STRUCTURAL, BIOCHEMICAL, AND BIOLOGICAL CHARACTERIZATION OF HIV NEF AND VPU

HIV NEF 和 VPU 的结构、生化和生物学特征

• 批准号: 3838332
• 负责人: J A LAUTENBERGER
• 金额: --
• 依托单位: DIVISION OF CANCER EPIDEMIOLOGY AND GENETICS
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3838332
• 关键词: DNA virus; T lymphocyte; cell line; chimeric proteins; electroporation; endoplasmic reticulum; gene induction /repression; genetic manipulation; human immunodeficiency virus 1; human immunodeficiency virus 2; human tissue; molecular cloning; molecular oncology; phosphorylation; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; transfection; viral carcinogenesis; virus protein

Structural studies have determined that the predicted Nef protein sequence of HIV-1, HIV-2 and SIV can be dissected into at least three regions: (a) leucine-zipper (dimerization domain); (b) autophosphorylation (kinase domain); and (c) acidic alpha-helical (transcriptional activation domain). Using molecular biological approaches, experiments have been designed aimed at defining functional domains of the Nef proteins of HIV-1 and HIV- 2. Genetic, biochemical and biological studies will be used to analyze the relationship between Nef structure and function. Preliminary results have shown that the HIV-2 (NIH-Z) Nef protein forms stable homo-oligomers in vitro and in vivo, and that phosphorylation appears to favor equilibrium towards the homodimer (50 kD) species. This finding implicates the conserved leucine repeat sequence motif in the oligomerization. In addition, glutathione S-transferase (GST) Nef fusions have been constructed, and have expressed the leucine repeat region of HIV-2 Nef and the acidic transcriptional activation-like region at the carboxy terminus of HIV-1 Nef in E. coli. These GST-Nef fusion proteins are used as Nef-affinity matrices bound to glutathione-Sepharose to identify T cell factors that bind to, and are potential in vivo targets for Nef. Using GAL4-Nef fusions and transient transfection/CAT assays, identification will be made of regions of the HIV-1 Nef protein that have transcriptional activation or repressive activity in COS-7 or HeLa cells. In addition, an endoplasmic reticulum (ER) retention signal sequence (I/VDDL) has been identified at the extreme carboxyl terminus of the HIV-1 Vpu protein. Site-specific mutagenesis was used to remove the terminal dipeptide sequence of the ER signal. Both WT and site-specific nef and vpu mutant genes were introduced by electroporation into H9 cells in the pCDNA-1 Neo vector for subsequent analysis of function and mechanism.

结构研究已经确定了预测的Nef蛋白序列