Astrocytic HIV Reservoirs: Molecular Mechanisms

尽管HAART降低了HIV-1感染者神经和神经行为功能障碍的发生率，但CNS可能是HIV-1在病毒抑制治疗期间的重要储存库或避难所。在特定目标I中，将检查星形胶质细胞中HIV-1持续存在的独特分子机制。我们最近已经证明，HIV-1 Rev结合，并在功能上相互作用，死亡盒解旋酶，DDX 1。该部分与Rev中的特定基序（称为NIS）结合，其涉及Rev的适当亚细胞定位（即，细胞核大于细胞质）。最近的数据表明，Rev是超定位在细胞质内的初级星形胶质细胞。因此，我们推测DDX 1细胞辅因子可能参与星形胶质细胞中低水平残留HIV-1复制。将探索人星形胶质细胞中DDX 1的存在和定量，以及与Rev的潜在共定位。将使用哺乳动物双杂交系统评估Rev+选定Rev突变体与DDX 1之间的直接相互作用。同样，星形胶质细胞中DDX 1的过表达和DDX 1 mRNA表达的反义和RNAi抑制将用于评估这种潜在的独特CNS细胞特异性机制，其诱导人类星形胶质细胞中的HIV-1持久性。合作研究将包括通过激光解剖显微镜对HIV-1感染细胞脑组织中的Rev：DDX 1轴进行体内分析。在Specific Aim II中，我们将以与Specific Aim I互补的方式评估星形胶质细胞中低水平病毒产生中Rev亚细胞区室化改变的潜在额外和/或替代机制。这将包括分析DDX 1的潜在剪接变体，以及假设的DDX 1与NIS结合的细胞抑制剂，其是星形胶质细胞特异性的。同样，将通过亲和色谱法和/或噬菌体肽展示文库分析可能改变亚细胞区室化的星形胶质细胞特异性NIS结合细胞蛋白的鉴定和纯化。因此，这些互补的方法将被用来分析，在分子水平上，HIV-1水库和持久性在人类星形胶质细胞的机制。

Although the frequency of neurological and neurobehavioral dysfunction in HIV-1-infected individuals has been reduced by HAART, nonetheless, the CNS may represent an important reservoir or sanctuary site for HIV-1 during virally-suppressive therapy. In Specific Aim I, unique molecular mechanism(s) of HTV-1 persistence in astrocytes will be examined. We have recently demonstrated that HIV-1 Rev binds to, and functionally interacts with, a DEAD-box helicase, DDX1. This moiety binds to a specific motif in Rev (entitled NIS), which is involved with proper subcellular localization of Rev (i.e., nuclear greater than cytoplasmic). Recent data suggest that Rev is hyper-localized in primary astrocytes within the cytoplasm. Thus, we hypothesize that the DDX1 cellular cofactor may be involved with low-level residual HTV-1 replication in astrocytes. Presence and quantitation of DDX1 in human astrocytes, as well as potential co-localization with Rev, will be explored. Direct interactions between Rev plus selected Rev mutants and DDX1 will be assessed with a mammalian two-hybrid system. As well, over-expression of DDX1 and anti-sense and RNAi inhibition of DDX1 mRNA expression in astrocytes will be used to evaluate this potentially unique CNS-cell-specific mechanism inducing HTV-1 persistence in human astrocytes. Collaborating studies will include in vivo analysis of the Rev:DDX1 axes in brain tissue of HTV-1 -infected cells via laser dissection microscopy. In Specific Aim II, we will evaluate, in a complementary fashion with Specific Aim I, the potential additional and/or alternative mechanisms for alterations of Rev subcellular compartmentalization in low-level viral production in astrocytes. This will include analysis of potential splice variants of DDX1, and a hypothesized cellular inhibitor of DDX1 binding to NIS which is astrocyte-specific. As well, identification and purification of astrocyte-specific NIS binding cellular proteins which may alter subcellular compartmentalization will be analyzed by affinity chromotography and/or phagepeptide display libraries. Thus, these complementary approaches will be utilized to analyze, on molecular levels, the mechanisms of HIV-1 reservoirs and persistence in human astrocytes.