DENV-2 NS1 promotes AMPK-LKB1 interaction to activate AMPK/ERK/mTOR signaling pathway to induce autophagy.

The global incidence of dengue fever has gradually increased in recent years, posing a serious threat to human health. In the absence of specific anti-dengue drugs, understanding the interaction of Dengue virus (DENV) with the host is essential for the development of effective therapeutic measures. Autophagy is often activated during DENV infection to promote viral replication, but the mechanism of how DENV's own proteins induce autophagy has not been clarified. In this study, we first preliminarily identified DENV-2 NS1 as the most likely viral protein for DENV-2-induced autophagy with the help of molecular docking techniques. Further experimental results confirmed that DENV-2 NS1 regulates DENV-2 infection of HUVEC-induced autophagy through the AMPK/ERK/mTOR signaling pathway. Mechanistically, DENV-2 NS1 mainly interacted with AMPK by means of its Wing structural domain, and NS1 bound to all three structural domains on the AMPKα subunit. Finally, the experimental results showed that DENV-2 NS1 promoted the interaction between LKB1 and AMPKα1 and thus activated AMPK by both increasing the expression of LKB1 and binding LKB1. In conclusion, the results of this study revealed that DENV-2 NS1 protein served as a platform for the interaction between AMPK and LKB1 after DENV-2 infection with HUVEC, and pulled AMPK and LKB1 together to form a complex. LKB1 to form a complex, promoting LKB1 action on the kinase structural domain of AMPKα1, which in turn promotes phosphorylation of the Thr172 site on the AMPK kinase structural domain and activates AMPK, thereby positively regulating the AMPK/ERK/mTOR signaling pathway and inducing autophagy. The present discovery improves our understanding of DENV-2-induced host autophagy and contributes to the development of anti-dengue drugs.

Mechanism of autophagy induced by activation of the AMPK/ERK/mTOR signaling pathway after TRIM22-mediated DENV-2 infection of HUVECs.

BACKGROUND: Dengue virus type 2 (DENV-2) was used to infect primary human umbilical vein endothelial cells (HUVECs) to examine autophagy induced by activation of the adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/mammalian target of rapamycin (mTOR) signaling pathway following tripartite motif-containing 22 (TRIM22)-mediated DENV-2 infection to further reveal the underlying pathogenic mechanism of DENV-2 infection. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to screen putative interference targets of TRIM22 and determine the knockdown efficiency. The effect of TRIM22 knockdown on HUVEC proliferation was determined using the CCK8 assay. Following TRIM22 knockdown, transmission electron microscopy (TEM) was used to determine the ultrastructure of HUVEC autophagosomes and expression of HUVEC autophagy and AMPK pathway-related genes were measured by qRT-PCR. Moreover, HUVEC autophagy and AMPK pathway-related protein expression levels were determined by western blot analysis. Cell cycle and apoptosis were assessed by flow cytometry (FCM) and the autophagosome structure of the HUVECs was observed by TEM. RESULTS: Western blot results indicated that TRIM22 protein expression levels increased significantly 36 h after DENV-2 infection, which was consistent with the proteomics prediction. The CCK8 assay revealed that HUVEC proliferation was reduced following TRIM22 knockdown (P < 0.001). The TEM results indicated that HUVEC autolysosomes increased and autophagy was inhibited after TRIM22 knockdown. The qRT-PCR results revealed that after TRIM22 knockdown, the expression levels of antithymocyte globulin 7 (ATG7), antithymocyte globulin 5 (ATG5), Beclin1, ERK, and mTOR genes decreased (P < 0.01); however, the expression of AMPK genes (P < 0.05) and P62 genes (P < 0.001) increased. FCM revealed that following TRIM22 knockdown, the percentage of HUVECs in the G2 phase increased (P < 0.001) along with cell apoptosis. The effect of TRIM22 overexpression on HUVEC autophagy induced by DENV-2 infection and AMPK pathways decreased after adding an autophagy inhibitor. CONCLUSIONS: In HUVECs, TRIM22 protein positively regulates autophagy and may affect autophagy through the AMPK/ERK/mTOR signaling pathway. Autophagy is induced by activation of the AMPK/ERK/mTOR signaling pathway following TRIM22-mediated DENV-2 infection of HUVECs.