Role of the HIV-1 envelope transmembrane domain in intracellular sorting

Abstract

Background: The envelope protein of lentiviruses are type I transmembrane proteins, and their transmembrane domain contains conserved potentially charged residues. This highly unusual feature would be expected to cause endoplasmic reticulum (ER) localization. The aim of this study was to determine by which means the HIV-1 Env protein is transported to the cell surface although its transmembrane domain contains a conserved arginine residue.

Results: We expressed various chimeric proteins and analyzed the influence of their transmembrane domain on their intracellular localization. The transmembrane domain of the HIV-1 Env protein does not cause ER retention. This is not due to the presence of conserved glycine residues, or to the position of the arginine residue, but to the length of the transmembrane domain. A shortened version of the Env transmembrane domain causes arginine-dependent ER targeting. Remarkably, the transmembrane domain of the HIV-1 Env protein, although it does not confer ER retention, interacts efficiently with negatively charged residues in the membrane.

Conclusion: These results suggest that the intrinsic properties of the HIV-1 Env transmembrane domain allow the protein to escape ER-retention mechanisms, while maintaining its ability to interact with cellular proteins and to influence cellular physiology.

Keywords: Endoplasmic reticulum; Envelope protein; HIV-1; Secretory pathway; Transmembrane domain; gp160.

Fig. 1

TMDs of lentiviral envelope proteins exhibit conserved charged residues. The sequence of the TMDs and surrounding region of several lentiviral envelope proteins are represented. Amino acid positions for each sequence are numbered in italic. The approximate position of the predicted TMDs is indicated. Potentially charged amino-acid residues are in red and glycine residues in italic. The Uniprot reference number of each protein is indicated

Fig. 2

The HIV-1 Env TMD allows efficient cell surface localization. a The T-H0 chimera is composed of the extracellular domain of the Tac protein, a 21-hydrophobic residue transmembrane domain (TMD), and a short cytosolic domain (CYT). In T-E14, the Tac extracellular domain is fused to the Env extracellular juxtamembrane segment followed by the Env TMD and its complete cytoplasmic domain. The T-E15 protein exhibits a short truncated cytosolic domain. In T-E26, the Tac extracellular domain is fused directly to the Env TMD and a short cytoplasmic tail. Detailed amino acid sequences are described in Table 1. b Hela cell expressing the indicated Tac chimeric protein were labeled by immunofluorescence before (Surface) and after (Total) permeabilization, using antibodies specific for the Tac extracellular domain. All pictures were taken with the same settings with a confocal microscope (LSM700, Zeiss). Scale bar: 10 μm. c The amount of each Tac chimeric protein present at the cell surface was determined by dividing the surface fluorescence by the total fluorescence. For calibration, this ratio is set to 100 arbitrary units (A.U.) for T-H0. T-E14 is significantly less localized at the cell surface than T-H0 (n = 4; one-way analysis of variance: p < 0.01; *: post-hoc Tukey-Kramer p < 0.05)

Fig. 3

Glycine residues in the HIV-1 Env TMD do not affect its intracellular targeting. a Fusion proteins composed of the extracellular domain of the Tac protein, the transmembrane domain of Env protein (TMD) and a short cytosolic tail (CYT) are derived from the T-E26 chimera. In T-E39 the arginine present in the Env TMD is mutated to leucine. In T-E59 glycine residues in the Env TMD are mutated to leucines. Both mutations are combined in T-E60 (see also Table 1). b The fraction of each chimeric protein present at the cell surface was determined as described in the legend to Fig. 2. Statistical analysis revealed no significant differences from T-H0 (n = 3; one-way analysis of variance: p = 0.246)

Fig. 4

The position of the arginine residue in the HIV-1 Env TMD does not affect its intracellular targeting. a In the T-E26 chimera, an arginine is present in the Env TMD at position 14. A series of chimeric proteins where the arginine was moved to other positions were constructed (T-E41 to T-E46) (see also Table 1). Positions within the sequence are numbered from the luminal to the cytoplasmic end of the TMD. b The fraction of each chimeric protein present at the cell surface was determined as described in the legend to Fig. 2. Statistical analysis revealed no significant differences from T-H0 (n = 3; one-way analysis of variance: p = 0.77)

Fig. 5

The intracellular targeting of the HIV-1 Env TMD is influenced by its length. a Compared to T-E26, two amino-acids were deleted from the Env TMD in T-E28, and four in T-E30. In addition, in T-E34 the arginine present in the TMD was mutated to a leucine residue (see also Table1). b Intracellular localization of each Tac chimeric protein was determined as described in the legend to Fig. 2. Scale bar: 10 μm. c The fraction of each chimeric protein present at the cell surface was determined as described in the legend to Fig. 2. T-E30 is significantly less localized at the cell surface than T-H0 (n = 4; one-way analysis of variance: p < 0.01; *: post-hoc Tukey-Kramer p < 0.05)

Fig. 6

A shortened HIV-1 Env TMD acts as an ER-targeting motif. a Immunofluorescence microscopy of HeLa cells co-expressing various Tac fusion proteins (stained with an anti-Tac antibody) and a marker of the endoplasmic reticulum (ER-YFP). All pictures were taken with a confocal microscope (LSM700, Zeiss). Scale bar: 10 μm. b The colocalization of Tac proteins with the ER was quantified by measuring the Pearson’s correlation coefficient with Imaris software. T-E30 and T-H0KKxx are significantly localized in the ER (n = 4; *: Student’s t-test p < 0.01)

Fig. 7

The HIV-1 Env TMD associates efficiently with a TMD containing a negative charge. a To reveal a putative interaction between TMDS, HeLa cells were co-transfected with plasmids encoding the δ chain of the CD3 receptor fused to the β-galactosidase (Gal) and various Tac fusion proteins. b Tac fusion proteins were immunoprecipitated and the amount of co-precipitated β-galactosidase activity assessed to reveal the degree of association with the δ chain of the CD3 receptor. The mean ± SEM of at least five independent experiments are indicated (one-way analysis of variance: p < 0.01; *: post-hoc Tukey-Kramer p < 0.05)