Rational site-directed mutations of the LLP-1 and LLP-2 lentivirus lytic peptide domains in the intracytoplasmic tail of human immunodeficiency virus type 1 gp41 indicate common functions in cell-cell fusion but distinct roles in virion envelope incorporation

Abstract

Two highly conserved cationic amphipathic alpha-helical motifs, designated lentivirus lytic peptides 1 and 2 (LLP-1 and LLP-2), have been characterized in the carboxyl terminus of the transmembrane (TM) envelope glycoprotein (Env) of lentiviruses. Although various properties have been attributed to these domains, their structural and functional significance is not clearly understood. To determine the specific contributions of the Env LLP domains to Env expression, processing, and incorporation and to viral replication and syncytium induction, site-directed LLP mutants of a primary dualtropic infectious human immunodeficiency virus type 1 (HIV-1) isolate (ME46) were examined. Substitutions were made for highly conserved arginine residues in either the LLP-1 or LLP-2 domain (MX1 or MX2, respectively) or in both domains (MX4). The HIV-1 mutants with altered LLP domains demonstrated distinct phenotypes. The LLP-1 mutants (MX1 and MX4) were replication defective and showed an average of 85% decrease in infectivity, which was associated with an evident decrease in gp41 incorporation into virions without a significant decrease in Env expression or processing in transfected 293T cells. In contrast, MX2 virus was replication competent and incorporated a full complement of Env into its virions, indicating a differential role for the LLP-1 domain in Env incorporation. Interestingly, the replication-competent MX2 virus was impaired in its ability to induce syncytia in T-cell lines. This defect in cell-cell fusion did not correlate with apparent defects in the levels of cell surface Env expression, oligomerization, or conformation. The lack of syncytium formation, however, correlated with a decrease of about 90% in MX2 Env fusogenicity compared to that of wild-type Env in quantitative luciferase-based cell-cell fusion assays. The LLP-1 mutant MX1 and MX4 Envs also exhibited an average of 80% decrease in fusogenicity. Altogether, these results demonstrate for the first time that the highly conserved LLP domains perform critical but distinct functions in Env incorporation and fusogenicity.

FIG. 1.

Diagram of gp41 with location and amino acid sequences of wild-type (WT) and mutant LLP domains. Mutations in the highly conserved LLP domains were engineered in the proviral clone of ME46. Helical wheel representations of LLP-1 and LLP-2 domains are depicted here with highly conserved arginine residues (shown by arrowheads) that were replaced with glutamate. Hydrophobic amino acids are shown as shaded circles; white circles represent hydrophilic amino acids. Conserved arginine residues in the wild-type sequence that were replaced with glutamate are shown in bold face. In the MX3 mutant, Δ147 refers to a deletion of 147 amino acids from the carboxyl-terminal end of gp41 by replacing the glutamine residue at position 715 [Q(715)] with a stop codon (*).

FIG. 2.

Effects of mutations in the LLP domains on Env expression and processing and gp120 incorporation into virions. (A and B) Two days after the transfection of 293T cells with wild-type (WT) and mutant proviral clones, proteins were labeled overnight with [³⁵S]methionine-cysteine, and viral proteins were immunoprecipitated thereafter. Immunoprecipitated viral proteins were resolved on SDS-polyacrylamide gel, and envelope gp160, gp120, and viral p24 from cell lysates (A) and from viral pellets (B) from the same experiment are shown. (C and D) Total Env expression (gp120+gp160/p24) (C) and Env processing (gp120/gp120+gp160) (D) were calculated after quantifying the individual proteins by densitometry. Results were normalized to wild-type levels in each experiment. Mock-transfected cells and supernatant from mock-transfected cultures were included as negative controls. Data are the averages of results from three independent experiments. Error bars show standard deviations.

FIG. 3.

Western blot analysis of gp41 and gp120 incorporation into virions. Virion lysates were prepared on day 3 from culture supernatants of 293T cells transfected with wild-type (WT) and mutant proviral clones after ultracentrifugation. (A) Samples were transferred to polyvinylidine difluoride membranes and blotted with anti-gp41 MAb (T32), polyclonal goat anti-gp120, anti-p24 MAb (AG3.0), and anti-RT MAb (8C4) (see Materials and Methods). (B) A longer exposure of a Western blot probed with 20 μg of T32/ml was used to confirm the lack of TM expression in the MX3 mutant. Molecular weights are indicated in thousands. (C and D) gp41, gp120, and p24 protein bands were also quantitated by densitometry, and Env incorporation was determined by calculating the ratios of gp41 to p24 (C) and gp120 to p24 (D) and normalizing to wild-type levels. The results shown are representative of results from duplicate experiments. Error bars indicate standard deviations.

FIG. 4.

Effects of mutations in the LLP domains on viral infectivity in a single-round assay in MAGI-R5 cells. Supernatants from transfected 293T cells were normalized for p24 levels and used to infect MAGI-R5 cells in duplicate. Cells were stained for β-galactosidase 48 h after infection. Blue foci were counted as the total number per well and normalized to the number of blue foci counted for wild-type virus (WT). Data represent results from at least two independent transfections, and each mutant was analyzed in duplicate. Error bars indicate standard deviations.

FIG. 5.

Effects of mutations in the LLP domains on viral replication. Mutations in the highly conserved LLP domains were engineered in the proviral clone, and the wild-type (WT) and different mutant clones were transfected into 293T cells to produce viral particles. (A) Two days posttransfection, supernatants from the transfections were filtered through a 0.45-μm-pore-size filter, normalized for p24 levels, and used to infect human PBMC stimulated with PHA. Fresh PHA-PBMC samples were added every 5 days by replacing half of the culture supernatants. Viral replication kinetics were followed by measuring p24 levels in culture supernatants. (B) Additionally, the replication properties of the mutant proviral constructs were compared with those of wild-type virus in H9 lymphoid cells. The cells were split 1:4 every 5 days following transfection, when the culture supernatant p24 levels were also determined.

FIG. 6.

Cytopathicity and syncytium induction properties of LLP-2 mutant virus. H9 cells (5 × 10⁶) were infected with the same TCID₅₀ of wild-type (WT) and MX2 virus. About 50,000 infected cells were plated in duplicate in a 96-well flat-bottom tissue culture plate and incubated at 37°C in a 5% CO₂ incubator. The remaining infected cells were maintained in T25 flasks and split 1:4 every 3 to 5 days. (A) Following incubation, the wells were scored for syncytium formation at 3- to 5-day intervals postinfection and cell death was determined by the trypan blue exclusion method. The total numbers of live cells were determined and normalized for cell growth by using mock-infected cultures. Induction of up to 5 syncytia per well was graded as +; ++ represents 6 to 10 syncytia, +++ represents 11 to 20 syncytia, and ++++ represents >20 syncytia. Excessive (>90%) cell death in virus-infected cultures is indicated by CPE (cytopathic effect). Data presented here are representative of results from at least three independent infections. (B) A 1-ml sample of supernatant from the infected cells described above was taken from each T25 flask every 3 to 5 days for measurement of tissue culture supernatant p24 levels. (C) H9 cells were also infected with MX2 virus stocks containing infectious doses of the virus that were equal to (1×) or about 10-fold higher than (10×) that of wild-type virus. Syncytia were scored 7 days postinfection, when p24 levels in the culture supernatant were also measured. Results are presented as the averages ± the standard deviations and represent results from three to four independent experiments.

FIG. 7.

Envelope glycoprotein expression on the surface of CD4⁺ T cells infected with wild-type (WT) or MX2 virus. H9 cells were infected with MX2 virus at infectious doses that were fivefold higher than those of wild-type virus. At 15 days postinfection, cells were simultaneously stained for cell surface envelope glycoprotein and intracellular p24 levels (by using KC57-RD1) and analyzed by flow cytometry. FITC-conjugated goat anti-mouse IgG and mouse anti-human IgG were used as secondary antibodies. A minimum of 50,000 gated-live events were acquired on a flow cytometer and analyzed by using FlowJo batch analysis software. (A to C) The percentage of p24-positive cells was determined for wild-type virus- (B) and MX2- (C) infected cells by using the uninfected control cells (A). (D to F) Env expression was then assessed in p24-gated cells by using antibodies reactive to gp41, T32 (D), 126-6 (E), and Md-1 (F). The solid lines represent the levels of expression of Env on wild-type virus-infected cells, and the broken lines represent the levels of expression on MX2-infected cells. The gray shaded peak in each graph represents the background level of staining for Env with the isotype control, anti-mouse IgG-FITC, or anti-human IgG-FITC in a p24-gated population of cells. Data presented here are representative of results from two independent infections. Mean fluorescence intensities of p24-PE and Env-FITC staining were determined for wild-type virus- and MX2-infected cells. FL₂-H and FL₁-H, fluorescence channels. (G to I) Ratios of mean fluorescence intensities (MFI) of p24-PE and Env-FITC were calculated as a measure of the levels of Env expressed relative to the level of p24 expressed per infected cell. Ratios are depicted graphically for antibodies T32 (G), 126-6 (H), and Md-1 (I) and are averages of results from two independent infections. Error bars indicate standard deviations.

FIG. 8.

Effects of mutations in the LLP domains on cell-cell fusion mediated by Env. ICT mutant Envs from MX1, MX2, MX3, and MX4 were cloned into pCDNA3 vector and transfected along with pCDNA3 vector alone (Mock) into 293T cells, which were also infected with recombinant vaccinia virus vTF1.1 expressing T7 polymerase. These effector cells were mixed with target quail QT6 cells transfected with CXCR4 and CD4 expression plasmids and the luciferase expression vector. The cells were allowed to fuse for at least 7 h, after which fusion was measured by quantitation of luciferase in cell lysates. The results shown are representative of results from two fusion experiments and are presented as relative light units (RLU) per microgram of protein in the cell lysate. Error bars indicate standard deviations. WT, wild type.