The Effect And Mechanism Of Cellular Autophagy Induction During Bluetongue Virus Infection

Bluetongue(BT) is an important insect-transmitted disease that is caused by Bluetongue virus(BTV). BT can cause ruminants, especially sheep, higher morbidity and mortality, posing a potential threat to the animal husbandry and the economic trade of livestock product. BTV, a member of the Orbivirus genus in the Reoviridae family, is an architecturally complex arbovirus. The virion is a nonenveloped particle consisting of the outermost double-capsids and ten genomic double-stranded RNA(dsRNA) segments. As a typical model of the Orbivirus genus, BTV has been the subject of extensive molecular biology studies, such as molecular structure, biochemical characterization, global epidemiology and diagnostic methods. Even so, the mechanisms guiding the interactions between the virus and host remain poorly understood, highlighting the need for in-depth exploring the pathogenic mechanism of BTV from the perspective of the host.As obligate intracellular parasites, viruses are extremely reliant on host cells and must interact with host response. Autophagy, as a highly conserved cellular adaptive response, plays important roles in keep homeostasis and promote metabolism. Autophagy is a dynamic process in eukaryotes that involves the formation of double-membrane autophagosomes, with damaged organelles or microbes wrapped. Then these substrates would be delivered for digestion and recycling via a lysosomal degradative pathway. Autophagy has intracellular antimicrobial properties. However, accumulating lines of evidence suggest that many viruses have evolved to evade, escape or exploit autophagy for their own benefit, such as influenza virus, hepatitis C virus(HCV), dengue virus and rotavirus. Regarding BTV, the relationship between BTV infection and autophagy has not been thoroughly examined and deserves to be explored.To begin with, three classical methods were adopted to examine whether BTV1 infection would induce autophagy in BSR cells, including the observation of autophagosomes formation by transmission electron microscopy(TEM), the detection of GFP-LC3 puncta accumulation by confocal microscopy and the analysis of LC3 conversion by Western Blot. Results showed that, compared with the negative control, BTV1 infection induced numerous autophagosome-like double-membrane vesicles in the cytoplasm and more GFP-LC3 dots accumulation. Besides, the LC3 conversion was significant after BTV infection. To further verify the result, primary lamb lingual epithelial cells were used in this experiment and similar results were obtained. In sum, based on these results, we concluded that notable autophagy could be triggered by BTV1 infection.Next, the autophagic flux in BSR cells based on the analysis of the autophagic degradation was measured. Autophagic flux is a continuous and complete process of autophagy and can be comprehensively assessed by p62 degradation, LC3-II turnover and colocalization analysis of autophagosomes and lysosomes. Immunoblotting results revealed significant progressive degradation of p62 in BTV-infected cells. And the accumulation of LC3-II and p62 was observed along with the employment of CQ in BTV-infected cells. Moreover, the colocalization of GFP-LC3-tagged autophagosomes and LysoTracker-stained lysosomes was detected in BTV1-infected BSR cells. Collectively, these results indicated that BTV1 induced autophagy initiation and autophagic degradation, proving that BTV1 infection induces the occurrence of enhanced autophagic flux.Besides, we speculated that whether the induction of autophagy was dependent on BTV1 effective replication. UV-inactivated BTV1 infection could not induce similar autophagy phenomenon in BSR cells, verifying our speculation. Further, the inhibition of autophagy by pharmacological inhibitors(3-MA, CQ) and RNA interference(siBeclin1) significantly decreased viral replication. In contrast, treating BSR cells with rapamycin, an inducer of autophagy, promoted BTV1 replication. These findings lead us to conclude that the effective replication of BTV1 is the critical factor for autophagy activation, and in turn autophagy is utilized for the virus replication.Finally, with a view to understanding the underlying mechanisms of autophagy initiation by BTV1 infection, the possible signaling pathways involved in BTV-induced autophagy were systematically dissected. We found that the activity of mTOR, a crucial pivot, was inhibited by BTV1 infection, subsequently leading to downstream p70S6 K suppression and autophagy initiation. We then explored the various upstream regulators of mTOR. First of all, we found BTV1-induced autophagy is independent of the ERK1/2 signaling pathway. We then assessed the possible role of PI3K/Akt pathway in autophagy via pharmacological treatment and mutant plasmids transfection. Results showed that the inhibition of PI3K/Akt was found to be partially responsible for mTOR inactivation. In contrast, enhanced Akt activity restored p-mTOR, leading to autophagy suppression. It indicated that Akt signaling pathway is involved in BTV1-induced autophagy activation. Furthermore, we found unexpectedly that AMPK seemed to play a more important role in BTV1-induced autophagy. We used drugs treatment and RNA interference to test the related molecules. Results showed that elevated [Ca2+]cyto-mediated activation of CaMKKβ exactly managed the activation of AMPK, which then positively regulated autophagy through suppressing mTOR. TSC2 is further verified a fatal mediator between upstream Akt or AMPK and downstream mTOR. Taken together, our data suggested that the BTV1-induced Akt inhibition and AMPK activation synergistically regulated downstream TSC2-mTOR pathway and then contributed to autophagy initiation and further favored virus replication.To sum up, this study firstly illustrates that BTV1-induced cellular autophagy plays an important role in the replication of BTV1. More importantly, we analyzed the signaling cascade network induced by BTV1 for autophagy initiation. Our achieved findings here identify a crucial step in driving the progress of interplay between BTV and autophagy. It will provide new insights into the pathogenesis of BTV and contribute to antiviral drugs development.