Mechanisms of efficient HIV infection through T cell virological synapses

描述（由申请人提供）：这个修订后的研究项目的目标是通过T细胞病毒学突触（VS）了解细胞间HIV-1感染的机制。T细胞VS是受感染细胞和未感染细胞之间形成的黏附结构，由Env和CD4分子结合引发。通过T细胞病毒学突触的传播是细胞培养中病毒传播的主要模式，在体内T细胞密度和运动性更明显的地方可能同样重要。通过VS有效的细胞间传播需要活跃的细胞过程来协调病毒组装和新生病毒粒子向受体细胞的转移。虽然相同的病毒蛋白介导无细胞病毒感染并通过VS，但VS将更多的病毒物质直接从一个细胞转移到另一个细胞，并且在此过程中避开了许多抗体反应。我们认为，通过Env细胞质尾（CT）的信号传导协调细胞-细胞感染，使Gag、Env和基因组rna作为感染性病毒一起组装和转移。利用新型Env成像技术结合试剂对RNA基因组包装进行成像，我们提出了机制研究，以测试病毒在VS中的组装和转移是如何协调的。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抵抗的结构决定因素。我们将测试一个“由内而外”的变构模型，即Env CT调节融合的能力也允许它抵抗抗体。通过对Env CT的系统诱变，我们发现突变体选择性地丧失了通过无细胞途径启动感染的能力，同时保留了通过细胞-细胞途径感染的能力，反之亦然。这些突变体将用于直接探测Env和Gag之间调节细胞-细胞传递的关键相互作用。此外，这些选择性突变体将使我们能够解剖细胞间传播与无细胞传播对HIV在原发性人类淋巴组织内有效传播的相对贡献。这项资助的主要假设是，Env CT协调HIV-1在T细胞中的组装和出芽，以增强感染性病毒的转移，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，因此确定最敏感的目标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.