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

After the transfection of TMV-RNA for 24, 48 and 72 h, cell lysates were harvested and used for the detection of proteins by Western blotting. The results indicate that TMV-RNA was translated into CP; CP markedly increased within 24 h in TMV-RNA-treated HeLa cells, and the accumulation of the protein increased further when the transfection time was extended to 72 h, whereas it was undetectable in HeLa cells not transfected with TMV-RNA ( Figure 4A ). Considering the high-expression level of CP in HeLa cells, we deduced that TMV might escape from immune defence. To determine whether TMV-RNA was translated in the ER, we isolated the ER from HeLa cells transfected with TMV-RNA for 24, 48 and 72 h. Analysis of CP in the ER fraction by Western blotting showed that the expression level of CP markedly increased when the time was extended to 72 h ( Figure 4A ). To further confirm whether CP was located in the ER and to observe the location of TMV-RNA, we performed immunofluorescence (IF) and FISH techniques [25] in succession. TMV-positive RNA and proteins were identified in situ by confocal microscopy in cells fixed by a protocol intended to retain native cell size and shape. The TMV-positive RNA was visualized by FISH with a strand-specific probe that was labelled with a Cy3 fluorochrome (red). CP was detected using a rabbit anti-CP antibody and a corresponding secondary antibody (Alexa Fluor ® 488 anti-rabbit antibody) (green), and the ER was stained with ER tracker (blue, Invitrogen). As shown in Figure 4 We observed that CP (green) indeed accumulated in the ER as small, irregularly sized granules, whereas TMV-RNA (red) rarely appeared in the ER but was enriched in the nucleolus. Neither CP nor TMV-positive RNA was found in cells not transfected with TMV-RNA ( Figure 4C-c) .