Kueck Tonya, Foster Toshana L, Weinelt Julia, Sumner Jonathan C, Pickering Suzanne, Neil Stuart J D
Department of Infectious Disease, King's College London School of Life Sciences and Medicine, Guy's Hospital, London, United Kingdom.
PLoS Pathog. 2015 Aug 28;11(8):e1005141. doi: 10.1371/journal.ppat.1005141. eCollection 2015 Aug.
HIV-1 Vpu prevents incorporation of tetherin (BST2/ CD317) into budding virions and targets it for ESCRT-dependent endosomal degradation via a clathrin-dependent process. This requires a variant acidic dileucine-sorting motif (ExxxLV) in Vpu. Structural studies demonstrate that recombinant Vpu/tetherin fusions can form a ternary complex with the clathrin adaptor AP-1. However, open questions still exist about Vpu's mechanism of action. Particularly, whether endosomal degradation and the recruitment of the E3 ubiquitin ligase SCFβTRCP1/2 to a conserved phosphorylated binding site, DSGNES, are required for antagonism. Re-evaluation of the phenotype of Vpu phosphorylation mutants and naturally occurring allelic variants reveals that the requirement for the Vpu phosphoserine motif in tetherin antagonism is dissociable from SCFβTRCP1/2 and ESCRT-dependent tetherin degradation. Vpu phospho-mutants phenocopy ExxxLV mutants, and can be rescued by direct clathrin interaction in the absence of SCFβTRCP1/2 recruitment. Moreover, we demonstrate physical interaction between Vpu and AP-1 or AP-2 in cells. This requires Vpu/tetherin transmembrane domain interactions as well as the ExxxLV motif. Importantly, it also requires the Vpu phosphoserine motif and adjacent acidic residues. Taken together these data explain the discordance between the role of SCFβTRCP1/2 and Vpu phosphorylation in tetherin antagonism, and indicate that phosphorylation of Vpu in Vpu/tetherin complexes regulates promiscuous recruitment of adaptors, implicating clathrin-dependent sorting as an essential first step in tetherin antagonism.
HIV-1 Vpu可阻止连接蛋白（BST2/CD317）掺入出芽的病毒体，并通过网格蛋白依赖的过程将其靶向内体进行ESCRT依赖的降解。这需要Vpu中一个可变的酸性双亮氨酸分选基序（ExxxLV）。结构研究表明，重组Vpu/连接蛋白融合体可与网格蛋白衔接蛋白AP-1形成三元复合物。然而，Vpu的作用机制仍存在一些悬而未决的问题。特别是，内体降解以及E3泛素连接酶SCFβTRCP1/2募集到保守的磷酸化结合位点DSGNES是否是拮抗作用所必需的。对Vpu磷酸化突变体和天然存在的等位基因变体的表型重新评估发现，Vpu磷酸丝氨酸基序在连接蛋白拮抗中的需求与SCFβTRCP1/2和ESCRT依赖的连接蛋白降解是可分离的。Vpu磷酸化突变体模拟ExxxLV突变体，并且在没有SCFβTRCP1/2募集的情况下可通过直接与网格蛋白相互作用来挽救。此外，我们证明了Vpu与细胞中的AP-1或AP-2之间存在物理相互作用。这需要Vpu/连接蛋白跨膜结构域相互作用以及ExxxLV基序。重要的是，这还需要Vpu磷酸丝氨酸基序和相邻的酸性残基。综合这些数据解释了SCFβTRCP1/2的作用与Vpu磷酸化在连接蛋白拮抗中的不一致，并表明Vpu/连接蛋白复合物中Vpu的磷酸化调节衔接蛋白的杂乱募集，这意味着网格蛋白依赖的分选是连接蛋白拮抗中必不可少的第一步。
