Single molecule super resolution microscopy reveals formation of Dengue NS2B3 cluster on mitochondrial network and its effect on fragmentation.

It is challenging to understand viral proteins at a single-molecule level and visualize their dynamics in a cellular environment. This calls for sophisticated microscopy techniques (such as,SMLM) and single-molecule analysis that can reveal details with the precision of a single molecule. In Dengue type-2(Denv - 2) infection, NS2B3 is recognized as a critical protein complex responsible for proteolytic activity and processing of viral polyprotein. We study the distribution of NS2B3 protein and its clusters. Accordingly, two distinct photoactivatable probes(mEos - NS2B3 and PAGFP - NS2B3) and a fluorescent probe (eGFP-NS2B3) were constructed. Results confirmed the formation of NS2B3 clusters in close vicinity to the mitochondrial network. Statistical analysis of single-molecule data (images) facilitated the estimation of critical cluster parameters(such as cluster density, number of molecules/cluster,and its spread). In addition, regional analysis suggests a correlation between NS2B3 cluster formation and fragmentation of mitochondrial network which is further supported by time-lapse confocal imaging. In other words, the presence of cluster depletes mitochondrial segment. To further demonstrate, time-lapse super-resolution imaging is carried out that enabled visualization of the formation of NS2B3 clusters. Understanding the underlying dynamics of NS2B3 clustering at the single-molecule level may facilitate the identification of potential drug targets,and methods to disrupt them.