The non-structural protein NS2B plays a critical role in the maturation of Dengue virus (DENV Type 2). The underlying mechanism and the role of NS2B are largely unknown due to the unavailability of its location of activity in the target organelle at single molecule level. This is largely due to the incapability of existing optical microscopes to resolve features beyond the diffraction limit of light ( \(\sim \lambda /2\) ), which is limited to a few hundred nanometers. Existing microscopy techniques are at best useful for ensemble study and details at the single molecule level remain hidden. To enable single-molecule resolution, we investigated the role of NS2B protein in a cellular system using scanning single molecule localization microscopy (scanSMLM). Accordingly, a photoactivable plasmid (Dendra2-NS2B) containing the gene-of-interest (NS2B) was constructed and the same is used to transfect NIH3T3 cells. Both super-resolution and confocal imaging studies suggest the accumulation of NS2B proteins in endoplasmic reticulum (ER). Subsequently, single-molecule analysis is carried out, where a total of \(\sim 41\) NS2B aggregates (both big and small) are noted with an area-spread of \(0.055 \pm 0.008 ~\upmu {\text {m}}^2\) , the molecular density of \(4358\pm 1170~mol/ \upmu {\text {m}}^2\) , and an average of \(174\pm 38\) NS2B molecules per cluster. Moreover, the super-resolved volume image revealed NS2B clusters spreading across several planes with a few extending up to 5 planes ( \(\sim 2.5 ~\upmu {\text {m}}\) from the coverslip). In addition, the collective dynamics of NS2B proteins leading to the formation of clusters is evident from time-lapse super-resolved data that provides conclusive evidence of NS2B accumulation, 24 hrs post-transfection. The present study revealed the dynamics of NS2B single viral protein molecule, and its accumulation at ER that may ultimately lead to organelle-specific drug targeting and help to reduce the rate of dengue infection by disrupting the NS2B accumulates.
