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Epitope tags beside the N-terminal cytoplasmic tail of human BST-2 alter its intracellular trafficking and HIV-1 restriction.

Lv M, Wang J, Zhang J, Zhang B, Wang X, Zhu Y, Zuo T, Liu D, Li X, Wu J, Zhang H, Yu B, Wu H, Zhao X, Kong W, Yu X - PLoS ONE (2014)

Bottom Line: Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function.These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function.These observations provide additional implications for the structure-function model of BST-2.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, P.R. China.

ABSTRACT
BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.

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Comparison of epitope tagged N-terminus of BST-2 ΔN20 with its native cytosolic region and orthologues.(A) Analysis of hydrophobicity in N-terminal peptides of BST-2 variants. The hydrophobic residues (M, F, W, V, L, I, P, A) were marked and the percentages were calculated to represent the total hydrophobicity. (B) Analysis of positive charged residues (K, R, H) in N-terminal regions of BST-2 variants. (C) The sequence alignment of BST-2 and its orthologues in the N-terminus. The conserved positive charged residues in the conjunctive region between the cytotail and the transmembrane domain were marked. (D) Indicated amount of BST-2 WT or BST-2 ΔKRK, a mutant with mutated above mentioned positive charged residues was co-transfected with 1 µg of the proviral plasmid pNL4-3 ΔVpu in 293T cells. At 48 h post-transfection, cultured supernatants were ultracentrifuged to concentrate the virus particles. Virions and cell lysates were analyzed by Western blotting.
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pone-0111422-g008: Comparison of epitope tagged N-terminus of BST-2 ΔN20 with its native cytosolic region and orthologues.(A) Analysis of hydrophobicity in N-terminal peptides of BST-2 variants. The hydrophobic residues (M, F, W, V, L, I, P, A) were marked and the percentages were calculated to represent the total hydrophobicity. (B) Analysis of positive charged residues (K, R, H) in N-terminal regions of BST-2 variants. (C) The sequence alignment of BST-2 and its orthologues in the N-terminus. The conserved positive charged residues in the conjunctive region between the cytotail and the transmembrane domain were marked. (D) Indicated amount of BST-2 WT or BST-2 ΔKRK, a mutant with mutated above mentioned positive charged residues was co-transfected with 1 µg of the proviral plasmid pNL4-3 ΔVpu in 293T cells. At 48 h post-transfection, cultured supernatants were ultracentrifuged to concentrate the virus particles. Virions and cell lysates were analyzed by Western blotting.

Mentions: Next we analyzed basic characteristics of these N-terminal tags and native cytosolic domain of human BST-2, attempting to draw parallels with the virion release restriction. As the cytosolic region usually appears as hydrophilic nature, we wondered if the dysfunction is related to the excessive hydrophobic residue content. The hydrophobic residues (M, F, W, V, L, I, P, A) were marked (Fig. 8A). The percentage of hydrophobic residues in NHA and NHARD is 40%, which is greater than that for NMyc (36.4%) and NFlag (11.1%). While the cytosolic region of BST-2 (BST-2 1–20) and short form BST-2 (BST-2 13–20) is 25% and 12.5%, respectively. Thus, the excessive proportion of hydrophobic residues in the N-terminus may impair the normal trafficking and the antiviral activity of BST-2.


Epitope tags beside the N-terminal cytoplasmic tail of human BST-2 alter its intracellular trafficking and HIV-1 restriction.

Lv M, Wang J, Zhang J, Zhang B, Wang X, Zhu Y, Zuo T, Liu D, Li X, Wu J, Zhang H, Yu B, Wu H, Zhao X, Kong W, Yu X - PLoS ONE (2014)

Comparison of epitope tagged N-terminus of BST-2 ΔN20 with its native cytosolic region and orthologues.(A) Analysis of hydrophobicity in N-terminal peptides of BST-2 variants. The hydrophobic residues (M, F, W, V, L, I, P, A) were marked and the percentages were calculated to represent the total hydrophobicity. (B) Analysis of positive charged residues (K, R, H) in N-terminal regions of BST-2 variants. (C) The sequence alignment of BST-2 and its orthologues in the N-terminus. The conserved positive charged residues in the conjunctive region between the cytotail and the transmembrane domain were marked. (D) Indicated amount of BST-2 WT or BST-2 ΔKRK, a mutant with mutated above mentioned positive charged residues was co-transfected with 1 µg of the proviral plasmid pNL4-3 ΔVpu in 293T cells. At 48 h post-transfection, cultured supernatants were ultracentrifuged to concentrate the virus particles. Virions and cell lysates were analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4210262&req=5

pone-0111422-g008: Comparison of epitope tagged N-terminus of BST-2 ΔN20 with its native cytosolic region and orthologues.(A) Analysis of hydrophobicity in N-terminal peptides of BST-2 variants. The hydrophobic residues (M, F, W, V, L, I, P, A) were marked and the percentages were calculated to represent the total hydrophobicity. (B) Analysis of positive charged residues (K, R, H) in N-terminal regions of BST-2 variants. (C) The sequence alignment of BST-2 and its orthologues in the N-terminus. The conserved positive charged residues in the conjunctive region between the cytotail and the transmembrane domain were marked. (D) Indicated amount of BST-2 WT or BST-2 ΔKRK, a mutant with mutated above mentioned positive charged residues was co-transfected with 1 µg of the proviral plasmid pNL4-3 ΔVpu in 293T cells. At 48 h post-transfection, cultured supernatants were ultracentrifuged to concentrate the virus particles. Virions and cell lysates were analyzed by Western blotting.
Mentions: Next we analyzed basic characteristics of these N-terminal tags and native cytosolic domain of human BST-2, attempting to draw parallels with the virion release restriction. As the cytosolic region usually appears as hydrophilic nature, we wondered if the dysfunction is related to the excessive hydrophobic residue content. The hydrophobic residues (M, F, W, V, L, I, P, A) were marked (Fig. 8A). The percentage of hydrophobic residues in NHA and NHARD is 40%, which is greater than that for NMyc (36.4%) and NFlag (11.1%). While the cytosolic region of BST-2 (BST-2 1–20) and short form BST-2 (BST-2 13–20) is 25% and 12.5%, respectively. Thus, the excessive proportion of hydrophobic residues in the N-terminus may impair the normal trafficking and the antiviral activity of BST-2.

Bottom Line: Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function.These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function.These observations provide additional implications for the structure-function model of BST-2.

View Article: PubMed Central - PubMed

Affiliation: National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, P.R. China.

ABSTRACT
BST-2 blocks the particle release of various enveloped viruses including HIV-1, and this antiviral activity is dependent on the topological arrangement of its four structural domains. Several functions of the cytoplasmic tail (CT) of BST-2 have been previously discussed, but the exact role of this domain remains to be clearly defined. In this study, we investigated the impact of truncation and commonly-used tags addition into the CT region of human BST-2 on its intracellular trafficking and signaling as well as its anti-HIV-1 function. The CT-truncated BST-2 exhibited potent inhibition on Vpu-defective HIV-1 and even wild-type HIV-1. However, the N-terminal HA-tagged CT-truncated BST-2 retained little antiviral activity and dramatically differed from its original protein in the cell surface level and intracellular localization. Further, we showed that the replacement of the CT domain with a hydrophobic tag altered BST-2 function possibly by preventing its normal vesicular trafficking. Notably, we demonstrated that a positive charged motif "KRXK" in the conjunctive region between the cytotail and the transmembrane domain which is conserved in primate BST-2 is important for the protein trafficking and the antiviral function. These results suggest that although the CT of BST-2 is not essential for its antiviral activity, the composition of residues in this region may play important roles in its normal trafficking which subsequently affected its function. These observations provide additional implications for the structure-function model of BST-2.

Show MeSH
Related in: MedlinePlus