Imaging of HTLV-1 by cryo-CLEM

人类t细胞白血病病毒（HTLV-1）估计在全世界感染了1500 - 2000万人，已知它是成人t细胞白血病/淋巴瘤（ATLL）、HTLV-1相关脊髓病/热带痉挛性麻痹（HAM/TSP）的炎症性疾病综合征以及肺、皮肤、眼睛和甲状腺病变的病因。HTLV-1因极难在细胞培养中繁殖而臭名昭著，这阻碍了对病毒复制的严格分析，包括逆转录病毒组装的步骤。已知HTLV-1传播严重依赖于病毒感染，涉及细胞间接触，形成所谓的病毒学突触（VS），这是病毒传播的主要途径，包括与口腔传播相关的事件。虽然HTLV-1之前已经研究过病毒通过细胞间接触传播，但关于病毒颗粒组装和通过vs传播的性质存在重大知识空白。病毒颗粒通过细胞-细胞接触传播增加了病毒颗粒感染事件的可能性，这些病毒颗粒可能在未形成vs的细胞中具有低颗粒感染性。先前的研究表明，低比例的成熟HTLV-1颗粒具有完整的衣壳核心，这表明异常的颗粒形态可能有助于解释无细胞HTLV-1的低感染性。为了解决目前在该领域的知识差距，我们在本探索性应用中提出开发最先进的生物工程和定量成像技术的工作流程，这些技术有望应用于HTLV-1颗粒组装和扩散的有效研究。我们将开发一个使用细胞微图技术的工作流程，以便可重复和有效地创建细胞-细胞接触，并研究病毒出芽在病毒传播中的作用。其次，我们建议建立一个有效的工作流程，其中我们可以利用高分辨率低温相关光和电子显微镜观察细胞-细胞接触，以研究宿主细胞蛋白在细胞-细胞接触处病毒组装中的作用。这些工作流程的发展将允许在细胞-细胞接触时对病毒颗粒的生物发生进行定量分析。这些技术在病毒学中具有广泛的适用性，这项研究的成功将适用于有关病毒复制和病毒-宿主细胞相互作用的各种问题。

Human T-cell leukemia virus (HTLV-1) has been estimated to infect 15-20 million individuals worldwide and is known to be the etiological agent of an adult T-cell leukemia/lymphoma (ATLL), an inflammatory disease syndrome known as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and pathologies of the lung, skin, eyes, and thyroid gland. HTLV-1 is notorious for being extremely difficult to propagate in cell culture, which has prohibited rigorous analyses of virus replication, including the steps involved in retrovirus assembly. HTLV-1 spread is known to be heavily reliant on virus infection involving cell-to-cell contacts that form what is termed the virological synapse (VS), which represents the primary means for virus spread, including events associated with oral transmission. While HTLV-1 has been previously studied in regard to virus spread via cell-cell contacts, a significant knowledge gap exists regarding the nature of virus particle assembly and transmission via the VS. In general, virus particle spread through cell-cell contacts increases the likelihood of an infection event of virus particles that may possess low particle infectivity in cells without formation of VS. Previous studies have indicated that a low proportion of mature HTLV-1 particles possess an intact capsid core, suggesting that aberrant particle morphology could help to explain the poorly infectious nature of cell-free HTLV- 1. In order to address the current knowledge gap in the field, we propose in this exploratory application to develop workflows for state-of-the-art bioengineering and quantitative imaging technologies that hold high promise in being applied to the efficient study of HTLV-1 particle assembly and spread at the VS. First, we will develop a workflow for the use of cell micropatterning technology in order to reproducibly and efficiently create cell-cell contacts and investigate the role virus budding in virus spread. Second, we propose to establish an efficient workflow in which we can view cell-cell contacts utilizing high-resolution cryo-correlative light and electron microscopy in order to investigate the role of host cell proteins in virus assembly at cell-cell contacts. Development of these workflows will allow for quantitative analysis of virus particle biogenesis at cell-cell contacts. These technologies have broad applicability in virology and the success of this research will be applicable to a variety of questions regarding virus replication and virus-host cell interactions.