Imaging of HTLV-1 by cryo-CLEM

研究项目 摘要 据估计，全球有1500万至2000万人感染人类T细胞白血病病毒(HTLV-1)，目前 已知是成人T细胞白血病/淋巴瘤(ATLL)的病原体，这是一种炎症性疾病 被称为HTLV-1相关性脊髓病/热带痉挛性瘫痪(HAM/TSP)的综合征和病理学 肺、皮肤、眼睛和甲状腺。HTLV-1因极难在细胞内传播而臭名昭著 培养，禁止对病毒复制进行严格分析，包括反转录病毒所涉及的步骤 集合。众所周知，HTLV-1的传播在很大程度上依赖于涉及细胞间接触的病毒感染，这种接触形成 所谓的病毒学突触(VS)，它代表了病毒传播的主要途径，包括 与口头传播相关的事件。虽然HTLV-1之前已经在病毒传播方面进行了研究 通过细胞-细胞接触，存在着关于病毒颗粒组装和性质的重大知识差距 通过VS传播。一般来说，病毒粒子通过细胞间的接触传播会增加 病毒颗粒在细胞中可能具有较低的颗粒传染性而不形成VS的感染事件。上一首 研究表明，较低比例的成熟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传染性较差的本质- 1.为了解决目前该领域的知识鸿沟，我们提出在这一探索性应用中开发 最先进的生物工程和定量成像技术的工作流程 用于HTLV-1病毒粒子在VS中聚集和扩散的有效研究。首先，我们将开发一种 用于使用细胞微图案化技术的工作流程，以便可重复且高效地产生细胞-细胞 接触并调查病毒萌芽在病毒传播中的作用。第二，我们建议建立一个高效的 我们可以利用高分辨率低温相关光和电子查看细胞-细胞接触的工作流程 显微镜下观察宿主细胞蛋白在细胞-细胞接触处病毒组装中的作用。 这些工作流程的开发将允许对细胞-细胞内病毒颗粒的生物发生进行定量分析 联系人。这些技术在病毒学中具有广泛的适用性，这项研究的成功将是 适用于各种有关病毒复制和病毒与宿主细胞相互作用的问题。

RESEARCH PROJECT Abstract 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.