生物谷報道：加拿大蒙特婁罕見疾病研究所（Institut de recherches cliniques de Montréal,，IRCM）Eric A. Cohen博士與其同事發(fā)現(xiàn)了一種研制治療HIV藥物的新機制。詳細內容刊登于7月13日PLoS Pathogens雜志,。
1型人類免疫缺陷病毒（HIV-1）通過耗盡人類宿主體內CD4+T淋巴細胞而破壞宿主的免疫系統(tǒng),，引發(fā)AIDS。此過程的中心環(huán)節(jié)是HIV蛋白——病毒蛋白R（viral protein R ,，Vpr）,，Vpr與控制細胞分裂的CD4＋T細胞蛋白相互作用，抑制受HIV感染的CD4＋T細胞分裂,，結果削弱了免疫功能,，同時，Vpr幫助HIV利用感染細胞的資源進行病毒復制,。
Cohen博士與其同事新發(fā)現(xiàn)一種HIV1 Vpr抑制受感染細胞分裂的新細胞蛋白復合體靶標,。這種被命名為DDB1-CUL4-VprBP的蛋白復合體，與泛素化過程有關,。泛素化過程中,，泛素蛋白與細胞蛋白相互作用，調節(jié)細胞蛋白的生物學活性或誘導細胞蛋白降解,。
研究人員證實,，Vpr與這種泛素復合體（又稱E3泛素連接酶復合體）之間的相互作用是Vpr誘導細胞分裂缺陷的一個重要因素。深入研究這種蛋白復合體的特征以及闡明其影響細胞分裂的機制,，為治療HIV感染開辟了新的道路,。
注：HIV是逆轉錄病毒，現(xiàn)在發(fā)現(xiàn)HIV-1型和HIV-2型,。根據(jù)HIV各型毒力的差異,，HIV-1型可分為M、N,、O亞型,，M亞型常見的又分為A,、B、C……K等11個亞型,，HIV-2比HIV-1傳播力弱,，感染者病毒載量也低、CD4細胞下降速度和秉承進展更緩慢,，垂直傳播發(fā)生率遠比HIV-1低,。根據(jù)每年的流行病學報告來看，我國的疫情以HIV-1型為絕對多數(shù),，其中又以M亞型下的B,、C亞型居多。
原始出處:
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.
