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

We next used an unrelated probe as a negative control to verify the location of TMV-positive RNA. Cells transfected with TMV-RNA for 24 and 48 h were fixed and analysed using the same IF and RNA-FISH techniques; the only difference was that instead of the ER staining step, cell nuclei were stained with DAPI (blue, Invitrogen). Thus blue fluorescence represented the location of the nucleus in Figure 5 . When we monitored the cells after TMV-RNA transfection for 24 h, TMV-positive RNA (red) was found throughout the cells as small, irregularly-sized granules ( Figure 5B , H.P.T. 24 h) that then accumulated in the nucleus as high-density granules after 48 h ( Figure 5C , H.P.T. 48 h). In addition, CP (green) was located around the nucleus and accumulated in the cytoplasm within 48 h. In contrast, TMV-positive RNA could not be detected in cells transfected with TMV-RNA that were hybridized with an unrelated probe ( Figure 5A-c, Con) . The above results suggest that TMV-RNA uses intracellular ri- bosomes to synthesize viral proteins in the ER and that the positive RNA localizes to the nucleus to perform other functions.