Figure 1

Dengue induces ER stress, especially activating the PERK pathway, accelerating its replication and protecting MDCK cells from toxicity of CPT. (a) A general marker of ER stress, calreticulin, increases during infection (dengue (Den)) or treatment with ER stress inducer tunicamycin (Tunica), whereas the inhibitor of ER stress response, salubrinal (Sal), decreases calreticulin even in infected cells (Den+Sal) and modestly suppresses the response to tunicamycin. Numbers below blot represent protein ratios to loading control. The cells were treated and/or infected for 24 h before the proteins were isolated for immunoblotting. (b) Dengue infection in MDCK significantly increases ATF4 gene expression at 12 h. (c) Transcription of GADD34, a downstream target of CHOP, increases at 12 HPI and remains high, relative to actin (control, illustrated in Figure 1e). (d) At 24 h of infection, salubrinal depresses the transcription of dengue NS4A gene by 42% (only 129-fold greater than mock for Den+Sal compared with 222-fold greater than mock for Den). Compared with mock, for transcription of NS4A, P<0.0001. Compared with infected cells without Sal, production of NS4A was 42% less, P<0.0004. (e) Verification of controls: transcription of β-actin in mock- and Den-infected MDCK cells after 12 and 24 h of infection. Infection has minimal and insignificant (n.s.) impact on the transcription of actin (f). In the presence of Sal, an inhibitor of the PERK pathway, there was higher cell death in CPT-treated and Den-infected MDCK cells after 24 h. In this and subsequent figures, *P<0.05; **P<0.01, ***P<0.005 and ****P<0.001 for the bracketed comparisons