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. 2005 Apr;79(8):4720-9.
doi: 10.1128/JVI.79.8.4720-4729.2005.

Role of the specific amino acid sequence of the membrane-spanning domain of human immunodeficiency virus type 1 in membrane fusion

Kosuke Miyauchi  1 , Jun Komano, Yoshiyuki Yokomaku, Wataru Sugiura, Naoki Yamamoto, Zene Matsuda
Affiliations

Role of the specific amino acid sequence of the membrane-spanning domain of human immunodeficiency virus type 1 in membrane fusion

Kosuke Miyauchi et al. J Virol. 2005 Apr.

Abstract

Fusion between cell and virus membranes mediated by gp41 initiates the life cycle of human immunodeficiency virus type 1. In contrast to the many studies that have elucidated the structure-function relationship of the ectodomain, the study of the membrane-spanning domain (MSD) has been rather limited. In particular, the role that the MSD's specific amino acid sequences may have in membrane fusion as well as other gp41 functions is not well understood. The MSD of gp41 contains well-conserved glycine residues that form the GXXXG motif (G, glycine; X, other amino acid residues), a motif often found at the helix-helix interface of membrane spanning alpha-helices. Here we examined the role that the specific amino acid sequence of the gp41 MSD has in gp41 function, particularly in membrane fusion, by making two types of MSD mutants: (i) glycine substitution mutants in which glycine residues of the MSD were mutated to alanine or leucine residues, and (ii) replacement mutants in which the entire MSD was replaced with one derived from glycophorin A or from vesicular stomatitis virus G. The substitution of glycines did not affect gp41 function. MSD-replacement mutants, however, showed severely impaired fusion activity. The assay using the Env expression vector revealed defects in membrane fusion after CD4 binding steps in the MSD-replacement mutants. In addition, the change in Env processing was noted for MSD-replacement mutants. These results suggest that the MSD of gp41 has a relatively wide but not unlimited tolerance for mutations and plays a critical role in membrane fusion as well as in other steps of Env biogenesis.

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Figures

FIG. 1.
FIG. 1.
Amino acid sequences of the MSD of HIV-1 isolates and several membrane proteins. (A) Amino acid sequences of the predicted MSD of HXB2, HIV-1 isolates, and several membrane proteins are shown. The sequence of HIV-1 isolates was according to HIV Sequence Compendium 2001 (17). The MSDs of CD4 and CD22 that could replace the MSD of gp41 without affecting gp41's function are also shown (41, 45). The capital letters indicate the amino acid sequences within the predicted MSD. The small letters indicate the amino acid sequences surrounding the MSD. Glycine residues in the GXXXG motif are underlined. (B) Amino acid sequences of MSD mutants. The mutated portions are underlined.
FIG. 2.
FIG. 2.
Protein profiles of cell and virus lysates of the WT and mutants. The cell and virus lysates for glycine substitution (A and B) and MSD replacement mutants (C and D) were prepared from COS7 transfected with proviral DNA. The Env proteins (gp160, gp120) were detected with anti-gp120 polyclonal antibody. Gag (p24, p17) and Pol (p66, p51) were detected using serum from an individual infected with HIV-1. The name of the provirus DNA is indicated below, and the bands corresponding to gp160, gp120, p66, p51, p24, and p17 are shown by arrows. (C and D) For MSD replacement mutants, two different amounts of lysates were loaded for each mutant. The second lane of each sample received an amount of the lysate that was half of the amount used for the preceding lane.
FIG. 3.
FIG. 3.
Relative fusion activity of MSD mutants in MAGI cell assay. Fusion activity of the WT and MSD mutants was expressed using a fusion index (fusion index = 2x + y, where x is the number of multinucleated cells [number of nuclei ≥ 5 in five visual fields] and y is the number of multinucleated cells [number of nuclei < 5 in five visual fields]). The fusion index was defined to reflect the number of nuclei in multinucleated cells. Fusion activities for each mutant are shown after normalization to that of the WT (the WT activity was set at 100%). Similar results were obtained in three independent experiments.
FIG. 4.
FIG. 4.
Replication profiles of the WT and MSD mutants in Jurkat cells. Viral stocks of the WT and each mutant were prepared from culture supernatant of COS7 cells transfected with proviral DNA, and Jurkat cells were infected with each virus adjusted by the amount of p24. Replication was monitored by measuring the amount of p24 in the culture supernatant at specific time points following infection. A representative result of three independent experiments is shown.
FIG. 5.
FIG. 5.
Evaluation of the cell surface expression and the CD4-binding capacity of Env of MSD mutants. (A) The map of the Env expression vector pElucEnv. pElucEnv supports the expression of HIV-1 env (gp120/gp41) and the gene of the EGFP-firefly luciferase (EGFP-fLuciferase) hybrid protein from two separate promoters. The NheI-BamHI fragments of pSP65HXB2RU3ΔN WT, MSD GpA, MSD GpAG83I, or MSD VSV-G were cloned into pElucEnv. 5′LTR, 5′ long terminal repeat of HIV-1; 3′LTR, 3′ long terminal repeat of HIV-1. tat, vpu, and rev represent tat, vpu, and rev of HIV-1. AmpR, beta-lactamase; SV40, simian virus 40 late promoter; ColE1, ColE1 replication origin; NheI and BamHI, restriction sites used for cloning of the mutated env. (B) Cell surface expression of Env. COS7 cells were transfected with each pElucEnv construct and subjected to flow cytometric analysis as described in the Materials and Methods section. The signal for each Env is shown with a gray line. The filled area depicts the signal obtained for the control vector, EnvKO. (C) CD4-binding capacity of Env of MSD mutants. The cell lysates prepared from the COS7 cells that had been transfected with the pElucEnv construct or the CD4 expression vector were mixed together, and CD4 was immunoprecipitated using OKT-4. The amount of gp120 coimmunoprecipitated with CD4 was evaluated. The upper panel shows the expression level of each Env mutant. Coimmunoprecipitated gp120 was detected using anti-gp120 polyclonal antibodies (shown in the lower panel).
FIG. 6.
FIG. 6.
Cell-cell fusion analysis. The results of a dye-transfer assay using a coculture system and COS and T cells are shown. The COS7 cells transiently transfected with each pElucEnv construct were cocultured with H9 cells that had been loaded with CellTracker CM-DiI (pseudo-red) and Calcein blue, AM (pseudo-blue). Pictures taken at 2 h after coculture are shown in panel A. Typical fused cells having green, red, or blue fluorescence are indicated by arrows. Original magnification, ×200. (B) Relative dye-transfer frequencies of MSDrep mutants. The number of Env-expressing cells (green cells) whose membranes or cytoplasms are labeled with CM-DiI (red) or Calcein blue, AM (blue), respectively, was counted in five, randomly selected fields. The value was established by setting WT at 100%. The averages of the results from four experiments are shown. (C) Results of T7 RNApol-transfer assay to determine fusion pore formation of MSD mutants. The COS7 cells were transfected with each pElucEnv construct and the T7 RNApol-responsive reporter plasmid, pTM3hRL. At 48 h after transfection, the transfected COS7 cells were cocultured with the 293CD4 cells that had been transfected with pCMMP T7RNApoliresGFP. At 12 h after coculture, the cells were lysed and firefly luciferase and renilla luciferase activity levels were measured. Pore formation efficiencies were calculated by comparing the induced renilla luciferase activity to the firefly luciferase activity. A representative result of four independent experiments is shown.
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