Herpes Simplex Virus 1 UL41 Protein Suppresses the IRE1/XBP1 Signal Pathway of the Unfolded Protein Response via Its RNase Activity

Abstract

During viral infection, accumulation of viral proteins can cause stress in the endoplasmic reticulum (ER) and trigger the unfolded protein response (UPR) to restore ER homeostasis. The inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved of the three UPR signal pathways. Upon activation, IRE1 splices out an intron from the unspliced inactive form of X box binding protein 1 [XBP1(u)] mRNA and produces a transcriptionally potent spliced form [XBP1(s)]. Previous studies have reported that the IRE1/XBP1 pathway is inhibited upon herpes simplex virus 1 (HSV-1) infection; however, the underlying molecular mechanism is still elusive. Here, we uncovered a role of the HSV-1 UL41 protein in inhibiting the IRE1/XBP1 signal pathway. Ectopic expression of UL41 decreased the expression of XBP1 and blocked XBP1 splicing activation induced by the ER stress inducer thapsigargin. Wild-type (WT) HSV-1, but not the UL41-null mutant HSV-1 (R2621), decreased XBP1 mRNA induced by thapsigargin. Nevertheless, infection with both WT HSV-1 and R2621 without drug pretreatment could reduce the mRNA and protein levels of XBP1(s), and additional mechanisms might contribute to this inhibition of XBP1(s) during R2621 infection. Taking these findings together, our results reveal XBP1 as a novel target of UL41 and provide insights into the mechanism by which HSV-1 modulates the IRE1/XBP1 pathway.

Importance: During viral infection, viruses hijack the host translation apparatus to produce large amounts of viral proteins, which leads to ER stress. To restore ER homeostasis, cells initiate the UPR to alleviate the effects of ER stress. The IRE1/XBP1 pathway is the most conserved UPR branch, and it activates ER-associated protein degradation (ERAD) to reduce the ER load. The IRE1/XBP1 branch is repressed during HSV-1 infection, but little is known about the underlying molecular mechanism. Our results show for the first time that UL41 suppresses the IRE1/XBP1 signal pathway by reducing the accumulation of XBP1 mRNA, and characterization of the underlying molecular mechanism provides new insight into the modulation of UPR by HSV-1.

Keywords: HSV-1; UL41; UPR; XBP1.

FIG 1

HSV-1 tegument protein UL41 inhibited the ectopic expression of XBP1. (A and B) HEK293T cells were cotransfected with XBP1(u)-HA (500 ng) or XBP1(s)-HA (500 ng) and UL41-HA (500 ng) or ICP0-flag (500 ng) plasmids. At 24 h posttransfection, the cells were harvested and subjected to WB analysis with antibodies against Flag, HA, or β-actin. The data represent the results of one of the triplicate experiments.

FIG 2

Ectopic expression of UL41 decreased the expression of endogenous XBP1 and blocked XBP1 splicing activation induced by thapsigargin. (A, C, D, and E) HEK293T cells were transfected with UL41-HA plasmid (300 ng [+] and 1,000 ng [++]) for 24 h, and then the transfected cells were treated with 20 nM thapsigargin. After 4 h of drug stimulation, cells were harvested and subjected to WB analysis with antibodies against XBP1, HA, or β-actin (A), semiquantitative RT-PCR (C), or qRT-PCR (D and E) analysis. (B) HEK293T cells were cotransfected with pXBP1u-FLuc reporter (250 ng), Renilla luciferase plasmid pRL-TK (150 ng), and pCMV-HA control vector or UL41-HA plasmid. At 24 h posttransfection, the cells were treated or not with 20 nM thapsigargin for an additional 4 h, followed by cell lysis. Firefly luciferase activity was determined by a dual-luciferase assay. (F and G) HEK293T cells were transfected with UL41-HA plasmid (300 ng [+] and 1000 ng [++]) for 24 h, and then the cells were harvested and subjected to WB analysis with antibodies against IRE1, HA, or β-actin (F) or semiquantitative RT-PCR analysis (G). The data represent the results of one of the triplicate experiments. The error bars represent mean standard deviations (SD) of three independent experiments. Statistical analysis was performed using Student's t test. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.

FIG 3

HSV-1 infection reduced the accumulation of XBP1 mRNA induced by thapsigargin via UL41. (A, B, and C) HeLa cells were pretreated or not with 100 nM thapsigargin; after 4 h, the medium was removed and the cells were mock infected or infected with WT HSV-1 or R2621 at MOI of 5 and 10. At 4 h postinfection, cells were harvested and subjected to semiquantitative RT-PCR (A) or qRT-PCR (B and C) analysis. The data represent the results of one of the triplicate experiments. The error bars indicate SD. **, 0.001 < P < 0.01; ***, P < 0.001.

FIG 4

HSV-1 infection suppressed the endogenous expression of XBP1(s). (A, B, and C) HeLa cells were infected with WT-HSV-1 or R2621 at an MOI of 5 or 10. At 10 h postinfection, cells were harvested and subjected to semiquantitative RT-PCR (A) or qRT-PCR (B and C) analysis. (D) HeLa cells were infected with WT-HSV-1 or R2621 at an MOI of 5 or 10. At 20 h postinfection, cells were harvested and subjected to WB analysis with antibodies against UL42, XBP1, or β-actin. The data represent the results of one of the triplicate experiments. The error bars indicate SD. *, 0.01 < P < 0.05; **, 0.001 < P < 0.01; ***, P < 0.001.