Molecular Mechanisms for gp160-Enhanced Apoptosis

艾滋病的特点是T细胞逐渐丧失，最终导致免疫瘫痪。尽管进行了积极的抗病毒治疗，HIV-1仍未被根除。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，是hiv -1介导的T细胞和其他艾滋病细胞损失的一种可能途径。将HIV-1外壳糖蛋白gp160转染到T细胞系中，通过增加钙调蛋白表达和钙调蛋白与gp41细胞内特定c端序列结合的机制，增强Fas介导的细胞凋亡。钙调素拮抗剂抑制gp160增强Fas介导的艾滋病患者CD4细胞凋亡和自发凋亡。gp160增强fas介导的细胞凋亡的潜在分子机制将首先通过两种新的试剂Jurkat细胞系来阐明，一种是稳定表达gp160，另一种是gp160在835位点发生A->W突变，在四环素关闭控制下消除钙调素结合。此外，这些实验将在HIV-1和艾滋病的背景下进行，研究来自HIV-1原代分离株和感染病毒的gp160变异中fas介导的凋亡，gp160的几个点突变包括A835W，这些突变破坏了钙调蛋白的结合。这些试剂对t细胞系的急性和慢性感染以及原发性淋巴细胞的感染被纳入这个综合项目的一部分，该项目正在研究艾滋病发病机制中关键的钙调蛋白依赖信号转导事件。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Ca=2+/钙调素相关信号的影响。2。利用从具有钙调蛋白结合域变异的HIV-1原代分离株中提取的gp160's，并利用具有钙调蛋白结合缺陷的gp160突变（包括gp160A835W）的感染性病毒，表征fas介导的细胞凋亡和病毒复制。3。表征160个表达细胞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.