Wang Xingtai, Grammatikakis Nicholas, Hu Jianming
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
J Biol Chem. 2002 Jul 5;277(27):24361-7. doi: 10.1074/jbc.M202198200. Epub 2002 May 1.
The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pull-down assays showed that p50 or p50deltaC, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50deltaC interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of protein-primed initiation of reverse transcription by p50deltaC in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50deltaC in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.
细胞伴侣Hsp90已被证明与鸭乙型肝炎病毒的逆转录酶（RT）相关联，并且是RT功能所必需的。然而，RT与Hsp90之间特异性相互作用的分子基础仍然未知。蛋白质组成特性的比较表明，RT与蛋白激酶c-Raf高度相关，后者通过伴侣蛋白p50（CDC37）与Hsp90相互作用。我们测试了RT是否像c-Raf一样被p50特异性识别。免疫沉淀和下拉实验表明，p50或p50deltaC（一种在Hsp90结合方面有缺陷的p50突变体）在体外和体内都能与RT特异性相互作用，这表明p50可以独立于Hsp90结合RT。此外，纯化的p50和p50deltaC与纯化的RT直接相互作用。1）体外p50deltaC对蛋白质引发的逆转录起始的抑制以及2）在转染细胞中p50对病毒DNA复制和RNA包装的刺激以及p50deltaC对它们的抑制，都强调了p50-RT相互作用对RT功能的重要性。这些结果表明，p50可以通过介导RT与Hsp90之间的相互作用，作为嗜肝DNA病毒RT的细胞辅助因子发挥作用。
