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

Understanding viral proteins at single-molecule level and visualizing their dynamics in a cellular environment is challenging. This calls for sophisticated microscopy techniques (such as SMLM) and image-based study that can reveal details with the precision of a single-molecule. Specifically, NS2B3 is recognized as a critical protein complex responsible for proteolytic activity and processing of viral polyprotein during dengue type 2 (Denv - 2) infection. Using single-molecule-based super-resolution imaging, we study the dynamics of NS2B3 protein, its kinetics and its interaction with cell organelles. Two distinct photoactivable recombinant plasmids (mEos-NS2B3 and PAGFP - NS2B3) and a fluorescent recombinant plasmid (eGFP-NS2B3) were constructed to exemplify the role of NS2B3 protein complex. The study was conducted on NIH3T3 cells and optimized transfection protocol was developed. Studies confirmed the formation of NS2B3 clusters on the mitochondrial network. Statistical analysis of super-resolution data (images) helped determine cluster dynamics and facilitated the estimation of critical biophysical parameters (such as, cluster density, number of molecules / cluster and its spread). Results indicate an average NS2B3 cluster area / spread of [~] 0.050 {micro}m2 with a density of 3500mol./ {micro}m2, and an average of [~] 120 NS2B3 molecules per cluster. In addition, regional analysis suggests a direct positive correlation between NS2B3 cluster formation (single-molecule localization microscopy study) and fragmentation of mitochondrial network (confocal microscopy study). To further exemplify, we have carried out time-lapse imaging to visualize formation of NS2B3 clusters and its dynamics. The corresponding cluster parameters (#clusters, #mol/cluster and cluster area) suggests an increase in average #mol/cluster and cluster area. For the first time, single-molecule-based super-resolution imaging study helped reveal the dynamics of NS2B3 clusters in a cellular system. Understanding the underlying dynamics of NS2B3 clustering at the single-molecule level may help to decipher potential drug targets and the ways to disrupt the NS2B3 clusters. The image-based study has immediate implications in the broad field of single molecule imaging, fluorescence microscopy and disease biology. Statement of SignificanceThe arrival of single-molecule super-resolution imaging techniques and image-based study have advanced the field of cell biology, and our understanding of sub-cellular processes with single-molecule precision. Here, we report the first ever application of super-resolution imaging to visualize NS2B3 clusters in a cellular system that directly links to the fragmentation of mitochondrial network. To facilitate the study, two new photoactivable probes (mEos - NS2B3 and P AGF P- NS2B3) with key protein complex, NS2B3 of dengue virus were developed. The study involves cell transfection studies followed by confocal and single-molecule imaging. The correlative biophysical study (comprising of confocal and single-molecule imaging) and estimated cluster parameters corroborates our findings.