XBP1-Mediated BiP/GRP78 Upregulation Copes with Oxidative Stress in Mosquito Cells during Dengue 2 Virus Infection

Figure 6

Association of BiP/GRP78 with synthesis of the dengue 2 virus (DENV2) envelope (E) protein in C6/36 cells. For the Co-IP assay, both an enhanced green fluorescent protein (eGFP) and human influenza hemagglutinin- (HA-) tagged BiP/GRP78-overexpressing vector were constructed and transfected into C6/36 cells. (a) The successful expression of BiP/GRP78 was validated with eGFP and HA antibodies in Western blot analysis. Nothing was seen in lane 1 which represents C6/36 cells neither overexpressing BiP/GRP78 nor infected with DENV2. BiP/GRP78 can be detected in the same position of the band by both antibodies in lane 2 which represents DENV2-infected C6/36 cells transfected with the BiP/GRP78-overexpressing vector tagged with eGFP and HA. Only eGFP can be detected as shown in lane 3 which represents C6/36 cells transfected with a vector only expressing eGFP. (b) Co-IP results revealed that overexpressed BiP/GRP78 in C6/36 cells eventually interacted with the viral E protein that was also detected in the lysate of cells infected with DENV2 for 24 h. Meanwhile, BiP/GRP78 was also identified in the same cells. Lane 1:  C6/36 cells that had neither BiP/GRP78 overexpression nor DENV2 infection (no treatment). Lane 2: DENV2-infected C6/36 cells transfected with BiP/GRP78 (cell lysate). Lane 3: IP results from DENV2-infected C6/36 cells transfected with BiP/GRP78 and infected with the DENV2 (Co-IP). (c) Confocal microscopy demonstrated that BiP/GRP78 was upregulated in C6/36 cells with DENV2 infection at 24 h postinfection (hpi) and colocalized with the viral envelope (E) protein, as shown in the merged image. Green: E protein of DENV; blue: DAPI; red: BiP/GRP78.

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