Garg Himanshu, Joshi Anjali, Freed Eric O, Blumenthal Robert
Membrane Structure and Function Section, Center for Cancer Research Nanobiology Program, NCI, National Institutes of Health, Frederick, Maryland 21702, USA.
J Biol Chem. 2007 Jun 8;282(23):16899-906. doi: 10.1074/jbc.M701701200. Epub 2007 Apr 6.
The loss of CD4(+) T cells in HIV-1 infections is hypothesized to be caused by apoptosis of bystander cells mediated by cell surface-expressed HIV-1 Env glycoprotein. However, the mechanism by which Env mediates this process remains controversial. Specifically, the role of HIV-1 gp120 binding to CD4 and CXCR4 versus the fusion process mediated by gp41 remains unresolved. Env-induced apoptosis in bystander cells has been shown to be gp41-dependent and correlates with the redistribution of membrane lipids between Env-expressing cells and target cells (hemifusion). Using a rational mutagenesis approach aimed at targeting Env function via the gp41 subunit, we examined the role of HIV gp41 in bystander apoptosis. A mutation in the fusion domain of gp41 (V513E) resulted in a fusion-defective Env that failed to induce apoptosis. A mutation in the gp41 N-terminal helix (G547D) reduced cell fusion capacity and apoptosis; conversely, an Env mutant with a deletion of the gp41 cytoplasmic tail (Ct Del) enhanced both cell-to-cell fusion and apoptosis. Most significantly, an Env mutant containing a substitution in the loop region of gp41 (D589L) mediated transfer of lipids (hemifusion) to bystander cells but was defective in cell-to-cell and to a lesser degree virus-to-cell fusion. This mutant was still able to induce apoptosis in bystander cells. Hence, we have provided the first direct evidence that gp41-mediated hemifusion is both required and sufficient for induction of apoptosis in bystander cells. These results may help to explain the mechanism of HIV-1 Env-induced T cell depletion.
据推测，HIV-1感染中CD4(+) T细胞的丧失是由细胞表面表达的HIV-1 Env糖蛋白介导的旁观者细胞凋亡所致。然而，Env介导这一过程的机制仍存在争议。具体而言，HIV-1 gp120与CD4和CXCR4结合的作用与gp41介导的融合过程之间的关系仍未解决。已证明Env诱导旁观者细胞凋亡是gp41依赖性的，并且与表达Env的细胞和靶细胞之间膜脂的重新分布（半融合）相关。我们采用一种合理的诱变方法，旨在通过gp41亚基靶向Env功能，研究了HIV gp41在旁观者细胞凋亡中的作用。gp41融合结构域中的一个突变（V513E）导致融合缺陷型Env，无法诱导细胞凋亡。gp41 N端螺旋中的一个突变（G547D）降低了细胞融合能力和细胞凋亡；相反，缺失gp41胞质尾（Ct Del）的Env突变体增强了细胞间融合和细胞凋亡。最重要的是，一个在gp41环区含有替代突变（D589L）的Env突变体介导了脂质向旁观者细胞的转移（半融合），但在细胞间融合以及在较小程度上的病毒与细胞融合方面存在缺陷。该突变体仍能够诱导旁观者细胞凋亡。因此，我们提供了首个直接证据，证明gp41介导的半融合对于诱导旁观者细胞凋亡既是必需的也是充分的。这些结果可能有助于解释HIV-1 Env诱导T细胞耗竭的机制。
