Imaging of HTLV-1 by cryo-CLEM

据估计，人类T细胞白血病病毒（HTLV-1）在全世界感染了1500万至2000万个体，并且已知是成人T细胞白血病/淋巴瘤（ATLL）、称为HTLV-1相关脊髓病/热带痉挛性下肢轻瘫（HAM/TSP）的炎性疾病综合征以及肺、皮肤、眼睛和甲状腺的病理的病原体。HTLV-1因在细胞培养中极难繁殖而臭名昭著，这禁止了对病毒复制的严格分析，包括逆转录病毒组装中涉及的步骤。已知HTLV-1传播严重依赖于病毒感染，涉及形成所谓的病毒学突触（VS）的细胞间接触，其代表病毒传播的主要方式，包括与口服传播相关的事件。虽然先前已经研究了HTLV-1通过细胞-细胞接触的病毒传播，但关于病毒颗粒组装和通过VS传播的性质存在显著的知识缺口。病毒颗粒通过细胞传播-细胞接触增加了病毒颗粒感染事件的可能性，所述病毒颗粒可能在细胞中具有低颗粒感染性而不形成VS。一部分成熟的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的感染性差的性质。为了解决该领域目前的知识差距，我们建议在这个探索性的应用程序中开发最先进的生物工程和定量成像技术的工作流程，这些技术在应用于HTLV-1粒子组装和在VS传播的有效研究方面具有很高的前景。我们将开发一种使用细胞微图案化技术的工作流程，以便可重复地和有效地创建细胞，细胞接触，并研究病毒出芽在病毒传播中的作用。第二，我们建议建立一个有效的工作流程，我们可以查看细胞接触，利用高分辨率的冷冻相关的光学和电子显微镜，以研究宿主细胞蛋白在病毒组装在细胞接触的作用。这些工作流程的开发将允许定量分析细胞-细胞接触时的病毒颗粒生物发生。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.