Macrobrachium rosenbergii Nodavirus (MrNV) causes white tail disease (WTD) in the giant freshwater prawn M. rosenbergii. In our past studies, we generated Macrobrachium rosenbergii Nodavirus virus-like particles (MrNV-VLPs) that were capable of being “nanocontainers” through the uptake of foreign plasmids after viral capsid disassembling/reassembling treatment. Nevertheless, the studies into the binding and internalization of this particular virus into susceptible host cells have been lacking; therefore our team has utilized live MrNV and recombinantly synthesized MrNV-VLPs as a tool to examine viral entry mechanisms in Sf9 insect cells; whereby revealing the dependence upon the caveolin-mediated endocytotic pathway for internalization and infection. Furthermore, viral capsid protein subunit modeling and capsid enzyme digestion were performed for revealing viral protrusions/ligands.
Figures:
MrNV VLPs (red) were localized using anti-MrNV. 3D reconstruction was generated from localizations showing VLPs on the cell membrane at T+0 min (B), submembranously and cytoplasmically at T+30 and T+120 min (C, D).Flow cytometric analyzes (I-L) of the internalized MrNV-VLPs indicate the shift of fluorescent intensity (arrow head).
MrNV RNA band of 1.1 kb appeared at 72-hr p.i. and at 120 hr p.i. (C). Colloidal gold reveal MrNV is confined to large vesicles (B), not seen in non-infected cells (A). MrNV mRNA levels (D, E) as well as an ELISA analysis of MrNV and CAV-1 protein levels (F) were performed on lysates under 3 conditions as seen in CLSM (G-J).
MrNV VLPs in pH 7.4 (A) reveal multi-spike projections from the surface like in pH 8.0 (B). MrNV-VLPs+ chymo remain spherical shaped (C). CLSM and flow cyto (G-I), MrNV-VLPs and those treated with chymotrypsin were incubated with Sf9 cells and stained with anti-MrNV (red).
C-terminal-truncated MrNV-VLPs showed an MW of 38 kDa (A). Negative TEM staining showed the change of surface topology in as capsid volume loss (D and E). Ribbon and space filling overlays of truncated MrNV-VLPs (B and C, arrow heads) depicts the missing area of surface topology.
To cite this abstract:
MONSICHA SOMRIT, ATTHABOON WATTHAMMAWUT, WATTANA WEERACHATYANUKUL, YANN GUERARDEL; Study of the interaction of Macrobrachium rosenbergii Nodavirus and its virus-like particles (MrNV-VLPs) in Sf9 insect cells: the dependence upon caveolin-mediated endocytosis and the possible existence of a capsid ligand domain. The 16th European Microscopy Congress, Lyon, France. https://emc-proceedings.com/abstract/study-of-the-interaction-of-macrobrachium-rosenbergii-nodavirus-and-its-virus-like-particles-mrnv-vlps-in-sf9-insect-cells-the-dependence-upon-caveolin-mediated-endocytosis-and-the-possible-existen/. Accessed: September 21, 2023« Back to The 16th European Microscopy Congress 2016
EMC Abstracts - https://emc-proceedings.com/abstract/study-of-the-interaction-of-macrobrachium-rosenbergii-nodavirus-and-its-virus-like-particles-mrnv-vlps-in-sf9-insect-cells-the-dependence-upon-caveolin-mediated-endocytosis-and-the-possible-existen/