The Dengue Virus Nonstructural Protein 1 (NS1) Is Secreted from Mosquito Cells in Association with the Intracellular Cholesterol Transporter Chaperone Caveolin Complex

Dengue virus (DENV) is a mosquito-borne virus of the familyFlaviviridae.The RNA viral genome encodes for a polyprotein that is co-translationally processed into three structural proteins and seven non-structural proteins. The non-structural protein 1 (NS1) is a multifunctional viral protein actively secreted in vertebrate and mosquito cells during DENV infection. In mosquito cells, NS1 is secreted in a caveolin-1 (CAV-1) dependent manner by an unconventional pathway. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40 and CyA which is responsible for cholesterol traffic inside the cell. In this work, we demonstrate that in infected mosquito cells, DENV NS1 is secreted by an early and unconventional route that bypasses the Golgi apparatus in close association with the CCC. Treatment of mosquito cells with classic secretion inhibitors such as brefeldin A, golgicide A and Fli-06 showed no effect on NS1 secretion, but significant reductions in recombinant luciferase secretion and virion release. Silencing the expression of CAV1, FKBP52 with siRNAs or the inhibition of CyA by cyclosporine A resulted in significant decrease in NS1 secretion without affecting virion release. Using co-localization, co-inmunoprecipitation and proximity ligation assays, NS1 was found to co-localize and interact with all the protein components of the CCC in mosquito infected cells. In addition, CAV-1 and FKBP52 expression was found augmented in DENV infected cells. Finally, the treatment of ZIKV infected mosquito cells with brefeldin A and golgicide A showed no effect on NS1 secretion, while affecting virion release. ZIKV NS1 was also found to co-localize with CAV-1 in infected mosquito cells. These results suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway observed for DENV NS1. The association of NS1 with the cholesterol transporter CCC agrees with the lipoprotein nature of secreted hexameric NS1.AUTHOR SUMMARYDengue protein NS1 is secreted in infected mosquito and vertebrate cells. In humans, secreted NS1 have been associated with pathogenesis. In mosquito cells, NS1 follows an unconventional secretion pathway that is dependent on Caveolin-1. This work shows that in mosquito cells, NS1 secretion is associated to the chaperone caveolin complex, a complex formed by caveolin-1 and several chaperones, in charge of cholesterol transport within the cells. Reduction of the expression or the activity of chaperone caveolin complex in mosquito infected cells, diminished the secretion of NS1 without affecting virion release. Direct interaction between NS1 and the chaperone caveolin complex proteins was demonstrated by several assays. Moreover, increased expression of the caveolin-1 and co-chaperone FKBP52 during dengue infection was found, presumably in response to the higher requirements of these proteins during dengue virus infection. Results obtained with ZIKV infected mosquito cells suggest that also ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. The functions of secreted NS1 within mosquito are unclear. However, giving the importance of the soluble NS1 in the vertebrate host, manipulation of the NS1 secretory route may prove a valuable strategy for dengue mosquito control and patient treatment.