The invasion of tobacco mosaic virus RNA induces endoplasmic reticulum stress-related autophagy in HeLa cells

Li, Li; Wang, Li; Xiao, Ruijing; Zhu, Guoguo; Li, Yan; Liu, Changxuan; Yang, Ru; Tang, Zhiqing; Li, Jie; Huang, Wei; Chen, Lang; Zheng, Xiaoling; He, Yuling; Tan, Jinquan

TMV-RNA is the key factor for triggering autophagy in HeLa cells Activation of autophagy genes is a critical step in autophagosome formation [17] . To examine whether the key autophagy gene (Beclin1) [21] was synergistically activated during TMV-RNA-induced autophagy, real-time PCR and Western blotting were performed to monitor the nucleic acid and protein levels, respectively, of Beclin1. Beclin1 mRNA increased markedly when the transfection time was extended to 72 h ( Figure 2A) . Simultaneously, Beclin1 protein markedly accumulated within 24 h in TMV-RNA-treated HeLa cells, and the accumulation of protein was much more pronounced when the transfection time was extended to 72 h ( Figure 2B ). Neither Beclin1 nucleic acid nor Beclin1 protein was found in HeLa cells not transfected with TMV-RNA. To verify that the activation of Beclin1 in HeLa cells was indeed associated with TMV-RNA-induced autophagy, a human breastcancer cell line in which Beclin1 is monoallelically deleted and thus expressed at reduced levels (MCF7 cells) [30] was transfected with TMV-RNA using the same protocol. Additionally, to interfere with the expression of the Beclin1 gene in cells we also transfected a Beclin1 siRNA (small interfering RNA) vector into HeLa cells. The Beclin1 siRNA vector was transfected into HeLa cells 24 h before the transfection of TMV-RNA. Analysis of the expression of Beclin1 and LC3 in MCF7 cells or HeLa cells by Western blotting showed that no Beclin1 was detected in MCF7 cells with or without TMV-RNA transfection, and the expression level of LC3 in MCF7 cells did not change after transfection ( Figure 2C) . Moreover, the expression of both LC3 and Beclin1 in HeLa cells transfected with both the Beclin1 siRNA vector and TMV-RNA was reduced compared with cells transfected with TMV-RNA only ( Figures 2B and 2C) . Taken together, the invasion of TMV-RNA directly results in autophagy, and Be-clin1 is the key autophagy gene in TMV-RNA-transfected HeLa cells.