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. 2004 Dec;78(24):13409-19.
doi: 10.1128/JVI.78.24.13409-13419.2004.

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain

Ling Ye  1 , Zhigao Bu, Andrei Vzorov, Dahnide Taylor, Richard W Compans, Chinglai Yang
Affiliations

Surface stability and immunogenicity of the human immunodeficiency virus envelope glycoprotein: role of the cytoplasmic domain

Ling Ye et al. J Virol. 2004 Dec.

Abstract

The effects of two functional domains, the membrane-proximal YXXPhi motif and the membrane-distal inhibitory sequence in the long cytoplasmic tail of the human immunodeficiency virus type 1 (HIV-1) envelope protein (Env), on immunogenicity of the envelope protein were investigated. Genes with codons optimized for mammalian expression were synthesized for the HIV 89.6 Env and a truncated Env with 50 amino acids in the cytoplasmic domain to delete the membrane distal inhibitory sequence for surface expression. Additional genes were generated in which the tyrosine residue in the YXXPhi motif was changed into a serine. Pulse-chase radioactive labeling and immunoprecipitation studies indicated that both domains can mediate endocytosis of the HIV Env, and removal of both domains is required to enhance HIV Env protein surface stability. Analysis of immune responses induced by DNA immunization of mice showed that the DNA construct for the mutant Env exhibiting enhanced surface stability induced significantly higher levels of antibody responses against the HIV Env protein. Our results suggest that the HIV Env cytoplasmic domain may play important roles in virus infection and pathogenesis by modulating its immunogenicity.

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Figures

FIG. 1.
FIG. 1.
Schematic diagram for the HIV 89.6 Env proteins encoded by synthetic genes. Codon-optimized genes for HIV 89.6 Env, Env/Y710S, Env750Tr, and Env750Tr/Y710S were synthesized as described in Materials and Methods. The positions for the Tyr residue in the YXXΦ motif and for the Ser residue in the mutant and the lengths of each construct are shown.
FIG. 2.
FIG. 2.
Mutation of the Tyr residue in the YXXΦ motif alone did not significantly affect HIV 89.6 Env protein surface expression. HeLa cells were transfected with the DNA constructs encoding HIV 89.6 Env and Env/Y710S by using Lipofectamine 2000. At 24 h posttransfection, the cells were labeled with [35S]Met-Cys labeling mix for 6 h, followed by surface biotinylation and immunoprecipitation as described in Materials and Methods. Protein samples were prepared and analyzed by SDS-PAGE. Lanes: 1, plasmid vector pCAGGS; 2, Env; 3, Env/Y710s; Pre, HIV Env precursor gp160; SU, surface subunit gp120; TM, transmembrane subunit gp41.
FIG. 3.
FIG. 3.
Combination of Tyr mutation and cytoplasmic domain truncation enhances HIV Env protein stability on cell surfaces. Protein expression was carried out by transfection of HeLa cells. At 24 h posttransfection, the cells were pulse-labeled with [35S]Met-Cys labeling mix for 1 h and then chased in complete medium for 2, 4, or 6 h, followed by surface biotinylation and immunoprecipitation as described in Materials and Methods. Protein samples were prepared and analyzed by SDS-PAGE. (A) Pulse-chase radioactive labeling analysis for expression of HIV 89.6 Env and Env/Y712S. Lanes: 1, plasmid vector pCAGGS; 2, Env, pulse only; 3, Env, 2-h chase; 4, Env, 4-h chase; 5, Env, 6-h chase; 6, Env/Y710S, pulse only; 7, Env/Y710S, 2-h chase; 8, Env/Y710S, 4-h chase; 9, Env/Y710S, 6-h chase. (B) Pulse-chase radioactive labeling analysis for expression of HIV 89.6 Env750Tr and Env750Tr/Y710S. Lanes: 1, plasmid vector pCAGGS; 2, Env750Tr, pulse only; 3, Env750Tr, 2-h chase; 4, Env750Tr, 4-h chase; 5, Env750Tr, 6-h chase; 6, Env750Tr/Y710S, pulse only; 7, Env750Tr/Y710S, 2-h chase; 8, Env750Tr/Y710S, 4-h chase; 9, Env750Tr/Y710S, 6-h chase; Pre, HIV Env precursor gp160; SU, surface subunit gp120; TM, transmembrane subunit gp41.
FIG. 4.
FIG. 4.
CD8 T-cell responses induced by DNA immunization against a dominant epitope in the HIV 89.6 Env protein were not affected by Tyr mutation and cytoplasmic domain truncation or their combination. Groups of BALB/c mice (six per group) were immunized by intramuscular injection with 100 μg of pCAGGS (vector control), Env, Env/Y710S, Env750Tr, or Env750Tr/Y710S at weeks 0 and 4. At week 6, mouse splenocytes were collected and stimulated with the peptide IGPGRAFYAR (10 μg/ml) for 6 h at 37°C. The cells were then stained for cell surface CD4 (PerCp) and CD8 (PE), as well as intracellular IFN-γ (APC), and analyzed by flow cytometry. Background IFN-γ-producing cells were determined by culturing mouse splenocytes with an irrelevant peptide (AMQMLKETI). Values in each box are the percentages of the gated population. (A) Gating of CD8 and CD4 lymphocytes and background IFN-γ production by CD8 or CD4 lymphocytes stimulated with peptide AMQMLKETI (control). (B) Representative results of FACS analysis for IFN-γ production by CD8 T cells from each immunization group stimulated with the peptide IGPGRAFYAR. Numbers in lower right boxes represent percentages of IFN-γ-positive CD8 T cells. (C) Percentages of IFN-γ-positive CD8 T cells for each immunization group after stimulation with the peptide IGPGRAFYAR. Error bars represent standard deviations for each group.
FIG. 5.
FIG. 5.
Combination of Tyr mutation and cytoplasmic domain truncation enhances induction of broad CD8 T-cell responses. Mouse splenocytes from each group were pooled, and the pooled splenocyte aliquots were stimulated for 16 h with each of the peptide pools covering the entire HIV 89.6 Env protein, followed by addition of brefeldin A, a further incubation of 5 h, and then stained for surface CD4 (PerCP) and CD8 (PE) and intracellular IFN-γ (APC). Numbers in lower right boxes represent percentages of IFN-γ-producing CD 8 T cells gated as shown in Fig. 4A. Peptide pools are 20-mers overlapping by 10 amino acids. Pool 1, amino acids 1 to 210; pool 2, amino acids 200 to 410; pool 3, amino acids 400 to 610; pool 4, amino acids 600 to 853.
FIG. 6.
FIG. 6.
Combination of Tyr mutation and cytoplasmic domain truncation enhances induction of CD4 T-cell responses. Mouse splenocytes from each group were pooled, and the pooled splenocyte aliquots (2 × 106 per well in 48-well plate) were stimulated for 16 h with each of the peptide pools covering the entire HIV 89.6 Env protein, followed by addition of brefeldin A, a further incubation of 5 h, and then staining for surface CD4 (PerCP) and CD8 (PE) and intracellular IFN-γ (APC). Numbers in lower right boxes represent percentages of IFN-γ-producing CD 4 T cells gated as shown in Fig. 4A. Peptide pools are 20-mers overlapping by 10 amino acids. Pool 1, amino acids 1 to 210; pool 2, amino acids 200 to 410; pool 3, amino acids 400 to 610; pool 4, amino acids 600 to 853.
FIG. 7.
FIG. 7.
The DNA construct for Env750Tr/Y710S is significantly more potent for inducing antibody responses. Sera from mice immunized with different DNA constructs were collected at 2 weeks postimmunization and analyzed for antibodies specific for HIV 89.6 Env protein by ELISA as described in Materials and Methods. The levels of antibody responses are expressed as the quantity of antibodies (total IgG, IgG1, or IgG2a) binding to HIV 89.6 gp120 in 1 ml of serum from each mouse. The results were analyzed with the Microsoft Excel program, and error bars indicate the standard deviations for each immunization group.
FIG. 8.
FIG. 8.
Neutralization of HIV 89.6 by sera from immunized mice. Sera from immunized mice were mixed with HIV 89.6 at 1:40 and 1:80 final dilutions, and neutralization of HIV 89.6 was analyzed in JC53-BL cells as described in Materials and Methods. The percentages of neutralization by each serum sample were calculated and compared. The average neutralization value and standard deviation are shown for each group.
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