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<p>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(<i>D</i><i>e</i><i>n</i><i>v</i> − 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(<i>m</i><i>E</i><i>o</i><i>s</i> − <i>N</i><i>S</i>2<i>B</i>3 and <i>P</i><i>A</i><i>G</i><i>F</i><i>P</i> − <i>N</i><i>S</i>2<i>B</i>3) 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月27日

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  • Jiby M. Varghese,
  • Prakash Joshi,
  • Aravinth S,
  • Partha Pratim Mondal

摘要

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.