Molecular Mechanisms for gp160-Enhanced Apoptosis

艾滋病的特征是T细胞的逐渐丧失，最终导致免疫瘫痪。尽管积极的抗病毒治疗，HIV-1并没有被根除。更好地了解HIV-1/宿主细胞相互作用对于确定新的治疗干预点至关重要。细胞凋亡，程序性细胞死亡，代表了艾滋病中HIV-1介导的T细胞和其他细胞损失的一种可能途径。将HIV-1外壳糖蛋白gp 160转染到T细胞系中，通过一种机制增强Fas介导的凋亡，该机制涉及钙调蛋白表达增加和钙调蛋白与gp 41的特异性C-末端细胞内序列结合。钙调素拮抗剂抑制gp 160增强的Fas介导的AIDS患者CD 4细胞凋亡和自发凋亡。gp 160增强Fas介导的细胞凋亡的潜在分子机制将首先使用两种新试剂Jurkat细胞系来阐明，所述细胞系具有稳定表达的gp 160和在835处具有A->W突变的gp 160，所述突变消除了在四环素关闭控制下的钙调蛋白结合。此外，这些实验将放置在HIV-1和艾滋病的背景下，通过研究Fas介导的细胞凋亡的gp 160变异体从主要的HIV-1分离株和感染性病毒与几个点突变的gp 160，包括A835 W，有受损的钙调蛋白结合。急性和慢性感染的T细胞系和感染的初级淋巴细胞与这些试剂被纳入作为这一综合计划的一部分，正在调查的关键钙调素依赖性信号转导事件在艾滋病发病机制。具体目标是：一。表征gp 160和钙调蛋白结合缺陷突变体（包括gp 160 A835 W）对Fas介导的细胞凋亡和Ca=2+/钙调蛋白相关信号传导的影响。二.使用来自钙调蛋白结合结构域变异的原代HIV-1分离株的gp 160，以及使用具有选定钙调蛋白结合缺陷gp 160突变（包括gp 160 A835 W）的感染性病毒，表征Fas介导的细胞凋亡和病毒复制。三.表征160个表达细胞中调节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.