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Duck enteritis virus activates CaMKK?-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium.

ABSTRACT: BACKGROUND:The results of our previous study showed that impaired cellular energy metabolism contributes to duck enteritis virus-induced autophagy via the 5`-adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2/mammalian target of rapamycin pathway in duck embryo fibroblast (DEF) cells. However, it remains unknown whether any other underlying mechanisms of AMPK activation are involved in autophagy induction. METHODS:The activity of CaMKK? and AMPK in DEF cells infected with DEV were evaluated.The Effect of inhibitory activity of CaMKK? on DEV-induced autophagy was investigated. In addtion to, the cytosolic calcium level in DEF cells infected with DEV were evaluated.The Effect of inhibitory cytosolic calcium level on DEV-induced autophagy was investigated. RESULTS:In this study, duck enteritis virus (DEV) infection activated CaMKK? and its substrate molecule AMPK at 36, 48, and 60 h post-infection (hpi). STO-609, a CaMKK? inhibitor, or CaMKK? siRNA significantly inhibited the activation of DEV to AMPK, LC3I to LC3II transformation, and GFP-LC3 puncta distribution. In addition, inhibition of CaMKK? activity also significantly reduced progeny DEV titer and gB protein expression. Besides, cytosolic calcium (Ca2+) was higher in DEV-infected cells than mock controls at 36, 48, and 60 hpi, respectively. Treatment of DEV-infected cells with 1,2-Bis (2-aminophenoxy) ethane-N, N, N', N-tetraacetic acid (BAPTA-AM) significantly reduced intracellular Ca2+ ion concentrations, as well as CaMKK? and AMPK activities, and subsequent autophagy, in addition to viral protein synthesis and viral titer. CONCLUSIONS:These results showed that elevated [Ca2+]cyto-mediated activation of CaMKK? managed the activation of AMPK, which then positively regulated autophagy, thereby providing further insight into DEV-host interactions.

SUBMITTER: Yin H

PROVIDER: S-EPMC6090797 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium.

Yin Haichang H Zhao Lili L Wang Yiping Y Li Siqi S Huo Hong H Chen Hongyan H

Virology journal 20180806 1

<h4>Background</h4>The results of our previous study showed that impaired cellular energy metabolism contributes to duck enteritis virus-induced autophagy via the 5`-adenosine monophosphate-activated protein kinase (AMPK)/tuberous sclerosis complex 2/mammalian target of rapamycin pathway in duck embryo fibroblast (DEF) cells. However, it remains unknown whether any other underlying mechanisms of AMPK activation are involved in autophagy induction.<h4>Methods</h4>The activity of CaMKKβ and AMPK i ...[more]

PMID: 30081955

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Project description:BackgroundDuck viral hepatitis (DVH) is a highly contagious viral disease affecting ducks. It can be caused by five agents, including duck hepatitis A virus genotypes 1 (DHAV-1), 2 (DHAV-2), and 3 (DHAV-3), as well as duck hepatitis virus 2 and duck hepatitis virus 3. Since 2007, DHAV-3 has been known to be the most prevalent in East and South Asia. So far, the information regarding the propagation of DHAV-3 in cultured cells is limited. In this study, we describe the comparative studies on the growth properties of DHAV-3 in primary duck embryo fibroblast (DEF) cells using two different strains: a virulent strain C-GY and an attenuated strain YDF120. The effect of fetal calf serum (FCS) and chick serum (CS) on DHAV-3 replication and the mechanism of the inhibitory effect conferred by FCS were also investigated.ResultsFollowing serial passages, both C-GY and YDF120 failed to produce cytopathic effect and plaques. The combined quantitative real-time PCR and indirect immunofluorescence staining methods showed that the two viruses could be propagated productively in DEF cells. Investigation of the viral growth kinetics revealed that the two viruses replicated in DEF cells with similar efficiencies, while the viral load of the virulent C-GY strain peaked more rapidly when compared with the attenuated YDF120 strain. Neutralization assay and time-of-drug-addition study indicated that FCS displayed inhibitory effect on DHAV-3 replication. Analysis on the mechanism of action of FCS against DHAV-3 demonstrated that the inhibitory effect was reflected at three steps of the DHAV-3 life cycle including adsorption, replication, and release.ConclusionsBoth virulent and attenuated DAHV-3 strains can establish noncytocidal, productive infections in DEF cells. The virulent strain replicates more rapidly than the attenuated strain in early infection period. FCS has an inhibitory effect on DHAV-3 replication, which may be attributed to action of a non-specific inhibitory factor present in FCS directly on the virus. These findings may provide new insights into the development of potential antiviral agents.

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Duck enteritis virus activates CaMKK?-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium.

Publications

Duck enteritis virus activates CaMKKβ-AMPK to trigger autophagy in duck embryo fibroblast cells via increased cytosolic calcium.

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