以in vitro及in vivo方式探討登革病毒二型所誘發自噬現象之特性

Abstract

Dengue virus (DV) causes about 100 million people infection every year. DV disease ranges from mild dengue fever (DF) to severe dengue haemorrhagic fever (DHF), dengue shock syndrome (DSS) and death. DV infection induces a cellular homeostasis response, autophagy, which is responsible for the degradation of long-lived proteins, aggregated proteins and damaged organelles. DV induced-autophagy regulates lipid metabolism of the host cell to enhance its replication. However, how DV induces autophagy, whether DV NS1 gene can induce autophagy and whether DV2 infection induces autophagy in vivo remain unclear. We demonstrated that DV2 infection triggered the complete autophagic flux by using a ptfLC3 (tandem fluorescent-tagged microtubule-associated protein 1 light chain 3) plasmid and a pRLucC124A-LC3wt plasmid. Furthermore, time lapse recording was conducted to demonstrate that DV2 infection induced multiple autophagic flux cycles including a short cycle (about 3 h) at early stage and a prolonged cycle (about 6 h) at the late stages of infection. Despite DV2 infection triggering complete autophagic progression, DV2 nonstructural protein 1 (NS1) protein was not degraded in the process. Previous study showed colocalization of NS1 and LC3 in autophagosomes in DV2 infected cells and NS1 is a component of DV replication complex. Furthermore, overexpression of NS1 (pCMVTaq3B-NS1) was not able to induce autophagy. DV has seven nonstructural proteins. We demonstrated that DV2 replicon (pD2R2A), which only contains nonstructural genes, induced autophagy, indicating that DV2 nonstructural proteins besides NS1 is involved in autophagy induction. Previous study showed that Hepatitis C virus induced autophagy via unfolded protein response (UPR). DV has been reported can induce ER stress. Our data showed that DV2 infection induced ER stress in Huh7 cells. After the treatment of an ER stress inhibitor 4-phenyl butyric acid (4-PBA), DV2 induced autophagy was inhibited demonstrated by decreased LC3 II expression, suggesting that DV2 infection induces ER stress which participates in DV2 induced autophagy. In animal study, DV2 was intracranially inoculated into six-day-old ICR suckling mice. Our data showed that in the DV-infected mouse brain, LC3 II expression was increased and NS1 was colocalized with inLC3 at day 5 post infections. Furthermore, in DV2 infected brain tissue the double-membrane autophagosome like vesicles were also detected under a transmission electronic microscope (TEM). It suggests that DV2 infection induces autophagy in vivo. Finally, we demonstrated that inhibition of autophagy by 3-methyladenine (3-MA) decreased the DV2 infection-related disease symptoms accompanied with decreased NS1 expression and viral titer. In summary, this study reveals that DV2 infection induces multiple autophagic flux cycles, ER stress participates in increased autophagy but NS1 does not, and finally DV2 infection induces autophagy in vivo, which affects disease symptoms.