FIG 5.
6-TG treatment upregulates UPR genes, and chemical chaperones can limit the UPR and restore viral glycoprotein accumulation. (A) A549 cells were infected with IAV-PR8 at an MOI of 1, washed, and overlaid with medium containing 6-MP, 6-TG, or TM. Cell lysates were collected at 24 hpi, and RNA was isolated and processed for RT-qPCR. Changes in CHOP, BiP, EDEM1, ERdj4, and HERPUD1 mRNA levels were calculated by the ΔΔCT method, normalized using 18S rRNA as a reference gene, and standardized to mock. Error bars represent standard deviations (n = 3). Circles represent individual replicates, and lines represent mean values. Statistical significance was calculated via two-way ANOVA followed by a Dunnett multiple-comparison test. (B and C) A549 cells were mock infected or infected with IAV-PR8 at an MOI of 1. After 1 h, cells were washed and incubated with 20 μM 6-TG or the vehicle control, with or without 10 μM 4-PBA. At 20 hpi, cell lysates were harvested and probed using a polyclonal IAV antibody that detects IAV HA, NP, and M1 proteins (B); IAV NA (C); and antibodies to cellular BiP and actin. Representative data from 3 independent experiments are shown.