Stress-Induced Cellular Responses During Dengue Virus Infection

• Main Authors: Yun-Wei Hsu, 許芸瑋
• Other Authors: Yi-Ling Lin
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/43020131115407072660

碩士 === 國防醫學院 === 微生物及免疫學研究所 === 91 === Dengue virus (DEN) might trigger two stress-related responses in the infected cells, the double-stranded RNA (dsRNA) that accumulates during viral replication and the endoplasmic reticulum (ER) stress induced by translating large amounts of viral proteins, both of which lead to protein synthesis shut-off due to phosphorylation of eukaryotic initiation factor eIF2α. Previously, it has been shown that cellular protein synthesis was not blocked during DEN infection, indicating that DEN might has evolved a mechanism to disrupt the dsRNA-dependent kinase (PKR)-mediated translational suppression. To identify the potential DEN proteins interfering with PKR activation, we used a yeast strain RY1-1, which contains two integrated copies of human pkr under the control of a galactose-inducible promoter. Individual DEN proteins were cloned to a yeast-bacterial shuttle plasmid, pYES2, in which the viral protein expression was also under a galactose-inducible promoter. Yeast strain RY1-1 grew poorly on synthetic galactose medium because PKR expression; and none of the DEN construct could rescue the growth of RY1-1 like the positive control, yeast expressing the HIV Tat. These results suggest that single dengue viral protein/RNA was insufficient to interrupt the PKR-mediated translation repression pathway. The status of PKR and its substrate eIF2α in virus infected mammalian cells was further characterized in baby hamster kidney endothelial cells (BHK-21), mouse neuroblastoma cell line (N18), human neuroblastoma cells (HTB-11) as well as human kidney epithelial cell line (293T). The results indicate that DEN infection dose not significantly altered the PKR protein level in rodent cell lines (BHK-21and N18) but the PKR expression was notably reduced in the human cell lines (HTB-11 and 293T). However, the phosphorylation of the eIF2α was observed in these cells especially during early times of infection, and subsided later on. The mechanism adapted by DEN to overcome the eIF2α phosphorylation on translational suppression remains elusive. For detection of ER stress during DEN infection, an ER stress marker, the X-box binding protein 1 (Xbp-1) mRNA processing was demonstrated in infected N18 cell. Not only Xbp-1 mRNA processing, but also the protein produced by spliced Xbp-1 mRNA was noticeable in DEN and JEV infected cells, indicating DEN infection induced an ER stress. To examine the role of XBP-1 on virus infection, we used RNA interference (RNAi) to block XBP-1 expression and no effect on the virus titer and the virus-induced cytotoxicity could be found by knocking out the XBP-1 expression in infected N18 cells. Overall, for the two cellular stress responses studied here, we conclude that DEN infection promoted a negative feedback loop to reverse eIF2α phosphorylation through an unknown mechanism, which could not be attributed to single DEN protein, and Xbp-1 mRNA splicing was triggered by ER stress induced by DEN infection.