Dengue News: Study Shows That DENV-2 NS1 Promotes AMPK-LKB1 Interaction To Activate AMPK/ERK/mTor Signaling Pathway To Induce Autophagy

Dengue News: Dengue fever, a mosquito-borne viral infection, has become a growing global concern. Among the five serotypes of the dengue virus, DENV-2 stands out as the most evolved and virulent strain that causes more disease severity. Its increasing prevalence poses a severe threat to human health, emphasizing the urgent need for effective therapeutic measures. While specific anti-dengue drugs are yet to be developed, understanding the virus's interaction with the host is essential for advancing potential treatments. In particular, this study by researchers from Guizhou Medical University-China explores the role of the DENV-2 non-structural protein 1 (NS1) in promoting autophagy, a cellular process often activated during DENV infection.


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

The Growing Threat of Dengue Fever
Over the past few decades, the number of dengue virus (DENV) cases has been on the rise, with an estimated 300-400 million people infected annually. Each outbreak not only leads to severe disease but also imposes a significant economic burden on affected regions.
 
This current year, 2023, Dengue News reports have shown that many countries are facing an unprecedented rise in dengue infections and also many cooler climate countries that previous never faced the dengue threat are now witnessing surges as well.
 
Unfortunately, specific drugs for treating dengue fever remain elusive, and the dengue vaccine's overall efficacy and safety have raised concerns. Therefore, a deeper understanding of the virus's pathogenesis is essential for developing effective therapeutic drugs and preventive measures.
 
DENV Infection and Autophagy
DENV infection initiates an innate immune response that produces antiviral effects. However, as the virus evolves, it can evade recognition or inhibit the production of antiviral states, ultimately exacerbating the disease. Autophagy, a cellular process that degrades and recycles damaged or unnecessary cellular components, is activated in DENV-infected cells. Most studies indicate that DENV-2-induced autophagy promotes the replication of viral RNA. In contrast, inhibiting autophagy significantly reduces viral replication. Autophagy's involvement in viral replication extends to energy metabolism, as DENV infection-induced autophagy increases cellular glucose uptake, transport, and glycolysis, thereby enhancing viral replication. It also promotes lipolysis to produce ATP, further supporting viral replication.
 
The Role of AMPK in Autophagy
Activated AMP-activated protein kinase (AMPK) plays a crucial role in autophagy. It inhibits mTORC1 activity, which, in turn, activates autophagy and triggers lipophagy, the breakdown of lipid droplets, to produce ATP for enhanced viral replication. Moreover, activated AMPK can promote autophagy independently of mTOR by directly phosphorylating ULK1/2 and phosphatidylinositol-3-kinase. Previous research by our team indicated that DENV-2 induces autophagy in human umbilical vein endothelial cells (HUVECs) through the AMPK/ERK/mTOR signaling pathway. However, the viral components and molecular mechanisms underlying DENV-2's regulation of the AMPK/ERK/mTOR signaling pathway to induce autophagy have not been fully elucidated.
 
DENV-2 NS1: A Key Player
DENV is an RNA virus that encodes both structural and non-structural proteins. Among these, the non-structural protein 1 (NS1) is vital for viral replication and immune evasion. NS1 is continuously secreted by infected host cells and can influence various aspects of DENV infection, including vascular leakage, coagulation disorders, and early events of viral RNA replication. While some DENV non-structural proteins have been studied in the context of autophagy, only NS1 and NS4A have been reported to have an autophagy-inducing role during DENV infection. However, there have been no reports on DENV's own proteins regulating autophagy through the AMPK pathway.
 
NS1's Role in Autophagy Activation
This study reveals a novel mechanism by which NS1 mediates DENV-2-induced autophagy. Through molecular docking techniques, we identified NS1 as the most likely non-structural protein to be involved in AMPK pathway-mediated autophagy. Further experiments confirmed that NS1 induces autophagy via the AMPK/ERK/mTOR signaling pathway.
 
Interaction Between NS1 and AMPK
Immunoprecipitation assays demonstrated that both DENV-2 NS1 and AMPK interact with each other, indicating a direct binding effect. Laser confocal microscopy revealed that NS1 and AMPK co-localize in the cytoplasm of DENV-2-infected HUVECs, further supporting their interaction.
 
The Crucial Role of NS1's Wing Structural Domain
NS1 contains three structural domains: β-roll, wing, and β-ladder. Deletion experiments showed that all three structural domains are essential for NS1's function, with the wing structural domain having the most significant impact on NS1's ability to activate autophagy. It appears that the wing structural domain may serve as a key structural domain for NS1's activation of autophagy. This interaction was further confirmed through Co-IP and GST pulldown experiments.
 
NS1's Interaction with AMPK Structural Domains
NS1 was found to bind to all three structural domains of the AMPKα subunit. This suggests that NS1 may promote AMPK phosphorylation through multiple mechanisms, but the kinase structural domains STC and C-terminal exhibited stronger binding to NS1. This implies that they may play a primary role in NS1's interaction with AMPK.
 
The Role of LKB1
LKB1 and CaMKKβ are the main upstream kinases responsible for phosphorylating Thr172 on the AMPK kinase structural domain. Molecular docking predictions suggested that LKB1 was more likely to bind to NS1, and subsequent experiments confirmed that NS1 promotes LKB1 expression. Knockdown of LKB1 reduced the activation of the AMPK/ERK/mTOR pathway by NS1, further underscoring LKB1's role in this process.
 
DENV-2 NS1 as an Assembly Platform
NS1 serves as an assembly platform for interactions between AMPK and LKB1, promoting their binding and subsequent activation of AMPK. These finding sheds light on the mechanisms behind AMPK activation by NS1, leading to the activation of the AMPK/ERK/mTOR signaling pathway and the induction of autophagy.
 
Conclusion
In summary, this study has provided new insights into the role of NS1 in DENV-induced autophagy and offers important evidence for the development of potential therapeutic strategies targeting NS1. As NS1 plays a multifaceted role in the pathogenesis of DENV, understanding its interactions and functions is critical in the quest to combat dengue fever. This research takes us one step closer to the development of effective drugs for this growing global health threat.
 
The study findings were published in the peer reviewed Virology Journal.
https://virologyj.biomedcentral.com/articles/10.1186/s12985-023-02166-0
 
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