Examining Trans-infection of HIV in Isolated Dendritic Cells

描述（由申请人提供）：人类免疫缺陷病毒（HIV）是一种破坏免疫系统的逆转录病毒，导致称为获得性免疫缺陷综合征（AIDS）的毁灭性疾病。为了达到这些效果，HIV破坏了宿主的防御，改变了正常的功能。树突状细胞（DC）是抗原呈递细胞，其捕获和处理进入的病原体，以便将抗原肽呈递给T淋巴细胞，从而引发免疫应答。由于树突状细胞是第一个遇到传入的艾滋病毒的细胞，病毒：细胞相互作用一直是相当多的研究的重点。最近的研究发现，DC能够捕获HIV并将病毒保持在感染状态，然后在共培养中增强病毒向靶细胞的递送，从而增加HIV感染性。然而，这些过程中的每一个所涉及的机制仍然未知。本项目将重点鉴定HIV使用的内吞途径，以避免DC内细胞内储存区室的降解和表征。为了实现这些目标，内吞途径将通过使用各种化学品和显性负结构来抑制。将通过使用荧光素酶报告病毒构建体来确定DC在每次处理后将感染性HIV反式传递至靶细胞的能力。高分辨率荧光显微镜将被用来详细检查细胞内储存区室。将通过使用荧光标记的病毒构建体来可视化HIV，并且将使用内吞隔室的各种标志物来表征储存隔室。还将检查用于阻断内吞途径的化学物质改变HIV细胞内运输的能力。此外，将通过上述方法检查DC成熟对内吞作用和细胞内储存的影响。相关性：目前全世界有4 000多万人感染艾滋病毒/艾滋病，因此不断需要开发更好的治疗方法和更有效的预防战略。由于树突状细胞是第一个遇到传入的艾滋病毒的细胞，直接靶向树突状细胞可能被证明是一种成功的疫苗开发方法。该提案将回答有关DC捕获和储存HIV的机制的重要问题，从而有助于对HIV：DC相互作用的全面理解。

DESCRIPTION (provided by applicant): The Human Immunodeficiency Virus (HIV) is a retrovirus that destroys the immune system, causing the devastating disease known as the acquired immune deficiency syndrome (AIDS). In order to accomplish these effects, HIV subverts host defenses, altering normal functions to its own advantage. Dendritic cells (DCs) are antigen presenting cells that capture and process incoming pathogens, in order to present antigenic peptides to T lymphocytes and thereby elicit an immune response. Since DCs are among the first cells to encounter incoming HIV, the virus:cell interaction has been the focus of considerable research. Recent studies have found DCs are capable of capturing HIV and retaining the virus in an infectious state, then enhancing viral delivery to target cells in co-culture thereby increasing HIV infectivity. However, the mechanism involved in each of these processes remains unknown. This project will focus on identification of the endocytic pathway used by HIV to avoid degradation and characterization of the intracellular storage compartment within DCs. To achieve these goals, endocytic pathways will be suppressed through use of various chemicals and dominant negative constructs. The ability of DCs to pass infectious HIV to target cells in trans following each treatment will be determined through use of a luciferase reporter viral construct. High resolution fluorescent microscopy will be employed to examine the intracellular storage compartment in great detail. HIV will be visualized through use of a fluorescently tagged viral construct, and various markers of endocytic compartments will be used to characterize the storage compartment. The chemicals used to block endocytic pathways will also be examined for their ability to alter intracellular trafficking of HIV. Additionally, the effect of DC maturation on endocytosis and intracellular storage will be examined through the methods detailed above. Relevance: With over 40 million people currently living with HIV/AIDS worldwide, there is a constant need for development of better therapeutics and more efficient prevention strategies. As DCs are the first cells to encounter incoming HIV, targeting DCs directly may prove to be a successful approach to vaccine development. This proposal will answer important questions about the mechanism of HIV capture and storage by DCs, thereby contributing to the overall understanding of the HIV:DC interaction.