Early steps of multi-receptor viral interactions dissected by high-density, multi-color single molecule mapping in living cells

Direct visualization of the early steps of multi-receptor viral interactions at the singlemolecule level has been largely impeded by the technical challenges associated to imaging individual multi-molecular systems at relevant spatial (nanometer) and temporal (millisecond) scales. Here, we present a four-color, high-density single-molecule spatiotemporal mapping methodology to capture real-time interactions between individual viruses and three different viral (co-)receptors on the membrane of living immune cells derived from donors. Together with quantitative tools, our approach revealed the existence of a coordinated spatiotemporal diffusion of the three different (co-)receptors prior to viral-engagement. By varying the temporal-windows of cumulated single-molecule localizations, we discovered that such a concerted diffusion impacts on the residence time of viruses on the host membrane and potential viral infectivity. Overall, our methodology opens a new door to the systematic analysis of the initial steps of viralhost interactions and paves the way to the investigation of other multi-molecular systems at the single-molecule level. ### Competing Interest Statement The authors have declared no competing interest.