Sarmento, M. J. | Search bioRxiv

bioRxiv2025

Dengue virus capsid protein interaction with nucleic acids

Dengue virus (DENV) transmission has greatly increased in the last decade, in part due to the geographical expansion of its vector, the Aedes spp. mosquitoes. These arthropods are now found even in temperate climates, including Europe, where outbreaks have already occurred. A better understanding of the virus life cycle is essential, as it may enable the development of specific treatments or therapies which are currently lacking. However, recent breakthroughs concerning the biologically relevant viral capsid (C) protein structure and function are encouraging. It is now clear the C protein binds both to host lipid droplets and to the viral genome, a set of interactions that is crucial for viral encapsidation and replication. Previously, we studied DENV C protein interaction with lipid droplets, at the molecular level. Here, we examine how the association of DENV C protein with the viral genome occurs. Using DENV C protein and single-stranded DNA (ssDNA) sequences analogous to previously identified important regions of the viral genome, we biophysically characterize their interactions. A decrease in fluorescence intensity and lifetime, an increase in both anisotropy and hydrodynamic diameter, as well as changes in the proteins secondary structure, were observed upon binding to ssDNA. These changes are consistent with molecular condensation and raise the possibility of liquid-liquid phase separation involvement in DENV C-nucleic acid complex formation. These findings may be useful for the development of future therapeutic strategies based on nucleic acids, not only for DENV but also for other related viruses.

biophysics↗

bioRxiv2019

Nanoscale distribution of nuclear sites analyzed by superresolution STED-ICCS

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet.\n\nHere we combine dual color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that STED-ICCS provides not only a value of colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the Proliferating cell nuclear antigen (PCNA) protein have a high level of proximity and are correlated at a nanometer distance which is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of nanoscale distribution of functional sites in the nucleus.\n\nStatement of significanceSeveral methods are available to quantify the proximity of two labeled molecules from dual color images. Among them, image cross-correlation spectroscopy (ICCS) is attractive as it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. Here, we combine for the first time ICCS with superresolution stimulated emission depletion (STED) microscopy (STED-ICCS) to quantify the spatial distribution of functional sites in the nucleus. Our results show that STED-ICCS, in addition to quantifying the colocalized fraction, detects characteristic nanometer distances associated to correlated nuclear sites. This work shows that STED-ICCS can be a powerful tool to quantify the nanoscale distribution of functional sites in the nucleus.

biophysics↗