Autophagy Promotes Duck Tembusu Virus Replication by Suppressing p62/SQSTM1-Mediated Innate Immune Responses In Vitro

Abstract

Duck Tembusu virus (DTMUV) has recently appeared in ducks in China and the key cellular determiners for DTMUV replication in host cells remain unknown. Autophagy is an evolutionarily conserved cellular process that has been reported to facilitate flavivirus replication. In this study, we utilized primary duck embryo fibroblast (DEF) as the cell model and found that DTMUV infection triggered LC3-II increase and polyubiquitin-binding protein sequestosome 1 (p62) decrease, confirming that complete autophagy occurred in DEF cells. The induction of autophagy by pharmacological treatment increased DTMUV replication in DEF cells, whereas the inhibition of autophagy with pharmacological treatments or RNA interference decreased DTMUV replication. Inhibiting autophagy enhanced the activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factor 7 (IRF7) pathways and increased the p62 protein level in DTMUV-infected cells. We further found that the overexpression of p62 decreased DTMUV replication and inhibited the activation of the NF-κB and IRF7 pathways, and changes in the NF-κB and IRF7 pathways were consistent with the level of phosphorylated TANK-binding kinase 1 (p-TBK1). Opposite results were found in p62 knockdown cells. In summary, we found that autophagy-mediated p62 degradation acted as a new strategy for DTMUV to evade host innate immunity.

Autophagy Is a Potential Therapeutic Target Against Duck Tembusu Virus Infection in vivo

“…Similar results are also found in ZIKV-infected mice with CQ treatment (Zhang et al, 2019a). Moreover, our lab's previous data invested that p62 regulated the innate antiviral response in DTMUV-infected cells (Hu et al, 2020). Hence, CQ treatments might also inhibit DTMUV replication by causing the accumulation of p62, and then affecting the host antiviral response in vivo.…”

“…Multiple evidence suggests that autophagy plays a crucial role in the life cycles of flaviviruses in vitro and in vivo (Ke, 2018). As we have investigated that autophagy promotes the replication of DTMUV in vitro (Hu et al, 2020), to further provide the clinical evidence on the effects of autophagy on DTMUV replication and pathogenesis, we utilized ducks as the animal model to study the role of autophagy in DTMUV-targeted organs.…”

“…3-MA inhibits autophagy by blocking autophagosome formation via the inhibition of class III PI3K (Klionsky et al, 2016) and has been used to inhibit the autophagy induced by various Flaviviruses, such as Zika Virus (ZIKV) in vitro (Cao et al, 2017), DENV in vitro (Lee et al, 2008) and in vivo (Lee et al, 2013), classical swine fever virus (CSFV) in vitro (Pei et al, 2014). And our previous data has shown 3-MA treatment successfully inhibits DTMUVinduced autophagy in vitro (Hu et al, 2020). However, one case shows that prolonged treatment with 3-MA promotes autophagy under nutrient-rich conditions (Wu et al, 2010).…”

New Insights into the Biology of the Emerging Tembusu Virus

“…Different types of avian cells have been used to evaluate TMUV biology and include BK3 cells, a transformed chicken B lymphocyte cell line, the chicken macrophage-derived cell line HD-11 and the DF1 cell line, isolated from chicken embryo fibroblasts. Besides specific usage for research, chicken embryo fibroblasts and duck and chicken eggs are routinely used for virus isolation and maintenance [ 3 , 4 , 5 , 26 , 30 , 93 , 94 ].…”

“…Despite the host immunity defense against several pathogen infections, autophagy can be hijacked by a range of viruses, including flaviviruses such as ZIKV [ 120 ], DENV [ 121 ] or JEV [ 122 ], to promote their own replication. Induction of autophagy by TMUV was confirmed in avian cells, acting as a viral strategy to evade the host immune response [ 94 ]. In these studies, stimulation of autophagy promoted TMUV replication, and, inversely, treatment with chemical inhibitors of autophagy led to a decrease in TMUV virions.…”