The HIV-1 accessory gene product Vpr can inhibit cell proliferation via cell cycle arrest at the G2 phase, and it can induce apoptosis after G2 arrest. We found recently that C81, a carboxy-terminally truncated form of Vpr, induced apoptosis via G1 arrest but did not induce G2 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 G2 arrest. We demonstrate here that Vpr-induced apoptosis occurs by a mechanism that does not necessarily require induction of G2 arrest. First, it was found that the extent of apoptosis reached a maximum even when few cells were arrested at the G2 phase of the cell cycle and was reduced in inverse proportion to the increased induction of G2 arrest. Thus, the extent of induction of G2 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 G2 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. (C) 2000 Academic Press.