Mechanisms of efficient HIV infection through T cell virological synapses

描述（由申请人提供）：本修订研究项目的目标是了解通过T细胞病毒学突触（VS）进行细胞间HIV-1感染的潜在机制。T细胞VS是由Env和CD 4的分子接合引发的感染和未感染细胞之间形成的粘附结构。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，并且在T细胞密度和运动性更明显的体内可能同样重要。 通过VS的有效的细胞间传播需要主动的细胞过程，该过程协调病毒组装和新生病毒体转移到受体细胞中。虽然相同的病毒蛋白介导无细胞病毒和通过VS的感染B，但VS将更多的病毒物质直接从细胞转移到细胞，并在逃避许多抗体应答的同时这样做。我们认为，通过Env胞质尾（CT）的信号转导协调细胞-细胞感染，使Gag，Env和基因组RNA组装和转移在一起，作为一个感染性病毒。 使用新的Env成像技术结合试剂成像RNA基因组包装，我们提出了机制的研究，以测试如何病毒组装和转移的病毒在整个VS的协调。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“由内而外”变构模型，其中Env CT调节融合的能力也允许其抵抗抗体。通过Env CT的系统诱变，我们已经鉴定了具有选择性丧失通过无细胞途径引发感染的能力同时保留其通过细胞间途径感染的能力的突变体，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剖析细胞间传播与无细胞传播的相对贡献，从而有效地促进HIV在人初级淋巴组织中的传播。该资助的首要假设是Env CT协调HIV-1在T细胞中的组装和萌芽，以增强传染性病毒的转移，并最大限度地减少细胞间感染期间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，因此确定最敏感的靶点对于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.