Mechanisms of efficient HIV infection through T cell virological synapses

描述(申请人提供)：这项修订的研究项目的目标是了解通过T细胞病毒学突触(VS)细胞对细胞感染HIV-1的潜在机制。T细胞VS是在感染细胞和未感染细胞之间形成的黏附结构，由Env和CD4的分子结合启动。通过T细胞病毒学突触传播是细胞培养中病毒传播的主要方式，在T细胞密度和运动性更明显的体内可能也同样重要。通过VS有效的细胞间传播需要活跃的细胞过程，以协调病毒组装和将新生病毒粒子转移到受体细胞中。虽然相同的病毒蛋白通过VS介导无细胞病毒的感染，但VS直接将更多的病毒物质从细胞转移到细胞，并在这样做的同时逃避许多抗体反应。我们认为，通过Env胞浆尾巴(CT)的信号协调细胞-细胞感染，从而使Gag、Env和基因组RNA作为感染性病毒组装和转移在一起。使用新的环境成像技术结合试剂对RNA基因组包装进行成像，我们建议进行机制研究，以测试病毒组装和病毒在VS之间的传输是如何协调的。我们已经了解到，通过VS感染可以抵抗中和抗体，并将检查可能支持这种耐药性的结构决定因素。我们将测试一个由内而外的变构模型，在该模型中，Env CT调节融合的能力也使其能够抵抗抗体。通过对Env CT的系统突变，我们已经鉴定出选择性丧失了通过无细胞途径启动感染的突变株，同时保留了通过CLL到细胞途径感染的能力，反之亦然。这些突变体将被用来直接探测调控细胞间传递的Env和GAG之间的关键相互作用。此外，这些选择性突变体将使我们能够剖析在人类初级淋巴组织中有效传播艾滋病毒的细胞间传播与非细胞传播的相对贡献。这笔赠款的主要假设是，Env CT协调T细胞中HIV-1的组装和萌发，以加强传染性病毒的转移，并在细胞-细胞感染期间最大限度地减少对敏感病毒表位的暴露。由于VS可能是抵抗体液免疫的有效体内传播的基础，因此识别最敏感的靶点可能对开发更有效的疫苗和药物至关重要。

DESCRIPTION (provided by applicant): The goal of this revised research project is to understand the mechanisms underlying cell-to-cell HIV-1 infection through T cell virological synapses (VS). T cell VS are adhesive structures formed between infected and uninfected cells that are initiated by the molecular engagement of Env and CD4. Transmission through T cell virological synapses is a predominant mode of viral spread in cell culture and likely is equally important in vivo where T cell density and motility are more pronounced. Efficient cell-to-cell transmission through VS requires active cellular processes that coordinate viral assembly with transfer of nascent virions into recipient cells. While the same viral proteins mediate infection b cell-free virus and through the VS, the VS transfers more viral material directly from cell to cell and does so while evading many antibody responses. We suggest that signaling through the Env cytoplasmic tail (CT) coordinates cell-cell infection so that Gag, Env and genomic RNAs are assembled and transferred together as an infectious virus. Using novel Env imaging techniques in conjunction with reagents to image RNA genome packaging, we propose mechanistic studies to test how viral assembly and transfer of virus across the VS are coordinated. We have learned that infection through the VS can resist neutralizing antibodies and will examine the structural determinants that may support such resistance. We will test an "inside out" allosteric model whereby the ability of the Env CT regulate fusion also allows it to resist antibodies. Through systematic mutagenesis of the Env CT, we have identified mutants with a selective loss of the capacity to initiate infection via cell-free route while preserving its ability to infect via the cll-to-cell route and vice versa. These mutants will be used to directly probe for key interactions between Env and Gag that regulate cell-cell transmission. In addition these selective mutants will allow us to dissect the relative contributions of cell-to-cell versus cell-free transmission fr efficient HIV spread within primary human lymphoid tissue. The overarching hypothesis of this grant is that the Env CT coordinates HIV-1 assembly and budding in T cells to enhance the transfer of infectious virus and to minimize exposure to sensitive viral epitopes during cell-cell infection. Because the VS may underlie efficient in vivo transmission that resists humoral immunity, identifying the most sensitive targets may be critical for developing more effective vaccines and drugs.