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Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infection<br>induces autophagy that enhances virus replication and vesicle release to evade immune system<br>surveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells and<br>showed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobility<br>group box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradient<br>centrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in the<br>cytoplasm of the infected cells were detected in the purified double-membrane autophagosome by<br>immunogold labeling under transmission electron microscopy. In DENV infected cells, the levels of<br>capsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramatic<br>accumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked by<br>chloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.<br>We further demonstrated that purified autophagosomes were infectious when co-cultured with<br>uninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrand<br>and full-length genomic RNAs, but no viral particles. It is possible that the infectivity of<br>the autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2<br>promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the first<br>to report that DENV2-induced double-membrane autophagosomes containing viral proteins and<br>full-length RNAs are infectious and not undergoing autophagic degradation. Our novel finding<br>warrants further validation of whether these intracellular vesicles undergo exocytosis to become<br>infectious autophagic vesicles.