Super-resolution Fluorescence Microscopy Reveals Densely Packed EboV Membrane Glycoprotein Clusters in the Cell Plasma-membrane During Ebola Infection

The glycoprotein (GP) of Ebola virus plays a central role in the viral entry into host cells. The detailed study of Ebola glycoprotein in the cellular system is unknown, mainly due to the resolution limit ([~]{lambda} /2) of optical fluorescence microscopes. To overcome this, we propose to use single-molecule localization microscopy (SMLM), which enables the study of the role of glycoproteins at both the single-molecule and ensemble levels. Accordingly, a photoactivable recombinant probe Dendra2 - EboV Glyc is developed by linking Ebola glycoprotein with Dendra2. The transient expression of photoactivable recombinant protein in NIH3T3 cells indicated the dominant presence of glycoprotein in the plasma membrane with regions of densely packed glycoproteins. Super-resolution single-molecule imaging gives direct proof of glycoprotein clusters in the plasma membrane. The corresponding single-molecule analysis using DBSCAN clustering algorithm reveals a dense packing of glycoprotein with an estimated average density of 5345 mol/{micro}m2 compared to the density in other regions of the membrane. The number of glycoproteins is estimated to be as high as 642 molecules per cluster and clusters spread area of {approx} 0.1219 {micro}m2 after 48 hrs post-transfection. Noting that glycoprotein is involved in cellular attachment, endosomal entry, and membrane fusion, the formation of glycoprotein clusters in the membrane is significant in the progression of Ebola pathogenesis. A drug capable of dispersing the glycoproteins clusters could be of therapeutic potential to lessen the intensity of infection and to slow down the disease progression. Statement of SignificanceThe fact that powerful fluorescence microscopes (such as SMLM) are available is a boon to disease biology, specifically to study the function of viral proteins at the cellular level. Ebola, one of the nocturnal viral diseases, is a threat and calls for study at the single-molecule level to understand the dynamics (kinetics and ensemble) of glycoprotein during viral infection. In this respect, the super-resolution microscopy techniques may help reveal the biological mechanism behind the infection process at the single-molecule level. A photoactivable fluorescent probe (Dendra2 - EboV Glyc) containing the gene-of-interest (EboVGP) is developed, and transfection studies are carried out, showing densely packed glycoprotein clusters in the cell plasma membrane with specific density, number, and size. We believe that dispersing these glycoprotein protein clusters could be of therapeutic potential.