HIV-1 Vpr-Induced Apoptosis Is Cell Cycle Dependent and Requires Bax but Not ANT

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Abstract
The HIV-1 accessory protein viral protein R (Vpr) causes G2 arrest and apoptosis in infected cells. We previously identified the DNA damage–signaling protein ATR as the cellular factor that mediates Vpr-induced G2 arrest and apoptosis. Here, we examine the mechanism of induction of apoptosis by Vpr and how it relates to induction of G2 arrest. We find that entry into G2 is a requirement for Vpr to induce apoptosis. We investigated the role of the mitochondrial permeability transition pore by knockdown of its essential component, the adenine nucleotide translocator. We found that Vpr-induced apoptosis was unaffected by knockdown of ANT. Instead, apoptosis is triggered through a different mitochondrial pore protein, Bax. In support of the idea that checkpoint activation and apoptosis induction are functionally linked, we show that Bax activation by Vpr was ablated when ATR or GADD45α was knocked down. Certain mutants of Vpr, such as R77Q and I74A, identified in long-term nonprogressors, have been proposed to inefficiently induce apoptosis while activating the G2 checkpoint in a normal manner. We tested the in vitro phenotypes of these mutants and found that their abilities to induce apoptosis and G2 arrest are indistinguishable from those of HIV-1NL4–3 vpr, providing additional support to the idea that G2 arrest and apoptosis induction are mechanistically linked. HIV-1 encodes a small gene known as vpr (viral protein regulatory) whose product is a 96–amino acid protein. HIV-1 infects cells of the immune system, such as CD4-positive lymphocytes. When cells become infected with HIV-1, two deleterious effects result from expression of the vpr gene. One effect of vpr is to manipulate the cell cycle by blocking the cells in G2 (the phase of the cell cycle immediately preceding mitosis). Thus, cells infected with HIV-1 cease to proliferate, due to the action of vpr. A second effect of vpr is the induction of cell death by a process known as apoptosis or programmed cell death. When cells die by apoptosis, they do so following activation of a cellular set of genes and proteins whose primary function is to inactivate various cellular functions that are needed in order to maintain cellular viability. In this study, Andersen et al. demonstrate that the above two effects of vpr are linked. In particular, the authors show that the blockade in cell proliferation in G2 is a requirement toward the onset of programmed cell death. Programmed cell death can be accomplished by a number of cellular proteins known as “executioners.” Various executioner proteins reside on the mitochondrial membranes and may trigger release of factors from the mitochondria, which in turn will precipitate the onset of apoptosis. In this work Anderson et al. identify the mitochondrial protein, Bax, as the key executioner of apoptosis in the context of HIV-1 vpr. The authors' findings provide important mechanistic understanding of how the vpr gene contributes to HIV-1–induced cell death.