NS2B/NS3 complex is a key protein complex essential for proteolytic activity and processing of viral polyprotein during Dengue (Denv-2) infection. The underlying mechanism involved in the early onset (first 24 hrs) of Dengue pathogenesis was studied using single molecule-based super-resolution studies to understand the Denv-2 infection. The study was conducted on transfected NIH3T3 cells using two distinct photoactivable fusion plasmid DNAs (mEos3.2-NS2B/NS3 and paGFP - NS2B/NS3). Studies demonstrated that the formation of NS2B/NS3 clusters (mEos3.2 - NS2B/NS3 and paGFP - NS2B3) on the mitochondrial network induces mitochondrial fragmentation. The NS2B/NS3 complex acts as a protease that clips specific sites of mitofusin (MFN1/2) proteins, responsible for fusion which holds the network together, disrupting the mitochondrial network. Statistical analysis of super-resolution data (images) estimates an average NS2B/NS3 cluster size of 250 ~nm with a density of 5.82 * 10^2 mol/ μm^2, and has an average of 164 molecules per cluster. Based on the present study, we hypothesize that the formation of clusters and the associated cluster related parameters are critical in promoting mitochondrial fragmentation. Overall, the single molecule-based super-resolution study helped reveal the basic mechanism of single-molecule (NS2B/NS3) clustering during the onset of Dengue viral infection. Understanding the underlying biophysical mechanism of NS2B/NS3 clustering at the single molecule level may help decipher potential drug targets and the mechanisms of action to disrupt the NS2B/NS3 clusters, which may ultimately usher the way to contain/treat Dengue viral infection.
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