Cellular Imaging Group, Wellcome Centre Human Genetics, University of Oxford, Oxford, United Kingdom.
HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, United States of America.
PLoS Pathog. 2020 Feb 21;16(2):e1008359. doi: 10.1371/journal.ppat.1008359. eCollection 2020 Feb.
There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically-encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 virions were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells.
目前，人们对于宿主代谢在病毒感染中的作用重新产生了兴趣，然而，解析单细胞代谢状态如何影响病毒进入和融合仍然未知。在这里，我们开发了一种新的测定方法，即将遗传编码的生物传感器与单病毒跟踪（SVT）相结合，以评估全局代谢过程对单个细胞中病毒进入成功率的影响。我们发现，在添加病毒之前具有较低 ATP:ADP 比的细胞对病毒融合和感染的许可性较低。这些结果表明宿主代谢状态与病毒-细胞融合发生的可能性之间存在关系。SVT 揭示了 HIV-1 病毒粒子在糖酵解活性低的细胞中处于半融合状态时被阻滞。有趣的是，急性用糖酵解抑制剂 2-脱氧葡萄糖（2-DG）处理的细胞对病毒感染具有抗性，并且表面膜胆固醇也较少。在这些糖酵解活性低的细胞中添加胆固醇可挽救半融合处的病毒进入阻断，并使 HIV-1 融合完成。使用基于 FRET 的膜张力和膜有序性报告器的进一步研究揭示了宿主细胞糖酵解活性与宿主膜有序性和张力之间的联系。实际上，用 2-DG 处理的细胞分别具有较低的质膜脂质有序性和较高的张力值。我们的新型成像方法结合寿命成像（FLIM）和 SVT，不仅揭示了病毒融合时质膜张力的变化，而且还表明 HIV 进入具有较高膜张力的区域的可能性较低。因此，我们已经确定了宿主细胞糖酵解活性与膜张力之间的联系，该联系在活细胞中单病毒融合水平上实时影响 HIV-1 融合。
