Co-evolution of HIV-1 Regulators in CNS Disease

描述（申请人提供）：免疫系统和中枢神经系统（CNS）中的HIV-1相关发病机制依赖于病毒复制，其可通过病毒结合和进入、受体/共受体使用、宿主细胞增殖和活化、细胞分化、抗逆转录病毒治疗状态、以及通过长末端重复序列（LTR）结合病毒和细胞调节蛋白调节病毒表达。调节LTR功能和病毒表达的病毒蛋白包括达特反式激活蛋白和病毒蛋白R（Vpr），其可以通过几种顺式作用LTR元件诱导LTR活性。HIV-1的复制能力进一步受到感染过程中病毒基因型（包括达特、vpr和LTR）进化的影响。这些研究的假设是，共同进化的病毒调节元件（CVRE）达特、Vpr和LTR改变病毒复制表型，影响病毒在区室化细胞类型之间的传播，并影响CNS疾病的病程和严重程度。本申请的具体目的是（1）建立CVRE序列数据库和HIV-1感染免疫细胞的DNA克隆库（活化T细胞、非活化T细胞、单核细胞）和CNS驻留细胞（小胶质细胞、血管周围巨噬细胞、星形胶质细胞）分离自HAART和痴呆状态不同的患者;（2）分析CVRE数据库中的患者内变异（细胞类型特异性或CNS或免疫系统区室化）和患者间变异（HAART和痴呆状态特异性）在共同进化的Vpr、达特和LTR序列中;（3）构建掺入选择的CVRE序列的感染性分子克隆（IMC），并分析每种IMC衍生的病毒的CD 4结合、共受体利用，和复制表型;和（4）确定来自脑源性CVRE克隆的共进化的Tat-LTR、Vpr-LTR和Tat-Vpr-LTR组合支持瞬时表达的能力，并建立瞬时LTR诱导、痴呆状态和HAART状态之间的相关性。这种独特的方法来研究调节HIV-1表达的背景下，中枢神经系统疾病和HAART将提供更深入的了解HIV-1相关的发病机制，并可能导致更有效的治疗HIV-1中枢神经系统疾病。

DESCRIPTION (provided by applicant): HIV-1-associated pathogenesis in the immune system and central nervous system (CNS) is dependent on viral replication, which can be controlled by viral binding and entry, receptor/co-receptor usage, host cell proliferation and activation, cellular differentiation, antiretroviral therapy status, and regulation of viral expression by the long terminal repeat (LTR) in conjunction with viral and cellular regulatory proteins. Viral proteins that regulate LTR function and viral expression include the Tat trans-activator protein and viral protein R (Vpr), which can induce LTR activity through several cis-acting LTR elements. The replicative capacity of HIV-1 is further affected by evolution of the viral genotype (including tat, vpr, and the LTR) during the course of infection. The hypothesis of these investigations is that co-evolved viral regulatory elements (CVRE) Tat, Vpr, and the LTR alter the viral replicative phenotype, affect the spread of virus between compartmentalized cell types, and affect the course and severity of CNS disease. The specific aims of this application are to (1) establish a CVRE sequence database and a bank of DNA clones derived from HIV-1-infected immune cells (activated T cells, non-activated T cells, monocytic cells) and CNS-resident cells (microglia, perivascular macrophages, astrocytes) isolated from patients that differ in HAART and dementia status; (2) analyze the CVRE database for intrapatient variations (specific to cell type or compartmentalized to the CNS or immune system) and interpatient variations (HAART and dementia status-specific) in co-evolved Vpr, Tat, and LTR sequences; (3) construct infectious molecular clones (IMCs) incorporating select CVRE sequences and analyze each IMC-derived virus for CD4 binding, co-receptor utilization, and replication phenotype in immune- and CNS-derived cell lines and primary cells; and (4) determine the ability of co-evolved Tat-LTR, Vpr-LTR, and Tat-Vpr-LTR combinations from brain-derived CVRE clones to support transient expression, and establish correlations between transient LTR induction, dementia status, and HAART status. This unique approach to studying regulated HIV-1 expression in the context of CNS disease and HAART will provide greater insights into HIV-1-associated pathogenesis and may lead to more efficacious treatments of HIV-1 CNS disease.