The dengue virus non-structural protein 1 (NS1) is secreted from mosquito cells in association with the intracellular cholesterol transporter chaperone caveolin complex

Romel Rosales Ramirez; Juan E. Ludert

V-1 [10]. Therefore, in the present work, we explored the association of NS1 with 307 proteins involved, together with CAV-1, in the intracellular transport of cholesterol and the 308 dependence of NS1 secretion on such association. The results revealed that NS1 is associated 309 to the chaperone caveolin complex (CAV-1, Cy40, FKBP52 and CyA) and that the integrity of 310 this complex is necessary for the secretion of NS1 in mosquito infected cells. Additional data 311 suggest that in virus infected mosquito cells, ZIKV NS1, but not yellow fever virus NS1, also 312 associates with CAV-1 and usurps the cholesterol traffic pathways for secretion. Thus, the lipid 313 content or molecular structure of the DENV NS1 seems to have taken advantage of mosquito 314 cell cholesterol transport to guarantee the efficient NS1 secretion in the mosquito vector. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/370932 doi: bioRxiv preprint 16 325 Few works have reported a luciferase secretion system in insect cell lines [26] . Yet, as an 326 additional tool to study classical secretion in mosquito cell lines, an efficient Gaussia luciferase 327 reporter system that enables the detection of Luciferase in cell supernatants 6 hours after 328 transfection was developed. The secretion of luciferase was significantly affected after treatment 329 with GCA and BFA (data not shown); these data, together with the observed inhibition of viral 330 particle release, indicates the existence of a robust classical secretion pathway in mosquito cells. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/370932 doi: bioRxiv preprint 18 371 (CSD) with a distance of 5Ǻ approximately. Further analysis also showed, high energy binding 372 through hydrophobic dynamics, between the NS1 -roll domain and FKBP52, CyA and Cy40 373 with distances under 6Ǻ. However, the model predicts all these proteins interacting at the same 374 domain in NS1 (data not shown), which seems unlikely, due to steric effects. Thus, we favor an 375 alternative molecular docking model, based on interaction energies, in which NS1 is bound 376 directly to CAV-1, and in turn FKBP52, CyA and Cy40 are bound to CAV-1 (Fig. 10D ). This 377 model still allowed interaction of NS1 with the CCC through membranes. However, if the NS1-378 CCC complex will traffic to the plasma membrane as a free-cytosolic complex or membrane 379 associated is totally unknown. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/370932 doi: bioRxiv preprint . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/370932 doi: bioRxiv preprint