Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, Mexico.
Department of Infectomics and Molecular Pathogenesis, Center for Research and Advanced Studies (CINVESTAV-IPN), Mexico City, Mexico
J Virol. 2019 Feb 5;93(4). doi: 10.1128/JVI.01985-18. Print 2019 Feb 15.
Dengue virus (DENV) is a mosquito-borne virus of the family The RNA viral genome encodes three structural and seven nonstructural proteins. Nonstructural protein 1 (NS1) is a multifunctional protein actively secreted in vertebrate and mosquito cells during infection. In mosquito cells, NS1 is secreted in a caveolin-1-dependent manner by an unconventional route. The caveolin chaperone complex (CCC) is a cytoplasmic complex formed by caveolin-1 and the chaperones FKBP52, Cy40, and CyA and is responsible for the cholesterol traffic inside the cell. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 associates with and relies on the CCC for secretion. 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 CAV-1 or FKBP52 with short interfering RNAs or the inhibition of CyA by cyclosporine resulted in significant decrease in NS1 secretion, again without affecting virion release. Colocalization, coimmunoprecipitation, and proximity ligation assays indicated that NS1 colocalizes and interacts with all proteins of the CCC. In addition, CAV-1 and FKBP52 expression was found augmented in DENV-infected cells. Results obtained with Zika virus-infected cells suggest that in mosquito cells, ZIKV NS1 follows the same secretory pathway as that observed for DENV NS1. These results uncover important differences in the dengue virus-cell interactions between the vertebrate host and the mosquito vector as well as novel functions for the chaperone caveolin complex. The dengue virus protein NS1 is secreted efficiently from both infected vertebrate and mosquito cells. Previously, our group reported that NS1 secretion in mosquito cells follows an unconventional secretion pathway dependent on caveolin-1. In this work, we demonstrate that in mosquito cells, but not in vertebrate cells, NS1 secretion takes place in association with the chaperone caveolin complex, a complex formed by caveolin-1 and the chaperones FKBP52, CyA, and Cy40, which are in charge of cholesterol transport inside the cell. Results obtained with ZIKV-infected mosquito cells suggest that ZIKV NS1 is released following an unconventional secretory route in association with the chaperone caveolin complex. These results uncover important differences in the virus-cell interactions between the vertebrate host and the mosquito vector, as well as novel functions for the chaperone caveolin complex. Moreover, manipulation of the NS1 secretory route may prove a valuable strategy to combat these two mosquito-borne diseases.
登革热病毒（DENV）是一种蚊媒病毒，属于黄病毒科。该 RNA 病毒基因组编码三种结构蛋白和七种非结构蛋白。非结构蛋白 1（NS1）是一种多功能蛋白，在感染期间在脊椎动物和蚊子细胞中被主动分泌。在蚊子细胞中，NS1 通过非经典途径依赖 caveolin-1 被分泌。Caveolin 伴侣复合物（CCC）是由 caveolin-1 和伴侣蛋白 FKBP52、Cy40 和 CyA 形成的细胞质复合物，负责细胞内胆固醇的运输。在这项工作中，我们证明在蚊子细胞中，但不在脊椎动物细胞中，NS1 与 CCC 结合并依赖 CCC 进行分泌。用经典分泌抑制剂处理蚊子细胞，如布雷菲德菌素 A、Golgicide A 和 Fli-06，对 NS1 分泌没有影响，但重组荧光素酶分泌和病毒粒子释放显著减少。用短发夹 RNA 沉默 CAV-1 或 FKBP52 的表达或用环孢素抑制 CyA 导致 NS1 分泌显著减少，但不影响病毒粒子释放。共定位、共免疫沉淀和邻近连接分析表明，NS1 与 CCC 的所有蛋白共定位并相互作用。此外，在感染 DENV 的细胞中发现 CAV-1 和 FKBP52 的表达增加。用 Zika 病毒感染细胞获得的结果表明，在蚊子细胞中，ZIKV NS1 遵循与 DENV NS1 观察到的相同的分泌途径。这些结果揭示了登革热病毒与脊椎动物宿主和蚊子媒介之间的细胞相互作用的重要差异，以及伴侣蛋白 caveolin 复合物的新功能。登革热病毒蛋白 NS1 从感染的脊椎动物和蚊子细胞中有效地分泌。我们的小组先前报道 NS1 在蚊子细胞中的分泌遵循一种依赖于 caveolin-1 的非经典分泌途径。在这项工作中，我们证明在蚊子细胞中，但不在脊椎动物细胞中，NS1 分泌与伴侣蛋白 caveolin 复合物有关，该复合物由 caveolin-1 和伴侣蛋白 FKBP52、CyA 和 Cy40 形成，负责细胞内胆固醇的运输。用感染 ZIKV 的蚊子细胞获得的结果表明，ZIKV NS1 通过与伴侣蛋白 caveolin 复合物相关的非经典分泌途径释放。这些结果揭示了登革热病毒与脊椎动物宿主和蚊子媒介之间的病毒细胞相互作用的重要差异，以及伴侣蛋白 caveolin 复合物的新功能。此外，操纵 NS1 分泌途径可能是对抗这两种蚊媒疾病的一种有价值的策略。
