Reprogramming the unfolded protein response for replication by porcine reproductive and respiratory syndrome virus
(A) MARC-145 cells were transfected with an siRNA targeting ATF4 (siATF4) or scrambled siRNA (siNC). At 36 h post transfection, the cells were infected with PRRSV strain JXwn06 at an MOI of 1. The abundance of viral RNA was assessed by RT-PCR of ORF7, normalized against the house-keeping gene GAPDH, and then compared to siNC control at 0 to 12 hpi. (B and C) The same as (A) except the abundance of positive- and negative stranded RNAs were analyzed by RT-qPCR at 0, 4, 8 and 12 hpi. (D) Control, rabbit IgG or antibodies specific for ATF4 were used for immunoprecipitations from lysates of infected MARC-145 cells (MOI = 0.1, 36 hpi). The presence of viral RNA was assayed by RT-qPCR targeting 5’-UTR sequence or (E) GAPDH as a negative control, and the fold enrichment was calculated against the normal rabbit IgG group. (F) GST-ATF4 or GST were purified from E. coli (left panel) and added to RNA extracted from PRRSV-infected MARC-145 cells in an in vitro binding assay. RT-qPCR with primers specific for a 5’UTR sequence was used to detect viral RNA (middle panel) and the fold of enrichment of viral RNA was expressed against the GST control. (G) In parallel, the pull down of cellular GAPDH mRNA was used as a negative control. (H) To look for ATF4 within viral replication compartments in infected MARC-145 cells (MOI = 0.1, 24 hpi), the negative-strand RNA was detected by the RNAscope in situ hybridization method, and ATF4 and nsp9 were detected by immunostaining. Data information: Statistical analysis was performed by two-tailed Student’s t-test, and error bars indicate standard deviations (SD) of means. Asterisks (*) indicate the statistical significance: ***, P < 0.001; ****, P<0.0001; NS, no significance (n = 3 experiments in each condition). Confocal images were acquired with Nikon A1 confocal microscope. Oil objective: 63 X; zoom in 1 X.