Nishizawa M, Kamata M, Katsumata R, Aida Y
Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), Ibaraki 305-0074, Japan.
J Virol. 2000 Jul;74(13):6058-67. doi: 10.1128/jvi.74.13.6058-6067.2000.
Viral protein R (Vpr) of human immunodeficiency virus type 1 inhibits cell proliferation by arresting the cell cycle at the G(2) phase and inducing to apoptosis after G(2) arrest. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation via a novel pathway that is distinct from G(2) arrest. However, the mechanism of this effect of C81 is unknown. We demonstrate here that C81 can induce apoptosis via G(1) arrest of the cell cycle. Immunostaining for various markers of stages of the cell cycle and flow cytometry analysis of DNA content showed that most HeLa cells that had been transiently transfected with a C81 expression vector were arrested at the G(1) phase and not at the G(2) or S phase of the cell cycle. Staining for annexin V, which binds phosphatidylserine on the plasma membrane, as an early indicator of apoptosis and measurement of the activity of caspase-3, a signaling molecule in apoptotic pathways, indicated that C81 is a strong inducer of apoptosis. Expression of C81 induced the condensation, fragmentation, and clumping of chromatin that are typical of apoptosis. Furthermore, the kinetics of the C81-induced G(1) arrest were closely correlated with changes in the number of annexin V-positive cells and the activity of caspase-3. Replacement of Ile or Leu residues by Pro at positions 60, 67, 74, and 81 within the leucine zipper-like domain of C81 revealed that Ile60, Leu67, and Ile74 play important roles both in the C81-induced G(1) arrest and in apoptosis. Thus, it appears that C81 induces apoptosis through pathways that are identical to those utilized for G(1) arrest of the cell cycle. It has been reported that Ile60, Leu67, and Ile74 also play an important role in the C81-induced suppression of growth. These results suggest that the suppression of growth induced by C81 result in apoptosis that is independent of G(2) arrest of the cell cycle.
1型人类免疫缺陷病毒的病毒蛋白R（Vpr）通过使细胞周期停滞在G2期并在G2期停滞后诱导细胞凋亡来抑制细胞增殖。我们之前报道过，C81是一种Vpr的羧基末端截短形式，它通过一条不同于G2期停滞的新途径干扰细胞增殖。然而，C81这种作用的机制尚不清楚。我们在此证明，C81可通过使细胞周期停滞在G1期来诱导细胞凋亡。对细胞周期各阶段的各种标志物进行免疫染色以及对DNA含量进行流式细胞术分析表明，大多数瞬时转染了C81表达载体的HeLa细胞停滞在细胞周期的G1期，而非G2期或S期。用膜联蛋白V进行染色，膜联蛋白V可结合质膜上的磷脂酰丝氨酸，作为细胞凋亡的早期指标，并检测凋亡途径中的信号分子半胱天冬酶-3的活性，结果表明C81是一种强大的细胞凋亡诱导剂。C81的表达诱导了典型的细胞凋亡染色质浓缩、碎片化和聚集。此外，C81诱导的G1期停滞的动力学与膜联蛋白V阳性细胞数量的变化以及半胱天冬酶-3的活性密切相关。在C81的亮氨酸拉链样结构域内第60、67、74和81位用脯氨酸取代异亮氨酸或亮氨酸残基，结果表明Ile60、Leu67和Ile74在C81诱导的G1期停滞和细胞凋亡中均起重要作用。因此，似乎C81通过与用于细胞周期G1期停滞相同的途径诱导细胞凋亡。据报道，Ile60、Leu67和Ile74在C81诱导的生长抑制中也起重要作用。这些结果表明，C81诱导的生长抑制导致了独立于细胞周期G2期停滞的细胞凋亡。
