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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 9 177 function of the CCC. Finally, the expression of Cy40 was significantly knock down at 48h post 178 siRNA transfection (Fig 4A) . A reduction in about 60% of lipid droplets counting per cell was 179 observed in transfected cells ( Fig 4B) . Yet, neither NS1 secretion nor virus release was affected 180 by Cy40 silencing (Fig 4C and 4D ). This result suggests that while Cy40 participates in 181 cholesterol traffic it may be dispensable for NS1 secretion. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 13 266 were detected in BHK-21 cells with ZIKV and YFV (data not shown). The secretion of YFV NS1 267 was found to be sensitive to BFA treatment in both mosquito and vertebrate cells (Fig. 9A) . In 268 contrast, the secretion of ZIKV NS1 was found to be insensitive to BFA and GCA treatment in 269 infected mosquito, but not in infected vertebrate cells (Fig 9B) . In addition, the secretion of ZIKV 270 NS1 was reduced in mosquito, but not in vertebrate cells, treated with the CyA inhibitor CsA (Fig 271 9C ). Co-localizations experiments carried out in C6/36, Aag2 and Vero-E6 cells infected with 272 ZIKV and YFV, showed a stronger, and a significantly different, co-localization between ZIKV 273 NS1 and CAV-1 than between YFV NS1 and CAV-1, in both mosquito cell lines. No co-274 localization of ZIKV or YFV NS1 with CAV-1 was observed in vertebrate cell line ( Fig 9D and 275 9E). Of note, the co-localization data support the predictions derived from the presence of a 276 conserved caveolin binding (CBD) domain in ZIKV (and DENV) but not in YFV NS1 (Fig 9F) . The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 14 289 10.2210/pdb2a65/pdb) with a p-value 2.69e-03. Molecular docking predicts a favorable 290 interaction between the NS1 hydrophobic domain including the grease fingers, where the CBD 291 is located, with a pocket in the CAV-1 scaffolding domain (CSD) with distances less 8Ǻ ( Fig 9A 292 and 9B). The molecular docking results obtained with CAV-1 and NS1 were used as a control to 293 carry on additional docking experiments between the DENV NS1 or CAV-1 and the CCC 294 chaperones FKBP52, Cy40 and CyA. Calculations of the energy binding of several docking 295 clusters showed that the DENV NS1 has the lowest and more favorable energy for CAV-1, 296 compared to the energy binding obtained for FKBP52, Cy40 or CyA. In addition, lower energies 297 were obtained for the binding of CAV-1 with the other chaperones of the CCC, than for the 298 binding of NS1 with those same chaperones ( Fig 9C) . Based in the free energies data, we predict 299 a model of interaction of NS1 with the CCC proteins in which FKBP52, Cy40 and CyA are directly 300 bound to CAV-1, and NS1 is bound to the CCC through the CAV-1 scaffolding domain ( Fig 9D) . 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 15 303 DISCUSSION 304 Our previous work demonstrated that DENV NS1 secretion in mosquito cell lines follows a BFA-305 insensitivity secretion pathway that depends on CAV-1; moreover, NS1 was found to interact 306 directly with CA