin by Western blotting. The effect of β-methyl cyclodextrin on p55^gag and envelope was tested on 293T cells cotransfected with pSVIIIenv and env⁻ pNL4.3.

FIG. 6.

Functionality, infectivity, and assembly of patient-derived NA420 envelopes. (A) Amino acids from position 758 to the C terminus (position 856) of NA420 B33 gp41 cytoplasmic domain were introduced into LN40 to produce the NA420 LN40 (B33^758-856) chimeric construct. (B) Functionality of LN40, B33, and NA420 LN40 (B33^758-856) was assessed by cell-cell fusion. (C) Envelope incorporation was measured by resolving virus pellets on SDS-polyacrylamide gels, followed by Western blotting. (D) Infectivity conferred by each of the patient-derived envelopes was measured in GHOST/CCR5 cells and plotted as infectivity (FFU) to particle (RT) ratios.

FIG. 7.

Lipid raft association of patient-derived envelopes. 293T cells expressing NA420 LN40, NA420 B33, and chimeric NA420 LN40 (B33^758-856) envelopes were lysed with cold Triton X-100, and the postnuclear supernatants were subjected to sucrose density gradient centrifugation. DRM and DSM fractions were immunoprecipitated with serum from a HIV patient as described in Materials and Methods and resolved by SDS-PAGE. Envelope and Gag proteins were detected by Western blotting using Chessie 8 and 183-H12-5C MAbs, respectively. Note that the substitution of the B33^758-856 sequence in the LN40 gp41 cytoplasmic domain fully rescued envelope recruitment to DRMs.