Formation and transmission of novel autophagy-associated vesicle in dengue infection

• Main Authors: Yan-WeiWu, 吳彥緯
• Other Authors: Yee-Shin Lin
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/99kke6

博士 === 國立成功大學 === 基礎醫學研究所 === 105 === Among globally infectious diseases, dengue disease is partly characterized by severe hemorrhage or shock syndrome. Dengue disease is caused by dengue virus (DENV). Aedes egypti or Aedes albopictus can transmit DENV in tropical and subtropical regions. In cell-to-cell transmission, DENV can spread by cell-free virions binding to specific receptors. Neutralizing antibodies (NAbs) can block such conventional transmission. However, even high titers of NAbs in patients’ sera may not prevent DENV infection and disease partly as a result of antibody-dependent enhancement (ADE). The low efficacy of NAbs in viral infection has already been described for hepatitis C virus (HCV) and human immunodeficiency virus (HIV). HCV can spread via mechanisms of NAb-resistant or exosome-mediated release. In the case of HIV, the close-contact between membranes of two neighboring cells avoids extracellular NAbs. The mechanism of transmission which can avoid NAbs for DENV has not been defined. We set up a transwell co-culture system using a pore size of 3 m to separate donor infected with DENV and recipient labeled with green fluorescent protein (GFP). After 24 h DENV infection, the recipient cells showed a lower infection rate than recipient cells which were close-contact co-cultured with donor cells. Adding NAbs in the medium of close-contact co-culture system had no effect on infection rate of recipient cells. To clarify the mechanism of DENV transmission in close-contact co-culture system, autophagy was considered because previous studies showed its important role in DENV replication. Autophagy is a highly conserved cellular pathway to degrade intracellular damaged organelles or protein aggregation. Besides, secretion of several proteins can be triggered by autophagy which is distinguished from endoplasmic reticulum (ER)-Golgi secretory pathway. Therefore, we tried to link autophagy-related unconventional secretion to the DENV transmission avoiding NAbs. First, we used autophagy-deficient donor cells such as Atg5 knockdown Huh7 and Atg5 knockout mouse embryonic fibroblast (MEF), then observed the reduction of DENV transmission in close-contact co-culture system. By analysis of confocal microscopy, autophagy marker LC3 and DENV envelope (E) proteins were observed to be secreted from donor cells. Those proteins formed vesicles and attached to recipient cells. Such dengue vesicle contained viral proteins E, nonstructural protein 1 (NS1), prM, and lipid droplet. Using NS1 primer to perform qRT-PCR, the DENV RNA was determined to be contained in vesicle. Treatment with RNAase eliminated the nonstructural protein 3 (NS3) expression in recipient cells transfected by RNA from dengue vesicle. These results imply that dengue vesicle relates to autophagosome and contains infectious DENV RNA. Furthermore, vesicle-mediated transmission could not be blocked by adding anti-dengue NAbs, suggesting that the infectious viral RNA was protected by a membrane. Such membrane directly fused to the plasma membrane of target cells to initiate a new round of viral infection. Importantly, we also detected dengue vesicles in a patient’s serum and showed that they were infectious ex vivo. The role of dengue vesicle for DENV spread in patient still need to be clarified. Due to the big size of dengue vesicle, membrane source is important for vesicle formation. Among Atg proteins family, Atg9 is the only one containing transmembrane domains for membrane trafficking. We found that Atg9 colocalized with NS1 in the cytosol and both of them were on the surface of dengue vesicle. Confocal microscopy and co-immunoprecipitation confirmed that Atg9 and NS1 were in an interaction complex. During early viral replication, NS1 is expressed on the ER. We confirmed that NS1 interacted with Atg9 to recruit it to ER via their interaction, and Atg9 mutant lost the interaction. It was caused by an abnormal distribution of Atg9 mutant in the cell. Besides, Atg9 deficient donor cells, such as Atg9 knockdown Huh7, Atg9 mutant-overexpressing Huh7 and Atg9 knockout MEF, had a decreased infection rate of recipient cells in the close-contact co-culture system. This was caused by the reduced numbers of vesicles in Atg9 deficient donor cells. These results show that Atg9 plays an important role in the formation of dengue vesicle. Our study clarifies that the induction of autophagy is not only related to DENV replication but also contributes to the formation and transmission of dengue vesicle. This discovery may explain the inefficiency of antibody neutralization upon DENV infection as a potential immune evasion mechanism in dengue patients.