Kuramitsu Madoka, Hashizume Chieko, Yamamoto Norio, Azuma Akihiko, Kamata Masakazu, Yamamoto Naoki, Tanaka Yoshimasa, Aida Yoko
Retrovirus Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Microbes Infect. 2005 Jul;7(9-10):1150-60. doi: 10.1016/j.micinf.2005.03.022. Epub 2005 Apr 19.
Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.
Vpr是人类免疫缺陷病毒1型（HIV-1）的辅助基因产物之一，它影响病毒和细胞增殖的多个方面，参与长末端重复序列（LTR）激活、使细胞周期停滞在G2期以及诱导细胞凋亡。我们发现Vpr在体内和体外均作为前体mRNA剪接的调节因子发挥新作用。通过逆转录聚合酶链反应（RT-PCR）和核糖核酸酶保护分析，我们发现Vpr在瞬时转染了Vpr表达载体的细胞中导致α-珠蛋白2和β-珠蛋白前体mRNA不完全剪接形式的积累。我们推测Vpr的这种新作用可能通过一条不同于使细胞周期停滞在G2期的途径发生。通过体外分析剪接反应，我们表明Vpr在体外抑制β-珠蛋白前体mRNA的剪接。Vpr适度抑制α-珠蛋白2前体mRNA内含子1的剪接，但内含子2的剪接未受影响。有趣的是，利用高滴度HIV-1/水疱性口炎病毒G蛋白的实验感染系统表明，由HIV-1前病毒表达的Vpr足以积累内源性α-珠蛋白2前体mRNA。因此，Vpr很可能有助于选择性抑制细胞前体mRNA的剪接。
