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Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway.

ABSTRACT: Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication.

Abbreviations

ATG14/Barkor: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; ER: endoplasmic reticulum; hpi: hours post-infection; IBDV: infectious bursal disease virus; IP: co-immunoprecipitation; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PDPK1: 3-phosphoinositid-dependent protein kinase-1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; vBCL2: viral BCL2 apoptosis regulator.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC8386615 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Autophagy is a lysosome-dependent degradation program to maintain cellular homeostasis in response to a variety of stressful conditions, such as long-lived or non-functional subcellular organelles, protein aggregates, nutrient limitation, and virus/bacteria infection. Accordingly, dysregulation of autophagy is closely associated with many human pathophysiological conditions, such as neurodegenerative diseases, aging, and cancer, and autophagy is highlighted as an important therapeutic target for these human diseases. In autophagy process, PIK3C3/VPS34 complex plays important roles in autophagosome biogenesis. Accumulating evidences that inhibition of PIK3C3/VPS34 complex successfully blocks autophagy make the complex as an attractive target for the development of autophagy-specific inhibitors. However, considering that various forms of PIK3C3/VPS34 complex exist and they are involved in many different cellular functions, the targeting of the pro-autophagy PIK3C3/VPS34 complex is required to specifically inhibit autophagy. To identify autophagy inhibitors targeting the pro-autophagy complex, we have performed the screening of a customized natural product library consisting of 35 herbal extracts which are widely used in the oriental medicine as anti-inflammation and/or anti-tumor reagents. We discovered that an alcoholic extract of Thuja orientalis L. leaves inhibits pro-autophagy complex formation by disrupting the interaction between autophagy-specific factor, ATG14L, and the complex core unit Vps34-Beclin 1 in vitro. Also, it inhibits the nutrient starvation induced autophagy and diminished pro-autophagy PIK3C3/VPS34 complex containing either ATG14L or UVRAG in several cell lines. Our results strongly suggest that Thuja orientalis L. leave extract functions as an autophagy-specific inhibitor not decreasing the complex activity nor the protein level, but preventing protein-protein interaction between autophagy-specific factor (ATG14L and UVRAG) and PIK3C3/VPS34 complex core unit, Vps34-Beclin 1, thereby specifically depleting the pro-autophagy complex to inhibit autophagy.

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Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway.

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