PLoS Pathogens：HIV Vpr抑制受感染细胞分裂的

    生物谷报道：加拿大蒙特娄罕见疾病研究所（Institut de recherches cliniques de Montréal，IRCM）Eric A. Cohen博士与其同事发现了1种研制治疗HIV药物的新机制。详细内容刊登于7月13日PLoS Pathogens杂志。

    1型人类免疫缺陷病毒（HIV-1）通过耗尽人类宿主体内CD4+T淋巴细胞而破坏宿主的免疫系统，引发AIDS。此过程的中心环节是HIV蛋白——病毒蛋白R（viral protein R ，Vpr），Vpr与控制细胞分裂的CD4＋T细胞蛋白相互作用，抑制受HIV感染的CD4＋T细胞分裂，结果削弱了免疫功能，同时，Vpr帮助HIV利用感染细胞的资源进行病毒复制。

   Cohen博士与其同事新发现1种HIV1 Vpr抑制受感染细胞分裂的新细胞蛋白复合体靶标。这种被命名为DDB1-CUL4-VprBP的蛋白复合体，与泛素化过程有关。泛素化过程中，泛素蛋白与细胞蛋白相互作用，调节细胞蛋白的生物学活性或诱导细胞蛋白降解。

    研究人员证实，Vpr与这种泛素复合体（又称E3泛素连接酶复合体）之间的相互作用是Vpr诱导细胞分裂缺陷的1个重要因素。深入研究这种蛋白复合体的特征以及阐明其影响细胞分裂的机制，为治疗HIV感染开辟了新的道路。

     注：HIV是逆转录病毒，现在发现HIV-1型和HIV-2型。根据HIV各型毒力的差异，HIV-1型可分为M、N、O亚型，M亚型常见的又分为A、B、C……K等11个亚型，HIV-2比HIV-1传播力弱，感染者病毒载量也低、CD4细胞下降速度和秉承进展更缓慢，垂直传播发生率远比HIV-1低。根据每年的流行病学报告来看，我国的疫情以HIV-1型为绝对多数，其中又以M亚型下的B、C亚型居多。

原始出处:

PLoS Pathogens

Received: January 12, 2007; Accepted: May 7, 2007; Published: July 13, 2007

HIV-1 Vpr-Mediated G2 Arrest Involves the DDB1-CUL4AVPRBP E3 Ubiquitin Ligase

Jean-Philippe Belzile1, Ghislaine Duisit2¤, Nicole Rougeau1, Johanne Mercier1, Andrés Finzi1, Éric A. Cohen1,2*

1 Laboratory of Human Retrovirology, Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada, 2 Department of Microbiology and Immunology, Université de Montréal, Montreal, Quebec, Canada

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)—components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4AVPRBP—were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest–defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4AVPRBP E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

 Figure 1.Immunoprecipitation of DDB1/Vpr and VPRBP/Vpr Complexes

(A) HEK293T cells were mock transfected (lanes 1) or transfected with either TAP (lanes 2) or TAP-Vpr–expressing plasmids (lanes 3). Two days later, immunoprecipitations of TAP tag were performed on cell lysates using IgG-coupled beads and purified complexes were eluted by cleavage with TEV protease. The levels of endogenous VPRBP and DDB1 were monitored in crude lysates and pulled-down fractions by Western blot using specific antibodies. TAP, TAP-Vpr, and cleaved Vpr were detected using a polyclonal rabbit antibody directed against a Vpr N-terminal peptide.

(B) HEK293T cells were mock transfected (lanes 1 and 2) or transfected with either TAP (lanes 3 and 5) or TAP-Vpr–expressing plasmids (lanes 4 and 6). Cells were transcomplemented with the empty vector (lanes 1, 3, and 4) or HA-DDB1–encoding plasmid (lanes 2, 5, and 6).

(C) HEK293T cells were mock transfected (lanes 1) or transfected with HA-Vpr–expressing plasmid (lanes 2). Immunoprecipitations using anti-HA antibodies were performed on cell extracts using protein A–sepharose beads. The levels of HA-Vpr and endogenous VPRBP were monitored in cell extracts as well as immunoprecipitated fractions by Western blot using specific antibodies.

(D) HEK293T cells were mock transfected (lanes 1 and 3) or transfected with a HA-Vpr–expressing plasmid (lanes 2 and 4). Cells were transcomplemented with the empty vector (lanes 1 and 2) or Myc-VPRBP–encoding plasmid (lanes 3 and 4). Anti-HA immunoprecipitations were performed as described above.

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