title: Dengue Virus Inhibition of Autophagic Flux and Dependency of Viral Replication on Proteasomal Degradation of the Autophagy Receptor p62.
abstract: Autophagic flux involves formation of autophagosomes and their degradation by lysosomes. Autophagy can either promote or restrict viral replication. In the case of Dengue virus (DENV), several studies report that autophagy supports the viral replication cycle, and describe an increase of autophagic vesicles (AVs) following infection. However, it is unknown how autophagic flux is altered to result in increased AVs. To address this question and gain insight into the role of autophagy during DENV infection, we established an unbiased, image-based flow cytometry approach to quantify autophagic flux under normal growth conditions and in response to activation by nutrient deprivation or them TOR inhibitor Torin1.We found that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Early after infection, basal and activated autophagic flux was enhanced. However, during established replication, basal and Torin1-activated autophagic flux was blocked, while autophagic flux activated by nutrient deprivation was reduced, indicating a block to AV formation and reduced AV degradation capacity. During late infection AV levels increased as a result of inefficient fusion of autophagosomes with lysosomes. In addition, endolysosomal trafficking was suppressed, while lysosomal activities were increased.We further determined that DENV infection progressively reduced levels of the autophagy receptor SQSTM1/p62 via proteasomal degradation. Importantly, stable overexpression of p62 significantly suppressed DENV replication, suggesting a novel role for p62 as a viral restriction factor. Overall, our findings indicate that in the course of DENV infection, autophagy shifts from a supporting to an antiviral role, which is countered by DENV.
  
  
  OBJECTIVE
  Autophagic flux is a dynamic process starting with the formation of autophagosomes and ending with their degradation after fusion with lysosomes. Autophagy impacts the replication cycle of many viruses. However, thus far the dynamics of autophagy in case of Dengue virus (DENV) infections has not been systematically quantified. Therefore, we used high-content, imaging-based flow cytometry to quantify autophagic flux and endolysosomal trafficking in response to DENV infection. We report that DENV induced an initial activation of autophagic flux, followed by inhibition of general and specific autophagy. Further, lysosomal activity was increased, but endolysosomal trafficking was suppressed confirming the block of autophagic flux. Importantly, we provide evidence that p62, an autophagy receptor, restrict DENV replication and was specifically depleted in DENV-infected cells via increased proteasomal degradation. These results suggest that during DENV infection autophagy shifts from a proviral to an antiviral cellular process, which is counteracted by the virus.
authors.forename: Philippe
authors.initials: P
authors.lastname: Metz
authors.forename: Abhilash
authors.initials: A
authors.lastname: Chiramel
authors.forename: Laurent
authors.initials: L
authors.lastname: Chatel-Chaix
authors.forename: Gualtiero
authors.initials: G
authors.lastname: Alvisi
authors.forename: Peter
authors.initials: P
authors.lastname: Bankhead
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Mora-Rodriguez
authors.forename: Gang
authors.initials: G
authors.lastname: Long
authors.forename: Anne
authors.initials: A
authors.lastname: Hamacher-Brady
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Brady
authors.forename: Ralf
authors.initials: R
authors.lastname: Bartenschlager
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy; D002460:Cell Line; D003715:Dengue / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology* / Q000821:virology; D003716:Dengue Virus / Q000235:genetics / Q000502:physiology*; D006801:Humans; D010588:Phagosomes / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis; D000071456:Sequestosome-1 Protein; D014779:Virus Replication
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C099718:SQSTM1 protein, human; D000071456:Sequestosome-1 Protein