The size and conservation of a coiled-coil structure in the ectodomain of human BST-2/tetherin is dispensable for inhibition of HIV-1 virion release

Abstract

BST-2/CD317/tetherin is a host factor that inhibits HIV-1 release and is counteracted by HIV-1 Vpu. Structural studies indicate that the BST-2 ectodomain assumes a coiled-coil conformation. Here we studied the role of the BST-2 ectodomain for tethering function. First, we addressed the importance of the length and structure of the ectodomain by adding or substituting heterologous coiled-coil or non-coiled-coil sequences. We found that extending or replacing the BST-2 ectodomain using non-coiled-coil sequences resulted in loss of BST-2 function. Doubling the size of the BST-2 ectodomain by insertion of a heterologous coiled-coil motif or substituting the BST-2 coiled-coil domain with a heterologous coiled-coil motif maintained tethering function. Reductions in the size of the BST-2 coiled-coil domain were tolerated as well. In fact, deletion of the C-terminal half of the BST-2 ectodomain, including a series of seven consecutive heptad motifs did not abolish tethering function. However, slight changes in the positioning of deletions affecting the relative placing of charged or hydrophobic residues on the helix severely impacted the functional properties of BST-2. Overall, we conclude that the size of the BST-2 ectodomain is highly flexible and can be reduced or extended as long as the positioning of residues important for the stability of the dimer interface is maintained.

FIGURE 1.

BST-2 ectodomain requires a coiled-coil sequence at the C terminus. A, schematic structure of BST-2 variants rCC1, rCC2, and rCD4 compared with HA-tagged BST-2. Transmembrane domains (TM1, TM2) and the position of the HA tag are indicated as black and shaded boxes, respectively. Numbers indicate amino acid positions and the total number of residues for each of the proteins is indicated on the right. B, expression and dimerization of BST-2 variants. 293T cells were transfected with 1 μg each of the indicated constructs together with 4 μg of empty vector DNA. Mock-transfected 293T cells were included as negative control. Cells were harvested 24 h later, washed once with PBS, and resuspended in 400 μl of PBS. Cell suspensions were divided into two equal aliquots (200 μl each) and either mixed with an equal volume of reducing sample buffer (4% sodium dodecyl sulfate, 125 mm Tris-HCl, pH 6.8, 10% 2-mercaptoethanol, 10% glycerol, 0.002% bromphenol blue) or non-reducing sample buffer lacking 2-mercaptoethanol. Samples were heated for 10 to 15 min at 95 °C with occasional vortexing of the samples to shear cellular DNA. Residual insoluble material was removed by centrifugation (2 min, 13,500 × g in an Eppendorf Minifuge). Cell lysates were subjected to SDS-PAGE (12.5% for reduced samples; 10% for non-reduced samples); proteins were transferred to PVDF membranes and reacted with a BST-2-specific polyclonal rabbit antibody. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) and proteins were visualized by enhanced chemiluminescence (ECL, GE Healthcare). C, 293T cells were transfected with 5 μg of NL4–3/Udel together with increasing amounts (0, 0.1 μg (50:1), 0.25 μg (20:1), or 0.5 μg (10:1)) of DNA encoding BST-2 wt (solid circles), rCC1, rCC2, or rCD4 chimera (open circles) as indicated. Virus-containing supernatants were collected 24 h after transfection and used for the infection of HeLa TZM-bl indicator cells. Virus-induced luciferase activity was recorded 48 h later and was used as measure of virus release. Values are expressed as mean ± S.E. of three to six experiments. The infectivity of NL4–3/Udel virus produced in the absence of BST-2 was defined as 100%.

FIGURE 2.

The length of the ectodomain of BST-2 is variable. A, schematic diagram of constructs used in this experiment. Transmembrane domains (TM1, TM2) and the position of the HA tag are indicated as black and shaded boxes, respectively. Numbers refer to amino acid positions and the total number of residues for each of the proteins is indicated on the right. B, expression and dimerization of BST-2 variants was tested as described for Fig. 1B. C, 293T cells were transfected with 5 μg of NL4–3/Udel together with varying amounts of BST-2 wt (closed circles), or BST-2 variants iCC1, iCC2, or iCD4 (open circles) as indicated. Virus-containing supernatants were collected 24 h later and used to infect HeLa TZM-bl indicator cells. Data analysis was performed as described in the legend to Fig. 1C. The signal produced by NL4–3/Udel virus in the absence of BST-2 was each defined as 100% infectivity.

FIGURE 3.

The N-terminal region of the ectodomain of BST-2 is flexible. A, an alignment of human BST-2 crystal structures was generated using the superpose function within YASARA 12.4.1. Structures aligned were 3MQ7 (27) (white), 3MQC (27) (magenta), 3MQB (27) (yellow), 3NWH (26) (red), and 2XG7 (26) (blue). B, schematic diagram of constructs used in this experiment. Transmembrane domains (TM1, TM2) and the position of the HA tag are indicated. Dashed boxes indicate deletions. Numbers refer to amino acid positions. C, expression and dimerization of BST-2 variants was tested as described in the legend to Fig. 1B. D, 293T cells were transfected with 5 μg each of NL4–3/Udel together with varying amounts of BST-2 wt (closed circles) or deletion mutants (open circles) as indicated. Virus-containing supernatants were collected 24 h later and used to infect HeLa TZM-bl indicator cells. Analysis of the data were performed as described in the legend to Fig. 1C. The signal produced by NL4–3/Udel virus in the absence of BST-2 was each defined as 100% infectivity.

FIGURE 4.

The functional properties of BST-2 deletions do not correlate with the size of the deletions. A, schematic diagram of constructs used in this experiment. Transmembrane domains (TM1, TM2) and the position of the HA tag are indicated as black and shaded boxes, respectively. Dashed boxes indicate deletions. Numbers refer to amino acid positions. B, expression and dimerization of BST-2 variants was tested as described in the legend to Fig. 1B. C, 293T cells were transfected with 5 μg each of NL4–3/Udel together with varying amounts of BST-2 wt (closed circles) or mutant DNA (open circles) as indicated. Virus-containing supernatants were collected 24 h later and used to infect HeLa TZM-bl indicator cells. Analysis of the data were performed as described in the legend to Fig. 1C. The signal produced by NL4–3/Udel virus in the absence of BST-2 was each defined as 100% infectivity.

FIGURE 5.

Heptad motifs in the C-terminal coiled-coil region of the BST-2 ectodomain are dispensable for tetherin function. A, amino acid alignment of BST-2 deletions Δ97–147 and Δ95–146 as compared with BST-2 wt. Heptad motifs are highlighted. Green highlights indicate motifs that perfectly match the HXXHEXE consensus motif where H is hydrophobic or uncharged and E is a charged residue; yellow highlights depict motifs in which one residue in the heptad sequence does not match the canonical motif. Black and shaded areas represent the transmembrane domains and the HA tag, respectively. B, expression and dimerization of BST-2 variants was tested as described in the legend to Fig. 1B. C, 293T cells were transfected with 5 μg each of NL4–3/Udel together with varying amounts of BST-2 wt (closed circles) or mutant DNA (open circles) as indicated. Virus-containing supernatants were collected 24 h later and used to infect HeLa TZM-bl indicator cells. Analysis of the data were performed as described in the legend to Fig. 1C. Values are expressed as mean ± S.E. of nine experiments. The signal produced by NL4–3/Udel virus in the absence of BST-2 was defined as 100%.

FIGURE 6.

The positioning of residues along the helix interface may determine the functional properties of BST-2 variants. A, sequence alignment of BST-2 deletion mutants Δ96–116, Δ96–118, Δ97–147, and Δ95–146 against wild type BST-2. Only the region encompassing positions 81–159 in wild type BST-2 is shown. Amino acids labeled in panel B are shown in red and marked as raised fonts. B, models of BST-2 Δ96–118 and Δ96–116 as well as Δ97–147 and Δ95–146 were created using the human BST-2 structure (PDB code 2XG7) (26) as a template using FOLDX within YASARA 12.4.1. Thr⁹⁸ in BST-2 Δ96–118 and the corresponding Thr¹⁰⁰ in Δ96–116 are indicated in orange. Similarly, Leu⁹⁸ in BST-2 Δ97–147 and the corresponding Leu⁹⁷ in Δ95–146 are indicated in orange. Charged residues Glu⁹⁸ and Arg⁹⁵ are indicated in red and blue, respectively. Cys⁹¹ in Δ97–147 and Δ95–146 is highlighted in purple.