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

TLR7 is an endosomal receptor that is expressed in endosomes, which fuse with lysosomes [38] . Autophagosomes are formed by the expansion of phagophores and fusion with lysosomes. Thus there should exist some relationship between TLR7 activation and autophagy. Delgado et al. [39] have indicated that TLRs control autophagy, and Iwasaki [18] has shown that VSV (vesicular-stomatitis virus) recognition via TLR7 requires cytosolic viral replication intermediates that are transported to the lysosome by means of autophagy. In our present study, we have shown by immunoelectron microscopy that TMV protein accumulated on autophagosomal membranes. Moreover, at 4 h post-transfection, we observed by confocal microscopy that TMV-RNA co-localized with autophagosomes on the ER (see Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320171add.htm). These observations indicate that both TMV-RNA and TMV protein might appear on autophagosomal membranes simultaneously and that they might be bound together as cytosolic viral replication intermediates to be transported by autophagosomes. Hence TMV-RNA and 'cytosolic viral replication intermediates' might be recognized by TLR7 during the maturation of autophagy. . Autophagy may function either as an antiviral pathway (that degrades viruses) or as a pro-viral pathway (that helps viruses replicate or exit from cells) [18, 40] . If TMV-RNA is recognized by TLR7, it would be engulfed by the host cell through the process of endocytosis and digested by lysosomal enzymes. Both TMV-RNA and TMV protein would therefore be removed through this pathway. However, here, we not only demonstrated the accumulation of CP in the ER, but we also unexpectedly observed that TMV-positive RNA moved from the cytoplasm to the nucleus. Even more surprisingly, we discovered suspected TMV virions in HeLa cells by TEM. Although this evidence is not sufficient by itself to determine whether TMV-RNA replicates in HeLa cells, we found that not all of TMV-RNA or TMV protein was digested during the process of autophagy. Thus we presume that autophagy functions as a pro-viral pathway after the invasion of TMV into HeLa cells, which might aid in TMV-RNA replication in HeLa cells. Some recent reports have demonstrated that viruses use accumulated autophagosomes for their replication. In these reports, an even more dramatic subversion of macroautophagy is induced by several ssRNA viruses that replicate in the cytoplasm. Picornaviruses (poliovirus and rhinoviruses), coronaviruses (mouse hepatitis virus and SARS) and one arterivirus (equine arteritis virus) replicate on the surface of autophagosomal membranes [40] [41] [42] . These viruses likely block the fusion of autophagosomes that carry the viral replication complexes with lysosomes. The generation of autophagic membranes as scaffolds for the replication machinery is essential because inhibition of macroautophagy by siRNA-mediated silencing of Atg12 or Atg8 (LC3) inhibits viral replication [40] [41] [42] . Based on these discoveries, we attempted to determine why TMV protein accumulated on autophagosomal membranes when the transfection time was extended and whether the translation of TMV-RNA was associated with the maturation of autophagosomes. To address these questions, we first sought to demonstrate the replication of TMV-RNA in HeLa cells; we need to find the production of TMV-negative RNA in HeLa cells, evidence of TMV particles isolated from HeLa cells by EM and the occurrence