Induction of apoptosis by the Vpr protein of human immunodeficiency virus type 1 occurs independently of G(2) arrest of the cell cycle

The HIV-I accessory gene product Vpr can inhibit cell proliferation via cel l cycle arrest at the G(2) phase, and it can induce apoptosis after G(2) ar rest 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 c ell cycle. Thus, it seemed possible that expression of Vpr in cells might c ause apoptosis independently of the ability of Vpr to induce G(2) arrest We demonstrate here that Vpr-induced apoptosis occurs by a mechanism that doe s 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 arreste d 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 o f 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 do main of Vpr with Ala or Pro and used cells that expressed the mutant protei n 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 leuci ne 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 elucida tion of the mechanism of apoptosis induced by expression of Vpr protein, (C ) 2000 Academic Press.