Molecular Mechanisms for gp160-Enhanced Apoptosis

艾滋病的特点是T细胞进行性丧失，最终导致免疫瘫痪。尽管进行了积极的抗病毒治疗，但艾滋病毒-1并未被根除。更好地了解HIV-1/宿主细胞的相互作用对于确定新的治疗干预可能点至关重要。细胞凋亡，即程序性细胞死亡，是HIV-1介导的T细胞和其他细胞在艾滋病中丢失的一种可能途径。将HIV-1外壳糖蛋白gp160导入T细胞系可增强Fas介导的细胞凋亡，其机制涉及增加钙调蛋白的表达和与gp41的特定C末端细胞内序列的结合。钙调素拮抗剂抑制gp160增强的Fas介导的艾滋病患者CD4细胞的自发性凋亡。首先将使用两个新试剂Jurkat细胞系阐明gp160增强Fas介导的细胞凋亡的潜在分子机制，其中gp160稳定表达gp160，gp160在835处发生A&gt；W突变，在四环素关闭控制下消除钙调蛋白结合。此外，这些实验将放在HIV-1和艾滋病的背景下，通过研究来自HIV-1初级分离株的gp160变异体和带有gp160几个点突变的传染病病毒的Fas介导的细胞凋亡，其中包括已损害钙调蛋白结合的A835W。T细胞系的急性和慢性感染以及这些试剂对初级淋巴细胞的感染是这一全面计划的一部分，该计划正在研究艾滋病发病机制中关键的钙调蛋白依赖的信号转导事件。具体目的是：1.研究gp160和钙调素结合缺陷突变体(包括gp160A835W)对Fas介导的细胞凋亡和钙=2/钙调素相关信号的影响。II.使用gp160‘S鉴定Fas介导的细胞凋亡和病毒复制，这些病毒来自具有钙调蛋白结合结构域变异的原始HIV-1分离株，并使用具有选定的钙调素结合缺陷gp160突变的感染性病毒，包括gp160A835W。研究160个表达细胞中调控钙调蛋白表达的分子机制。

AIDS is characterized by, progressive loss of T cells with ultimate immune paralysis. Despite aggressive antiviral therapy, HIV-1 is not eradicated. A better understanding of HIV-1/host cell interactions is critical for identifying new possible points for therapeutic intervention. Apoptosis, programmed cell death, represents one possible pathway for HIV-1-mediated loss of T cells and other cells in AIDS. Transfection of the HIV-1 coat glycoprotein, gp160, into T cell lines enhances Fas- mediated apoptosis by a mechanism that involves increased calmodulin expression and calmodulin binding to a specific C-terminal intracellular sequence of gp41. Calmodulin antagonists inhibit gp160-enhanced Fas- mediated apoptosis and spontaneous apoptosis of CD4 cells obtained from AIDS patients. The underlying molecular mechanism for gp160 enhanced Fas-mediated apoptosis will be elucidated first using two new reagent Jurkat cell lines, with stably expressing gp160, and gp160 with an A->W mutation at 835 that eliminates calmodulin binding under tetracycline-off control. Furthermore, these experiments will be placed in the context of HIV-1 and AIDS by investigating Fas-mediated apoptosis in gp160 variants from primary HIV-1 isolates and infectious virus with several point mutations of gp160 including A835W that have impaired calmodulin binding. Acute and chronic infection of T-cell lines and infection of primary lymphocytes with these reagents are incorporated as part of this comprehensive program that is investigating the key calmodulin- dependent signal transduction events in AIDS pathogenesis. The Specific Aims are: I. Characterize the effects of gp160 and calmodulin-binding deficient mutants, including gp160A835W, on Fas-mediated apoptosis and Ca=2+/calmodulin related signaling. II. Characterize Fas-mediated apoptosis and viral replication using gp160's from primary HIV-1 isolates with variations in the calmodulin- binding domain and using infectious virus with selected calmodulin- binding deficient gp160 mutations, including gp160A835W. III. Characterize the molecular mechanisms regulating calmodulin expression in 160 expressing cells.