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HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT.

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 GADD45alpha 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.

SUBMITTER: Andersen JL

PROVIDER: S-EPMC1665652 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT.

Andersen Joshua L JL DeHart Jason L JL Zimmerman Erik S ES Ardon Orly O Kim Baek B Jacquot Guillaume G Benichou Serge S Planelles Vicente V

PLoS pathogens 20061201 12

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 kno ...[more]

PMID: 17140287

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Project description:Lentiviruses encode a number of multi-functional accessory proteins, however, the primary role of the accessory protein Vpr remains unclear. As Vpr engages the host DNA damage response (DDR) at multiple steps, modulation of the DDR is considered central to the function(s) of Vpr. Vpr activates ataxia telangiectasia and Rad3 (ATR)-mediated DDR signaling, resulting in cell cycle arrest. However, the cellular consequences of Vpr-induced DNA damage, and the connection of Vpr-induced DNA damage to other Vpr functions, are unknown. Here, we determined that HIV-1 Vpr-induced DNA damage activates the ATM-NF-κB essential modulator (NEMO) pathway and alters cellular transcription via NF-κB/RelA. Through RNA-sequencing (RNA-seq) of cells expressing Vpr or mutants that separate the ability of Vpr to induce DNA damage from other DDR phenotypes, we identified that Vpr alters the transcriptome independent of cell cycle arrest. In tissue-cultured U2OS cells and primary human monocyte-derived macrophages (MDMs), we showed Vpr activates both ataxia telangiectasia mutated (ATM) and NF-κB/RelA signaling cascades. While inhibition of NEMO did not affect Vpr-induced DNA damage, it prevented NF-κB activation by Vpr, highlighting the importance of NEMO in Vpr-mediated transcriptional reprogramming. Virion-delivered Vpr was sufficient to induce DNA damage and activate ATM-NEMO dependent NF-κB transcription, suggesting that engagement of the DDR and transcriptional changes can occur early during viral replication. Together, our data uncover cellular consequences of Vpr-induced DNA damage and provide a mechanism for how Vpr activates NF-κB through DNA damage and ATM-NEMO signaling, which occur independent of cell cycle arrest. We propose this is essential to overcoming restrictive environments, such as in macrophages, to enhance viral replication.IMPORTANCEThe HIV accessory protein Vpr is multi-functional and required for viral replication in vivo, yet how Vpr enhances viral replication is unknown. Emerging literature suggests that a conserved function of Vpr is the engagement of the host DNA damage response (DDR). For example, Vpr activates DDR signaling, causes DDR-dependent cell cycle arrest, promotes degradation of various DDR proteins, and alters cellular consequences of DDR activation. However, a central understanding of how these phenotypes connect and how they affect HIV-infected cells remains unknown. Here, we found that Vpr-induced DNA damage alters the host transcriptome by activating an essential transcription pathway, NF-κB. This occurs early during the infection of primary human immune cells, suggesting NF-κB activation and transcriptome remodeling are important for establishing productive HIV-1 infection. Together, our study provides novel insights into how Vpr alters the host environment through the DDR, and what roles Vpr and the DDR play to enhance HIV replication.

Project description:Vpr is an accessory protein of human immunodeficiency virus type 1 (HIV-1) with multiple functions. The induction of G2 arrest by Vpr plays a particularly important role in efficient viral replication because the transcriptional activity of the HIV-1 long terminal repeat is most active in G2 phase. The regulation of apoptosis by Vpr is also important for immune suppression and pathogenesis during HIV infection. However, it is not known whether Vpr-induced apoptosis depends on the ability of Vpr to induce G2 arrest, and the dynamics of Vpr-induced G2 arrest and apoptosis have not been visualized. We performed time-lapse imaging to examine the temporal relationship between Vpr-induced G2 arrest and apoptosis using HeLa cells containing the fluorescent ubiquitination-based cell cycle indicator2 (Fucci2). The dynamics of G2 arrest and subsequent long-term mitotic cell rounding in cells transfected with the Vpr-expression vector were visualized. These cells underwent nuclear mis-segregation after prolonged mitotic processes and then entered G1 phase. Some cells subsequently displayed evidence of apoptosis after prolonged mitotic processes and nuclear mis-segregation. Interestingly, Vpr-induced apoptosis was seldom observed in S or G2 phase. Likewise, visualization of synchronized HeLa/Fucci2 cells infected with an adenoviral vector expressing Vpr clearly showed that Vpr arrests the cell cycle at G2 phase, but does not induce apoptosis at S or G2 phase. Furthermore, time-lapse imaging of HeLa/Fucci2 cells expressing SCAT3.1, a caspase-3-sensitive fusion protein, clearly demonstrated that Vpr induces caspase-3-dependent apoptosis. Finally, to examine whether the effects of Vpr on G2 arrest and apoptosis were reversible, we performed live-cell imaging of a destabilizing domain fusion Vpr, which enabled rapid stabilization and destabilization by Shield1. The effects of Vpr on G2 arrest and subsequent apoptosis were reversible. This study is the first to characterize the dynamics of the morphological changes that occur during Vpr-induced G2 arrest and apoptosis.

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HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT.

Publications

HIV-1 Vpr-induced apoptosis is cell cycle dependent and requires Bax but not ANT.

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