Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum
Abstract The non-structural protein NS2B plays a critical role in the maturation of Dengue virus (DENV Type 2). The underlying mechanism and the role of NS2B are largely unknown due to the unavailability of its location of activity in the target organelle at single molecule level. This is largely du...
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Nature Portfolio
2025-03-01
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| Series: | Scientific Reports |
| Online Access: | https://doi.org/10.1038/s41598-025-94135-6 |
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| author | S. Aravinth Jiby Mary Varghese Neeraj Pant Partha Pratim Mondal |
| author_facet | S. Aravinth Jiby Mary Varghese Neeraj Pant Partha Pratim Mondal |
| author_sort | S. Aravinth |
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| description | Abstract The non-structural protein NS2B plays a critical role in the maturation of Dengue virus (DENV Type 2). The underlying mechanism and the role of NS2B are largely unknown due to the unavailability of its location of activity in the target organelle at single molecule level. This is largely due to the incapability of existing optical microscopes to resolve features beyond the diffraction limit of light ( $$\sim \lambda /2$$ ), which is limited to a few hundred nanometers. Existing microscopy techniques are at best useful for ensemble study and details at the single molecule level remain hidden. To enable single-molecule resolution, we investigated the role of NS2B protein in a cellular system using scanning single molecule localization microscopy (scanSMLM). Accordingly, a photoactivable plasmid (Dendra2-NS2B) containing the gene-of-interest (NS2B) was constructed and the same is used to transfect NIH3T3 cells. Both super-resolution and confocal imaging studies suggest the accumulation of NS2B proteins in endoplasmic reticulum (ER). Subsequently, single-molecule analysis is carried out, where a total of $$\sim 41$$ NS2B aggregates (both big and small) are noted with an area-spread of $$0.055 \pm 0.008 ~\upmu {\text {m}}^2$$ , the molecular density of $$4358\pm 1170~mol/ \upmu {\text {m}}^2$$ , and an average of $$174\pm 38$$ NS2B molecules per cluster. Moreover, the super-resolved volume image revealed NS2B clusters spreading across several planes with a few extending up to 5 planes ( $$\sim 2.5 ~\upmu {\text {m}}$$ from the coverslip). In addition, the collective dynamics of NS2B proteins leading to the formation of clusters is evident from time-lapse super-resolved data that provides conclusive evidence of NS2B accumulation, 24 hrs post-transfection. The present study revealed the dynamics of NS2B single viral protein molecule, and its accumulation at ER that may ultimately lead to organelle-specific drug targeting and help to reduce the rate of dengue infection by disrupting the NS2B accumulates. |
| format | Article |
| id | doaj-art-0b3e6ff500cf4aa7ae47c2be641cd8a4 |
| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Scientific Reports |
| spelling | doaj-art-0b3e6ff500cf4aa7ae47c2be641cd8a42025-08-20T02:49:26ZengNature PortfolioScientific Reports2045-23222025-03-0115111110.1038/s41598-025-94135-6Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulumS. Aravinth0Jiby Mary Varghese1Neeraj Pant2Partha Pratim Mondal3Department of Instrumentation and Applied Physics, Indian Institute of ScienceDepartment of Instrumentation and Applied Physics, Indian Institute of ScienceDepartment of Instrumentation and Applied Physics, Indian Institute of ScienceDepartment of Instrumentation and Applied Physics, Indian Institute of ScienceAbstract The non-structural protein NS2B plays a critical role in the maturation of Dengue virus (DENV Type 2). The underlying mechanism and the role of NS2B are largely unknown due to the unavailability of its location of activity in the target organelle at single molecule level. This is largely due to the incapability of existing optical microscopes to resolve features beyond the diffraction limit of light ( $$\sim \lambda /2$$ ), which is limited to a few hundred nanometers. Existing microscopy techniques are at best useful for ensemble study and details at the single molecule level remain hidden. To enable single-molecule resolution, we investigated the role of NS2B protein in a cellular system using scanning single molecule localization microscopy (scanSMLM). Accordingly, a photoactivable plasmid (Dendra2-NS2B) containing the gene-of-interest (NS2B) was constructed and the same is used to transfect NIH3T3 cells. Both super-resolution and confocal imaging studies suggest the accumulation of NS2B proteins in endoplasmic reticulum (ER). Subsequently, single-molecule analysis is carried out, where a total of $$\sim 41$$ NS2B aggregates (both big and small) are noted with an area-spread of $$0.055 \pm 0.008 ~\upmu {\text {m}}^2$$ , the molecular density of $$4358\pm 1170~mol/ \upmu {\text {m}}^2$$ , and an average of $$174\pm 38$$ NS2B molecules per cluster. Moreover, the super-resolved volume image revealed NS2B clusters spreading across several planes with a few extending up to 5 planes ( $$\sim 2.5 ~\upmu {\text {m}}$$ from the coverslip). In addition, the collective dynamics of NS2B proteins leading to the formation of clusters is evident from time-lapse super-resolved data that provides conclusive evidence of NS2B accumulation, 24 hrs post-transfection. The present study revealed the dynamics of NS2B single viral protein molecule, and its accumulation at ER that may ultimately lead to organelle-specific drug targeting and help to reduce the rate of dengue infection by disrupting the NS2B accumulates.https://doi.org/10.1038/s41598-025-94135-6 |
| spellingShingle | S. Aravinth Jiby Mary Varghese Neeraj Pant Partha Pratim Mondal Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum Scientific Reports |
| title | Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum |
| title_full | Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum |
| title_fullStr | Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum |
| title_full_unstemmed | Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum |
| title_short | Super-resolution optical microscopy reveals accumulation of photoactivable dengue protein (Dendra2-NS2B) in the endoplasmic reticulum |
| title_sort | super resolution optical microscopy reveals accumulation of photoactivable dengue protein dendra2 ns2b in the endoplasmic reticulum |
| url | https://doi.org/10.1038/s41598-025-94135-6 |
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