Imaging of HTLV-1 by cryo-CLEM

通过冷冻 CLEM 进行 HTLV-1 成像

基本信息

批准号：
10675383
负责人：
Louis M Mansky
金额：
$ 23.25万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10675383
关键词：
2019-nCoV Address Adult T-Cell Leukemia/Lymphoma Behavior Biogenesis Biomedical Engineering Capsid Cell Communication Cell Culture Techniques Cells Cellular biology Cryoelectron Microscopy Development Disease Electron Microscope Electron Microscopy Engineering Etiology Event Eye Fluorescence Microscopy Goals Human Human Characteristics Human T-Cell Leukemia Viruses Human T-lymphotropic virus 1 Image Imaging technology Individual Infection Inflammatory Knowledge Lasers Malignant Neoplasms Morphology Nature Oral Organ Pattern Preparation Proteins Pulmonary Pathology Reproducibility Research Resolution Retroviridae Role Sampling Skin Spinal Cord Diseases Syndrome Technology Testing Thyroid Gland Tissues Tropical Spastic Paraparesis Viral Virion Virus Virus Assembly Virus Diseases Virus Replication experimental study fluorescence imaging high resolution imaging human imaging imaging study improved insight instrument light microscopy live cell imaging microscopic imaging mutant new technology novel particle permissiveness quantitative imaging success superresolution microscopy transmission process viral transmission virological synapse virology

项目摘要

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.

据估计，人类 T 细胞白血病病毒 (HTLV-1) 会感染全世界 15-2000 万人，并且是成人 T 细胞白血病/淋巴瘤 (ATLL) 的病原体，ATLL 是一种称为 HTLV 的炎症性疾病综合征-1 相关的脊髓病/热带痉挛性截瘫 (HAM/TSP) 以及肺、皮肤、眼睛和甲状腺的病变。 HTLV-1 因在细胞培养中极难繁殖而臭名昭著，这阻碍了对病毒复制的严格分析，包括逆转录病毒组装所涉及的步骤。众所周知，HTLV-1 的传播严重依赖于涉及细胞间接触的病毒感染，这种接触形成了所谓的病毒学突触 (VS)，它代表了病毒传播的主要手段，包括与口腔传播相关的事件。虽然 HTLV-1 之前已通过细胞间接触传播病毒进行了研究，但关于病毒颗粒组装和通过 VS 传播的性质仍存在重大知识空白。一般来说，病毒颗粒通过细胞与细胞接触传播增加了病毒颗粒感染事件的可能性，该病毒颗粒在细胞中可能具有低颗粒感染性而没有形成VS。先前的研究表明，一小部分成熟的 HTLV-1 颗粒拥有完整的衣壳核心，这表明异常的颗粒形态可能有助于解释无细胞 HTLV-1 感染性差的本质。在这一领域，我们建议在这个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，这些技术在应用于 HTLV-1 颗粒组装和在 VS 传播的有效研究方面具有很高的前景。首先，我们将开发一个使用细胞微图案技术的工作流程，以便可重复且高效地创建细胞与细胞的接触，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，在该工作流程中，我们可以利用高分辨率冷冻相关光学和电子显微镜观察细胞与细胞的接触，以研究宿主细胞蛋白在细胞与细胞接触处的病毒组装中的作用。这些工作流程的开发将允许对细胞与细胞接触处的病毒颗粒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与宿主细胞相互作用的各种问题。