Liang X H, Kleeman L K, Jiang H H, Gordon G, Goldman J E, Berry G, Herman B, Levine B
Departments of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
J Virol. 1998 Nov;72(11):8586-96. doi: 10.1128/JVI.72.11.8586-8596.1998.
bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis. To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library. We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy. To examine the role of Beclin in Sindbis virus pathogenesis, we constructed recombinant Sindbis virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinDeltaBcl-2BD), or Beclin containing a premature stop codon near the 5' terminus (SIN/beclinstop). The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinDeltaBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001). The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinDeltaBcl-2BD or SIN/beclinstop. These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense.
bcl-2是细胞抗凋亡基因的原型，它可减少辛德毕斯病毒在小鼠脑中的复制以及辛德毕斯病毒诱导的细胞凋亡，从而预防致死性脑炎。为了研究Bcl-2预防中枢神经系统辛德毕斯病毒感染的潜在机制，我们进行了酵母双杂交筛选，以在成年小鼠脑文库中鉴定与Bcl-2相互作用的基因产物。我们鉴定出一种新的60 kDa卷曲螺旋蛋白Beclin，利用荧光共振能量转移显微镜，我们证实它在哺乳动物细胞中与Bcl-2相互作用。为了研究Beclin在辛德毕斯病毒发病机制中的作用，我们构建了表达全长人Beclin的重组辛德毕斯病毒嵌合体（SIN/beclin）、缺失假定的Bcl-2结合结构域的Beclin（SIN/beclinDeltaBcl-2BD）或在5'末端附近含有提前终止密码子的Beclin（SIN/beclinstop）。感染SIN/beclin的小鼠存活率（71%）显著高于感染SIN/beclinDeltaBcl-2BD（9%）或SIN/beclinstop（7%）的小鼠（P < 0.001）。与感染SIN/beclinDeltaBcl-2BD或SIN/beclinstop的小鼠脑相比，感染SIN/beclin的小鼠脑中辛德毕斯病毒RNA阳性细胞更少、凋亡细胞更少且病毒滴度更低。这些发现表明，Beclin是一种新的与Bcl-2相互作用的细胞蛋白，可能在抗病毒宿主防御中发挥作用。
