Progressive Rotavirus Infection Downregulates Redox-Sensitive Transcription Factor Nrf2 and Nrf2-Driven Transcription Units

Figure 1

RV infection triggers gradual decline of Nrf2 protein levels beyond an initial upsurge. (a) MA104 cells were infected with either RV-SA11, UV-inactivated RV-SA11, or kept mock infected for indicated time points. Whole cell extracts were subsequently prepared and subjected to SDS-PAGE followed by immunoblotting to assess protein levels of Nrf2. Absence of viral protein (VP6 and NSP3) expression in UV-treated RV-SA11 confirmed functional inactivation of the virus. Relative fold change of Nrf2 is represented; the symbols “ns” and “” represent comparisons with respect to the first lane; “#” represents comparison with respect to the RV-SA11 group infected for 3 hours. (b) Nrf2 protein levels were assessed from whole cellular extracts as well as purified nuclear isolates and cytosolic fractions of RV-SA11/UV-irradiated RV-SA11-infected MA104 cells by immunoblot analysis. Relative fold change of Nrf2 is represented; the symbols “ns” and “” represent comparisons with respect to the first lane; “#” represents comparison with respect to the RV-SA11 group infected for 3 hours. (c) Whole cell, purified nuclear, and cytosolic extracts of MA104 cells treated with an indicated concentration of Brusatol for 3 hours were resolved on SDS-PAGE, and Nrf2 protein levels were checked by immunoblotting. Relative fold change of Nrf2 is represented; “” represents comparison with respect to a Brusatol-untreated (vehicle treated) group. (d) Mock-infected/RV-SA11-infected and (g) DMSO- (vehicle)/Brusatol- (0.4 μM) treated (3 hours exposure) MA104 cells were fixed at indicated time point post infection/treatment and were further processed for confocal microscopy; scale bar, 20 μM. (e, h) Quantification of background-normalized Nrf2 fluorescence in (e) infected and (h) Brusatol-treated set is represented as CTCF. “ns” and “” represent comparison with respect to the first lane; “#” represents comparison with respect to the RV-SA11 group infected for 3 hours. (f, i) Quotient of nuclear hollowing (NH_Q) of Nrf2 in (f) RV-SA11-infected cells at 3, 6, and 9 hours post infection and (i) Brusatol-treated cells after 3 hours of exposure was calculated.

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