Role of the membrane-spanning domain of human immunodeficiency virus type 1 envelope glycoprotein in cell-cell fusion and virus infection

Abstract

The membrane-spanning domain (MSD) of the human immunodeficiency virus type 1 (HIV-1) gp41 glycoprotein is critical for its biological activity. Previous C-terminal truncation studies have predicted an almost invariant core structure of 12 amino acid residues flanked by basic amino acids in the HIV-1 MSD that function to anchor the glycoprotein in the lipid bilayer. To further understand the role of specific amino acids within the MSD core, we initially replaced the core region with 12 leucine residues and then constructed recovery-of-function mutants in which specific amino acid residues (including a GGXXG motif) were reintroduced. We show here that conservation of the MSD core sequence is not required for normal expression, processing, intracellular transport, and incorporation into virions of the envelope glycoprotein (Env). However, the amino acid composition of the MSD core does influence the ability of Env to mediate cell-cell fusion and plays a critical role in the infectivity of HIV-1. Replacement of conserved amino acid residues with leucine blocked virus-to-cell fusion and subsequent viral entry into target cells. This restriction could not be released by C-terminal truncation of the gp41 glycoprotein. These studies imply that the highly conserved core residues of the HIV Env MSD, in addition to serving as a membrane anchor, play an important role in mediating membrane fusion during viral entry.

FIG. 1.

Amino acid sequences of HIV-1 MSDs. The consensus sequence of the HIV-1 MSD was generated by the alignment of the Env MSD sequences of all M and N group HIV-1 isolates from the Los Alamos HIV sequence database (updated to June 2007). The percentages of isolates with the conserved amino acid residue are shown above the consensus sequence as a bar chart. The amino acid residues of the putative MSD region are shown as uppercase letters. The flanking sequences of the HIV-1 MSD are shown as lowercase letters. The position of the MSD in the sequence of HIV-1 NL4-3 is marked above the positively charged amino acid residues. The mutated portions of the HIV-1 MSD are underlined.

FIG. 2.

Expression profiles of HIV-1 Env glycoprotein. (A) COS-1 cells transiently transfected with SV40-based Env expression vectors were metabolically labeled and chased over 5 h. Env glycoprotein was immunoprecipitated, followed by 8% SDS-PAGE analysis and autoradiography (lane 1, pSRHS WT; lane 2, pSRHS L12; lane 3, pSRHS L12G; lane 4, pSRHS L12G2; lane 5, empty vector; lane 6, mock). The bands of gp160 precursors (B), gp120 (C), and gp41 (D) in cell lysates at each time point were quantified by phosphorimager calculation, and the intensity of bands is shown as digital light units (DLU) (squares, pSRHS WT; circles, pSRHS L12; upward triangles, pSRHS L12G; downward triangles, pSRHS L12G2; leftward triangles, empty vector; rightward triangles, mock). Results are from four independent experiments; error bars represent standard deviations from the means.

FIG. 3.

Cell surface level of Env glycoprotein and cell-cell fusion examined using SV40-based Env expression vectors. (A) COS-1 cells transfected with pSRHS expression vectors were fixed and labeled with Alexa 647-conjugated anti-gp120 MAb b12. Cells were then permeabilized and stained with Alexa 488-conjugated anti-gp160 MAb Chessie 13-39.1 as a transfection efficiency control. (B) Cell-cell fusion assay. COS-1 cells transfected with the pSRHS Env expression vectors were cocultured with JC53BL indicator cells. Cell mixtures were lysed, and luciferase activity was measured after 6 h, 12 h, and 24 h of incubation. (C) Cell-cell fusion assay. 293T cells transfected with pCDNA3.1 Env expression vectors were cocultured with JC53BL indicator cells. Cell mixtures were lysed, and luciferase activity was measured after 6 h, 12 h, and 24 h of incubation. Results are from three independent experiments; error bars represent standard deviations from the means. EV, empty vector.

FIG. 4.

Infectivity of HIV-1 Env mutants. (A and C) HIV-1 viruses containing the Env MSD core mutations (A) and viruses containing truncated Env plus the MSD core mutations (C) were produced by transfecting proviral DNA into 293T cells. The p24-normalized cell culture supernatant was used to infect JC53BL indicator cells. Cells were lysed and luciferase enzyme activity was assayed after 48 h of incubation. (B and D) Single-round infectivity assay. Env-defective proviral construct pSG3Δenv was cotransfected into 293T cells with WT and mutant Env expression vectors (B) and cytoplasmic domain-truncated Envs (D). Pseudotyped viruses in the cell culture supernatant were quantitated using a p24 ELISA assay. The p24-normalized supernatant was then used to infect JC53BL indicator cells. Infectivity of MSD mutants is shown as relative luciferase activity compared to that of the WT. Results are from three independent experiments; error bars represent standard deviations from the means.

FIG. 5.

Cell surface level of Env glycoprotein and cell-cell fusion examined in the context of provirus expression. (A) 293T cells transfected with pNL4-3 proviral vectors were fixed and were labeled with Alexa 647-conjugated anti-gp120 MAb b12. Cells were then permeabilized and stained with Alexa 488-conjugated anti-p24 MAb 183-H12-5C as a transfection efficiency control. (B) The surface levels of Env glycoprotein are shown as relative mean fluorescence index (MFI) compared to that of WT. (C) Cell-cell fusion in the context of viruses. 293T cells transfected with proviral DNA were mixed with JC53BL indicator cells and cocultured for 6 h, 12 h, and 24 h before the measurement of luciferase activity. P values were calculated using Student's t test. Results are from three independent experiments; error bars represent standard deviations from the means.

FIG. 6.

Incorporation of Env into virions. (A) 293T cells transfected with proviral DNA were pulse-labeled with [³⁵S]methionine and [³⁵S]cysteine and chased over 24 h. Viral particles in medium were pelleted through a 25% sucrose cushion by ultracentrifugation. Viral proteins were immunoprecipitated with pooled AIDS patient sera and analyzed by 8% SDS-PAGE followed by autoradiography. (B) The viral gp120 and p24 bands in viral pellets and supernatants after ultracentrifugation were quantified by phosphorimaging analysis. The efficiency of incorporation of Env into viral particles is shown as the ratio of the intensities of gp120 bands in virions to those of p24 in virions. (C) The relative amount of gp120 shed into medium is shown as the ratio of the intensities of gp120 bands in supernatant after ultracentrifugation to those of p24 in virions. (D) Unlabeled viral pellets and supernatants were subjected to gp120 ELISA and p24 ELISA. The incorporation efficiency is shown as the ratio of the concentration of gp120 to that of p24 in virions for both full-length (left) and truncated (right) Envs. (E) The degree of shedding of gp120 is shown as the ratio of the amount of gp120 in supernatant after centrifugation to that of p24 in virions for both full-length (left) and truncated (right) Envs. The standard deviations of three repeats for each experiment are shown as error bars.

FIG. 7.

Virus-cell fusion assay. (A) Proviral constructs were cotransfected into 293T cells with pCMV-BlaM-Vpr vectors. Viruses containing the BlaM-Vpr fusion protein were pelleted by ultracentrifugation through 25% sucrose. Viral pellets normalized by p24 ELISA were used to infect JC53BL indicator cells. Then cells were loaded with fluorescent dye CCF2-AM. Blue cells were counted by flow cytometry analysis after 16 h of incubation. (B and C) The ability of the Env MSD mutants (B) and the truncation mutants (C) to mediate virus-cell fusion is shown as relative numbers of blue cells compared to those of the WT. The experiment was repeated at least three times; standard deviations are shown as error bars.