sirolimus and Dengue

The mechanistic target of rapamycin (mTOR) functions in two distinct complexes: mTORC1, and mTORC2. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. Activation of mTORC2 occurs upon infection with some viruses, but its functional role in viral pathogenesis remains poorly understood. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

Topics: Apoptosis; Dengue; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Multiprotein Complexes; RNA, Small Interfering; Sirolimus; TOR Serine-Threonine Kinases; Virus Replication

Mosquito-borne dengue virus (DENV) causes major disease worldwide, impacting 50-100 million people every year, and is spread by the major mosquito vector Aedes aegypti. Understanding mosquito physiology, including antiviral mechanisms, and developing new control strategies have become an important step towards the elimination of DENV disease. In the study reported here, we focused on autophagy, a pathway suggested as having a positive influence on virus replication in humans, as a potential antiviral target in the mosquito.. To understand the role played by autophagy in Ae. aegypti, we examined the activation of this pathway in Aag-2 cells, an Ae. aegypti-derived cell line, infected with DENV. Rapamycin and 3-methyladenine, two small molecules that have been shown to affect the function of the autophagy pathway, were used to activate or suppress, respectively, the autophagy pathway.. At 1-day post-DENV infection in Aag-2 cells, transcript levels of both the microtubule-associated protein light chain 3-phosphatidylethanolamine conjugate (LC3-II) and autophagy-related protein 1 (ATG1) increased. Rapamycin treatment activated the autophagy pathway as early as 1-h post-treatment, and the virus titer had decreased in the Aag-2 cells at 2 days post-infection; in contrast, the 3-methyladenine treatment did not significantly affect the DENV titer. Treatment with these small molecules also impacted the ATG12 transcript levels in DENV-infected cells.. Our studies revealed that activation of the autophagy pathway through rapamycin treatment altered DENV infection in the mosquito cells, suggesting that this pathway could be a possible antiviral mechanism in the mosquito system. Here we provide fundamental information needed to proceed with future experiments and to improve our understanding of the mosquito's immune response against DENV.

Topics: Adenine; Aedes; Animals; Autophagy; Cell Line; Dengue; Dengue Virus; Mosquito Vectors; Sirolimus; Virus Replication

Antibody-dependent enhancement (ADE) of dengue virus (DENV) infection is identified as the main risk factor of severe dengue diseases. The underlying mechanisms leading to severe dengue fever remain unclear.. THP-1 cells were treated with an autophagy inducer (rapamycin) or inhibitor (3-methyladenine [3-MA]) and infected with DENV and DENV-ADE. In order to investigate the expression profile of autophagy-related genes in DENV-ADE and DENV direct infection of THP-1 cells, the PCR array including 84 autophagy-related genes was selected to detect the expression of related genes, and then heat map and clustergram were established by analysis software to compare the expression differences of these genes between the DENV-ADE and DENV direct infection.. Autophagy-inducing complex related genes ATG5 and ATG12 were upregulated, and autophagosomes were also observed by transmission electron microscopy among DENV-ADE- and DENV-infected THP-1 cells, which indicated that autophagy was involved in dengue infection. The results show that 3-MA has a significant inhibitory effect on ATG12 in THP-1 cells; on the contrary, the expression of ATG12 was upreg-ulated in THP-1 cells that were treated with rapamycin. The autophagy-related genes ESR1, INS, BNIP3, FAS, TGM2, ATG9B, and DAPK1 exhibited significant differences between DENV-ADE and DENV direct infection groups.. In the present study, an additional mechanism of autophagy was inhibited by the autophagy inhibitor (3-MA) in DENV- and DENV-ADE-infected THP-1 cells. Our finding provided a clear link between autophagy and antibody-enhanced infection of DENV.

Topics: Adenine; Antibodies, Viral; Antibody-Dependent Enhancement; Autophagosomes; Autophagy; Autophagy-Related Protein 12; Autophagy-Related Proteins; Dengue; Dengue Virus; Humans; Sirolimus; THP-1 Cells; Transcriptome

Dengue virus (DENV) is one of the major infectious pathogens worldwide. DENV infection is a highly dynamic process. Currently, no antiviral drug is available for treating DENV-induced diseases since little is known regarding how the virus interacts with host cells during infection. Advanced molecular imaging technologies are powerful tools to understand the dynamics of intracellular interactions and molecular trafficking. This study exploited a single-virus particle tracking technology to address whether DENV interacts with autophagy machinery during the early stage of infection. Using confocal microscopy and three-dimensional image analysis, we showed that DENV triggered the formation of green fluorescence protein-fused microtubule-associated protein 1A/1B-light chain 3 (GFP-LC3) puncta, and DENV-induced autophagosomes engulfed DENV particles within 15-min postinfection. Moreover, single-virus particle tracking revealed that both DENV particles and autophagosomes traveled together during the viral infection. Finally, in the presence of autophagy suppressor 3-methyladenine, the replication of DENV was inhibited and the location of DENV particles spread in cytoplasma. In contrast, the numbers of newly synthesized DENV were elevated and the co-localization of DENV particles and autophagosomes was detected while the cells were treated with autophagy inducer rapamycin. Taken together, we propose that DENV particles interact with autophagosomes at the early stage of viral infection, which promotes the replication of DENV.

Topics: Adenine; Autophagy; Cell Line, Tumor; Dengue; Dengue Virus; Host-Pathogen Interactions; Humans; Molecular Imaging; Phagosomes; Sirolimus; Virion

We and others have reported that autophagy is induced by dengue viruses (DVs) in various cell lines, and that it plays a supportive role in DV replication. This study intended to clarify whether DV infection could induce autophagy in vivo. Furthermore, the effect of DV induced autophagy on viral replication and DV-related pathogenesis was investigated.. The physiopathological parameters were evaluated after DV2 was intracranially injected into 6-day-old ICR suckling mice. Autophagy-related markers were monitored by immunohistochemical/immunofluorescent staining and Western blotting. Double-membrane autophagic vesicles were investigated by transmission-electron-microscopy. DV non-structural-protein-1 (NS1) expression (indicating DV infection) was detected in the cerebrum, medulla and midbrain of the infected mice. In these infected tissues, increased LC3 puncta formation, LC3-II expression, double-membrane autophagosome-like vesicles (autophagosome), amphisome, and decreased p62 accumulation were observed, indicating that DV2 induces the autophagic progression in vivo. Amphisome formation was demonstrated by colocalization of DV2-NS1 protein or LC3 puncta and mannose-6-phosphate receptor (MPR, endosome marker) in DV2-infected brain tissues. We further manipulated DV-induced autophagy by the inducer rapamycin and the inhibitor 3-methyladenine (3MA), which accordingly promoted or suppressed the disease symptoms and virus load in the brain of the infected mice.We demonstrated that DV2 infection of the suckling mice induces autophagy, which plays a promoting role in DV replication and pathogenesis.

Topics: Adenine; Animals; Animals, Newborn; Antimetabolites; Autophagy; Blotting, Western; Dengue; Dengue Virus; Fluorescent Antibody Technique; Immunochemistry; Immunosuppressive Agents; Mice; Mice, Inbred ICR; Microscopy, Electron, Transmission; Sirolimus; Viral Load; Virus Replication