Visualizing Vpr-Induced G2 Arrest and Apoptosis

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.

Vpr是人类免疫缺陷病毒1型（human immunodeficiency virus type 1, HIV-1）的辅助蛋白，具有多种生物学功能。Vpr介导的G2期阻滞（G2 arrest）在病毒高效复制过程中发挥关键作用，这是由于HIV-1长末端重复序列（long terminal repeat, LTR）的转录活性在G2期达到峰值。Vpr对细胞凋亡的调控同样参与HIV感染过程中的免疫抑制与致病进程。然而目前仍不清楚Vpr诱导的细胞凋亡是否依赖于其引发G2期阻滞的能力，且Vpr诱导的G2期阻滞与细胞凋亡的动态变化尚未被实时可视化观测。本研究借助携带基于泛素化的荧光细胞周期指示剂2（Fucci2）的HeLa细胞，通过延时活细胞成像技术探究Vpr诱导的G2期阻滞与细胞凋亡之间的时序关联。研究人员成功观测到转染Vpr表达载体的细胞中，G2期阻滞及后续长期有丝分裂细胞变圆的动态过程。此类细胞在经历延长的有丝分裂进程后出现核染色体分离异常，随后进入G1期。部分细胞在经历延长的有丝分裂过程与核染色体分离异常后，呈现出细胞凋亡的特征。值得注意的是，Vpr诱导的细胞凋亡极少在S期或G2期被观测到。同样地，对感染表达Vpr的腺病毒载体的同步化HeLa/Fucci2细胞进行可视化观测，结果明确显示Vpr可使细胞周期阻滞于G2期，但不会在S期或G2期诱导细胞凋亡。此外，借助表达半胱天冬酶-3（caspase-3）敏感融合蛋白SCAT3.1的HeLa/Fucci2细胞的延时活细胞成像，研究明确证实Vpr可诱导半胱天冬酶-3依赖型细胞凋亡。为探究Vpr对G2期阻滞与细胞凋亡的影响是否可逆，本研究对不稳定结构域融合Vpr（destabilizing domain fusion Vpr）开展活细胞成像——该融合蛋白可通过Shield1实现快速稳定与去稳定。结果显示Vpr对G2期阻滞及后续细胞凋亡的影响具备可逆性。本研究首次系统表征了Vpr诱导的G2期阻滞与细胞凋亡过程中的形态变化动态特征。