Nishizawa M, Kamata M, Mojin T, Nakai Y, Aida Y
RIKEN Tsukuba Institute, 3-1-1 Koyadai, Tsukuba, Ibaraki, 305-0074, USA.
Virology. 2000 Oct 10;276(1):16-26. doi: 10.1006/viro.2000.0534.
The HIV-1 accessory gene product Vpr can inhibit cell proliferation via cell cycle arrest at the G(2) phase, and it can induce apoptosis after G(2) arrest. We found recently that C81, a carboxy-terminally truncated form of Vpr, induced apoptosis via G(1) arrest but did not induce G(2) arrest of the cell cycle. Thus, it seemed possible that expression of Vpr in cells might cause apoptosis independently of the ability of Vpr to induce G(2) arrest. We demonstrate here that Vpr-induced apoptosis occurs by a mechanism that does not necessarily require induction of G(2) arrest. First, it was found that the extent of apoptosis reached a maximum even when few cells were arrested at the G(2) phase of the cell cycle and was reduced in inverse proportion to the increased induction of G(2) arrest. Thus, the extent of induction of G(2) arrest was not correlated with the extent of Vpr-induced apoptosis. Furthermore, we replaced the Ile/Leu residues in the leucine zipper-like domain of Vpr with Ala or Pro and used cells that expressed the mutant protein to demonstrate that Vpr caused apoptosis in a manner that was independent of G(2) arrest. Finally, replacement of Ile/Leu by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of wild-type Vpr and C81 revealed that the Ile/Leu residues at positions 60, 67, and 74 in the leucine zipper-like domain were indispensable for induction of apoptosis induced by Vpr and by C81 and confirmed, in addition, that both processes might be regulated by the same pathway. C81 appears to be a useful tool for elucidation of the mechanism of apoptosis induced by expression of Vpr protein.
HIV-1辅助基因产物Vpr可通过使细胞周期停滞在G2期来抑制细胞增殖，并且在G2期停滞之后可诱导细胞凋亡。我们最近发现，Vpr的一种羧基末端截短形式C81通过使G1期停滞来诱导细胞凋亡，但不会诱导细胞周期的G2期停滞。因此，细胞中Vpr的表达似乎有可能独立于Vpr诱导G2期停滞的能力而导致细胞凋亡。我们在此证明，Vpr诱导的细胞凋亡是通过一种不一定需要诱导G2期停滞的机制发生的。首先，发现即使很少有细胞停滞在细胞周期的G2期，细胞凋亡的程度也会达到最大值，并且与G2期停滞诱导的增加成反比降低。因此，G2期停滞的诱导程度与Vpr诱导的细胞凋亡程度无关。此外，我们将Vpr亮氨酸拉链样结构域中的异亮氨酸/亮氨酸残基替换为丙氨酸或脯氨酸，并使用表达突变蛋白的细胞来证明Vpr以独立于G2期停滞的方式导致细胞凋亡。最后，在野生型Vpr和C81的亮氨酸拉链样结构域内的第60、67、74和81位将异亮氨酸/亮氨酸替换为脯氨酸，结果表明亮氨酸拉链样结构域中第60、67和74位的异亮氨酸/亮氨酸残基对于Vpr和C81诱导的细胞凋亡是不可或缺的，此外还证实这两个过程可能受同一途径调控。C81似乎是阐明Vpr蛋白表达诱导细胞凋亡机制的有用工具。
