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. 2014 Nov;88(22):13086–13098. doi: 10.1128/JVI.02156-14

FIG 6.

FIG 6

GCA enhances adenovirus infection through IRE-1 and XBP-1. (A) Effect of knockdown of ER stress sensors on GCA-mediated infection boost. A549 cells were reverse transfected with control nontargeting siRNAs (NT) or pooled siRNAs against ER stress sensors ATF-6A, ATF-6B, PERK, and IRE-1alpha. At 43 h posttransfection, cells were preincubated with DMSO or GCA for 5 h (no addition indicates no pretreatment) and inoculated with HAdV-C5-dE1_GFP, and the average nuclear GFP signal was analyzed at 18 h p.i. (B) IRE-1 knockdown by siP RNAs reduces the HAdV-C5-dE1_GFP infection boost in GCA-treated A549 cells. siP Neg, nontargeting negative-control siP RNAs. Intracellular IRE-1 levels were determined by Western blotting using β-tubulin as a loading control. (C) GBF-1 inhibition by GCA induces ER stress and activates IRE-1 nuclease and splicing of XBP-1 mRNA. A549 cells were treated with GCA or the ER stress activator thapsigargin (Tg) for 5 h, and IRE-1 activation was analyzed by PstI digestion of XBP1 cDNA amplicons. The spliced XBP-1 cDNA amplicon lacks a PstI site (1S), whereas the unspliced one retains the site and is cleaved into two fragments (2U and 3U) upon PstI digestion. The uppermost band (*) is a spliced/unspliced XBP-1 hybrid amplicon (69). XBP-1 splicing was inhibited by the IRE-1 nuclease inhibitor 4μ8C in GCA- and thapsigargin-treated cells. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) cDNA amplicons were used as a loading control. Fwd, forward; Rev, reverse; nt, nucleotides. (D) GCA-induced HAdV-C5-dE1_GFP infection boost in A549 cells requires IRE-1 endonuclease activation. Cells were preincubated with GCA or thapsigargin for 5 h with or without 4μ8C, inoculated with HAdV-C5-dE1_GFP, and analyzed for GFP expression at 18 h p.i. contin. inc., continuous incubation. (E) Reduced GCA infection boost in XBP-1−/− mouse embryo fibroblasts. XBP-1+/+ or XBP-1−/− MEFs were preincubated with GCA, inoculated with HAdV-C5-dE1_GFP, and analyzed for GFP expression at 18 h p.i. (F) XBP-1 knockdown by siP RNAs reduces the HAdV-C5-dE1_GFP infection boost in GCA-treated A549 cells. Cells were reverse transfected with siP RNAs against XBP-1 or nontargeting negative-control siP RNA (siP Neg), at 72 h posttransfection preincubated with GCA or DMSO for 5 h, inoculated with HAdV-C5-dE1_GFP, and analyzed for GFP expression at 18 h p.i. Levels of knockdown of the unspliced XBP-1 protein (XBP-1u) were controlled by Western blotting using β-tubulin as a loading control.