Abstract
Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infection induces autophagy that enhances virus replication and vesicle release to evade immune system surveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells and showed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobility group box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradient centrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in the cytoplasm of the infected cells were detected in the purified double-membrane autophagosome by immunogold labeling under transmission electron microscopy. In DENV infected cells, the levels of capsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramatic accumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked by chloroquine, indicating that these proteins are not regulated by autophagic degradation machinery. We further demonstrated that purified autophagosomes were infectious when co-cultured with uninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negative-strand and full-length genomic RNAs, but no viral particles. It is possible that the infectivity of the autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2 promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the first to report that DENV2-induced double-membrane autophagosomes containing viral proteins and full-length RNAs are infectious and not undergoing autophagic degradation. Our novel finding warrants further validation of whether these intracellular vesicles undergo exocytosis to become infectious autophagic vesicles.
Highlights
Dengue is an arboviral disease caused by the dengue virus (DENV), which is transmitted to humans by mosquitoes of the Aedes family
We previously reported that DENV2 and Enterovirus 71 (EV71) infection-induced autophagy promotes viral replication and pathogenesis [17,18,19,20,21,22]
We previously reported that Dengue virus (DENV) induces autophagy in liver cancer Huh7 cells and plays a promoting role in viral replication [22,37]
Summary
Dengue is an arboviral disease caused by the dengue virus (DENV), which is transmitted to humans by mosquitoes of the Aedes family. NS4B is involved in blocking IFN signaling and inducing autophagosome formation [10] All of these structural and non-structural proteins of DENV play diverse roles in DENV pathogenesis. Autophagy is functionally classified as conventional degradative autophagy and unconventional secretory autophagy The former regulates cellular metabolism to protect the cell from stresses caused by damage through degradative machinery [11]. DENV can activate the AMP kinase-mTOR axis to stimulate proviral lipophagy [26] These findings imply that autophagy degradative machinery plays a pivotal role in DENV replication. Based on the findings of Li et al, mature DENV exits the cell through the Lyn-dependent (a src family tyrosine kinase) secretory autophagy machinery, implying that the intracellular infectious autophagosomes carrying DENV proteins and genomic RNAs may use the similar pathway to exit the cargoes
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