Epitope Tags beside the N-Terminal Cytoplasmic Tail of Human BST-2 Alter Its Intracellular Trafficking and HIV-1 Restriction

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.

BST-2可阻断包括HIV-1在内的多种包膜病毒的颗粒释放，其抗病毒活性依赖于四个结构域的拓扑排布。此前已有多项研究探讨了BST-2胞质尾区（cytoplasmic tail, CT）的多项功能，但该结构域的确切作用仍有待明确阐明。本研究中，我们针对人源BST-2胞质尾区的截短操作与常用标签修饰，探究其对该蛋白细胞内运输、信号转导及抗HIV-1活性的影响。截短胞质尾区的BST-2对缺失Vpu的HIV-1乃至野生型HIV-1均展现出强效抑制活性。然而，经N端HA标签修饰的截短胞质尾区BST-2几乎丧失抗病毒活性，且在细胞表面表达水平与细胞内定位方面，与原型蛋白存在显著差异。进一步研究证实，将胞质尾区替换为疏水标签，或通过干扰其正常囊泡运输改变BST-2的功能。值得注意的是，我们证实了灵长类BST-2中保守存在的、位于胞质尾区与跨膜结构域（transmembrane domain）连接区域的带正电基序"KRXK"，对该蛋白的运输过程与抗病毒功能至关重要。上述结果表明，尽管BST-2的胞质尾区并非其抗病毒活性所必需，但该区域的氨基酸残基组成或可通过影响其正常运输过程，进而对其功能发挥重要调控作用。本研究结果为BST-2的结构-功能模型提供了新的补充阐释。