The role of secretory autophagy in the expression of dengue virus proteins and HMGB1

Abstract

Dengue virus (DENV) causes over 390 million people infection every year Dengue disease ranges from mild dengue fever (DF) to severe dengue hemorrhagic fever (DHF) dengue shock syndrome (DSS) and death DENV is an enveloped single-stranded positive-sense RNA virus DENV genome consists of three structural proteins capsid protein (C) premembrane/ membrane protein (prM/M) envelope protein (E) and seven nonstructural proteins (NS1 to NS5) These proteins play diverse roles from viral RNA replication to protein synthesis We previously reported that DENV infection induces autophagy to enhance virus replication Others further reported that DENV infection triggered autophagy related vesicle releasing to evade immune system surveillance Autophagic machinery is involved in selective and non-selective cargoes degradation process and participates in extracellular delivery of a number of pathogen particles and cytosolic proteins such as high mobility group box 1 (HMGB1) which was significantly increased in DENV-infected patients This study clarified the relationship among autophagosome HMGB1 and DENV proteins including which DENV-protein can be selectively recruited to the autophagosome and whether secretory autophagy is involved in the release of these recruited proteins Initially we showed that autophagy is induced by DENV2 infection of lung cancer A549 cells We then purified the autophagosome from DENV2-infected Huh7 cell and A549 cells We further reveal that DENV-C E NS1 NS3 NS4B and HMGB1 proteins are existed in the purified autophagosomes by Western blotting We confirmed that C NS1 and NS3 proteins are indeed in the autophagosome by immunogold labeling transmission electron microscopy We also clarified that DENV-C E NS1 NS3 NS4B and HMGB1 can be selectively recruited to the autophagosome Furthermore the levels of C E NS1 and NS3 proteins increased alone with infection time indicating that these proteins were not degraded We further used chloroquine (CQ) to block lysosome fusion at 48 h p i The levels of DENV-C E and NS1 proteins decreased compared to the LC3-II protein levels in DENV2 infected A549 cells indicating that an unconventional secretory autophagy was triggered which leads to the exocytosis of these three proteins under CQ treatment conditions The increase release of HMGB1 after CQ treatment support our speculation that secretory autophagy was promoted when degradative autophagic process was inhibition In conclusion this study provide the evidence that an unconventional secretory autophagy can be induced during under DENV infection when autophagy degradation was suppression

Date of Award	2017 Mar 9
Original language	English
Supervisor	Hsiao-Sheng Liu (Supervisor)