Quantitative Single-Cell Assessment of Lentivirus Susceptibility Determinants

自2017年2月开始工作以来，已经招募了一名通过美国国立卫生研究院-牛津大学-剑桥大学学者项目共享的医学博士/博士生（Chad Coomer），他取得了重大进展。在头12个月的大部分时间里，Chad都在做以下工作：1)技术获取和中试；2)生化试剂获取和中试。他于2018年开始了该项目的实验部分，在使用质粒编码的生物传感器测量细胞内代谢物的同时，制作和测试假设。通过研究HIV暴露后和病毒与细胞融合特定阶段的生物传感器活性，他发现糖酵解活性与HIV进入细胞呈正相关。糖酵解活性由报告ATP与ADP比值的生物传感器测量。此外，使用糖酵解抑制剂，2-脱氧葡萄糖，抑制hiv细胞融合。有趣的是，当HIV通过包膜糖蛋白假型修饰进入途径时，对2-脱氧葡萄糖的敏感性发生了改变。我们发现糖酵解活性诱导的胆固醇水平对于HIV-1与人类细胞的有效融合至关重要（Coomer等人，PLoS Pathog. 16: e1008359, 2020）。该项目对代谢物的关注使其与Z1A BC 011756项目（细胞代谢和抗病毒状态之间的内在联系）以及癌症生物学相关。

Since work has begun in February 2017, a shared MD/PhD student through the NIH-Oxford-Cambridge Scholars program has been recruited (Chad Coomer) and he has made significant progress. For much of the first 12 months, Chad worked on the following: 1) technology acquisition and pilot testing, and 2) biochemical reagent acquisition and pilot testing. He began the experimental side of the project in 2018, crafting and testing hypotheses while making use of plasmid-encoded biosensors that measure intracellular metabolites. By studying biosensor activity following HIV exposure and during the specific stage of virus-cell fusion, he has found that glycolytic activity is positively associated with HIV entry into cells. Glycolytic activity is measured by a biosensor reporting the ratio of ATP to ADP. Additionally, the use of an inhibitor of glycolysis, 2-deoxyglucose, inhibits HIV-cell fusion. Interestingly, when the entry pathway used by HIV was modified through envelope glycoprotein pseudotyping, sensitivity to 2-deoxyglucose was altered. We identified that cholesterol levels induced by glycolytic activity are critical for efficient HIV-1 fusion with humans cells (Coomer et al., PLoS Pathog. 16: e1008359, 2020). The focus on metabolites in this project makes it relevant to work being done in Project Z1A BC 011756 (An Intrinsic Link between the Metabolic and Antiviral States of the Cell), as well as to cancer biology.