Single-Molecule Super-Resolution Microscopy Reveals Formation of NS2B3 Protein Clusters on Mitochondrial Network Leading to its Fragmentation during the Onset of Dengue (Denv-2) Viral Infection

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 × 102#mol./μm2, and 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.