Genetic Characterization of HIV Type 1 Nef-Induced Vesicle Secretion
Genetic Characterization of HIV Type 1 Nef-Induced Vesicle Secretion
复制标题
DOI：
10.1089/aid.2009.0068
复制
发表时间：
2010-02-01
影响因子：
1.5
通讯作者：
Bond, Vincent C.
中科院分区：
文献类型：
--
作者：
Ali, Syed A.;Huang, Ming-Bo;Bond, Vincent C.
已知HIV-1 Nef蛋白是分泌的，我们的研究小组已经证明Nef是从nef转染和HIV-1感染的细胞分泌的，在小的外泌体样囊泡中（d. 40-100 nm）。分泌Nef的作用仍有待充分表征。因此，重要的是表征Nef分泌的性质和调节机制。我们假设Nef蛋白上的特定结构域与内体运输机制的组分相互作用，将Nef分选成多泡体（MVB）并将其包装在外泌体样囊泡中。为了鉴定这些结构域，制备了一系列跨越整个nef序列的突变体，并克隆到表达载体pQB 1中，其将突变体表达为Nef-GFP融合蛋白。这些构建体用于瞬时转染测定以鉴定Nef-GFP融合蛋白分泌所必需的序列。N-末端结构域被鉴定为Nef诱导的囊泡分泌的关键：（1）四个精氨酸残基（aa 17、19、21、22）的碱性簇，（2）磷酸弗林酸性簇序列（PACS; Glu 62 -65），和（3）先前未表征的跨越氨基酸残基66-70的结构域（VGFPV），我们将其命名为分泌修饰区（SMR）。在HIV-1 Nef中鉴定了另外的氨基酸P25、29 GVG 31和T44作为调节其分泌。这些残基与其他报道的Nef功能无关。豆蔻酰化结构域，泛素化赖氨酸残基，和C-末端部分的Nef（氨基酸71-206）的分泌没有影响。一个最小的HIV-1 Nef序列，包括所确定的图案，是足够的Nef诱导的囊泡分泌。
The HIV-1 Nef protein is known to be secreted, and our group has shown that Nef is secreted from nef-transfected and HIV-1-infected cells in small exosome-like vesicles (d. 40-100 nm). The role of secreted Nef remains to be fully characterized. Thus, it is important to characterize the nature of and the mechanisms regulating Nef secretion. We hypothesized that specific structural domains on the Nef protein interact with components of the endosomal trafficking machinery, sorting Nef into multivesicular bodies (MVB) and packaging it in exosome-like vesicles. To identify those domains, a series of mutants spanning the entire nef sequence were made and cloned into the expression vector pQB1, which expresses the mutants as Nef-GFP fusion proteins. These constructs were used in transient transfection assays to identify sequences necessary for secretion of the Nef-GFP fusion protein. N-terminal domains were identified as critical for Nef-induced vesicle secretion: (1) a basic cluster of four arginine residues (aa 17, 19, 21, 22), (2) the phosphofurin acidic cluster sequence (PACS; Glu62-65), and (3) a previously uncharacterized domain spanning amino acid residues 66-70 (VGFPV), which we named the secretion modification region (SMR). Additional amino acids P25, 29GVG31, and T44 were identified in HIV-1 Nef as regulating its secretion. These residues have not been associated with other reported Nef functions. The myristoylation domain, ubiquitination lysine residues, and the C-terminal portion of Nef (aa 71-206) had no effect on secretion. A minimal HIV-1 Nef sequence, comprising the identified motifs, was sufficient for Nef-induced vesicle secretion.