Paragraph: In response to the endoplasmic reticulum (ER) stress induced by herpes simplex virus type 1 (HSV-1) infection, host cells activate the unfolded protein response (UPR) to reduce the protein-folding burden in the ER. The regulation of UPR upon HSV-1 infection is complex, and the downstream effectors can be detrimental to viral replication. Therefore, HSV-1 copes with the UPR to create a beneficial environment for its replication. UPR has three branches, including protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activated transcription factor 6 (ATF6). IRE1α is the most conserved branch of UPR which has both RNase and kinase activities. Previous studies have shown that IRE1α RNase activity was inactivated during HSV-1 infection. However, the effect of the two activities of IRE1α on HSV-1 replication remains unknown. Results in this study showed that IRE1α expression was up-regulated during HSV-1 infection. We found that in HEC-1-A cells, increasing RNase activity, or inhibiting kinase activity of IRE1α led to viral suppression, indicating that the kinase activity of IRE1α was beneficial, while the RNase activity was detrimental to viral replication. Further evidence showed that the kinase activity of IRE1α leads to the activation of the JNK (c-Jun N-terminal kinases) pathway, which enhances viral replication. Taken together, our evidence suggests that IRE1α is involved in HSV-1 replication, and its RNase and kinase activities play differential roles during viral infection. Keywords: endoplasmic reticulum (ER); herpes simplex virus 1 (HSV-1); inositol-requiring enzyme 1 (IRE1); unfolded protein response (UPR); X-box binding protein 1 (XBP1) Viruses 2017, 9, 235 9 of 17 3.3. The Effect of IRE1α Kinase Activity on HSV-1 Replication Next, we investigated the effect of kinase activity of IRE1α on viral replication. IRE1 phosphorylation is generally considered as a reliable indicator of IRE1α kinase activity [45, [49] [50] [51] . Kinase activity of IRE1α was induced at a later stage of viral infection, as indicated by the phosphorylation of IRE1α from 12 h p.i. (Figure 3A ). Viruses 2017, 9, 235 9 of 17 Next, we investigated the effect of kinase activity of IRE1α on viral replication. IRE1 phosphorylation is generally considered as a reliable indicator of IRE1α kinase activity [45, [49] [50] [51] . Kinase activity of IRE1α was induced at a later stage of viral infection, as indicated by the phosphorylation of IRE1α from 12 h p.i. (Figure 3A) . Figure 3. HSV-1 replication is regulated by kinase activity of IRE1α. (A) HSV-1 infection stimulated kinase activity of IRE1α in HEC-1-A cells. HEC-1-A cells were infected with HSV-1 (HF) (moi = 1). Cell lysates were prepared at the times indicated and subject to Western blot analysis using antibodies against total IRE1α, phosphorylated IRE1α, gD-1 and GAPDH; (B) XBP1 splicing induced by APY29 was attenuated by STF-083010 in a dose dependent manner. HEC-1-A cells were treated with both a serial concentration of STF-083010 and 3.125 μM of APY29, and then incubated for 24 h. Total RNA was prepared for measuring the expression of spliced and unspliced XBP1using reverse transcription-PCR analysis; (C,D) Viral replication was inhibited by APY29; (C) Confluent HEC-1-A cells cultured in a 96-well plate were pretreated with serial concentrations of APY29 and then infected with HSV-1 (HF) (moi = 1) for 24 h; (D) Before HEC-1-A cells were infected with HSV-1 (HF) (moi = 1), cells were incubated with both serial concentration of STF-083010 and 3.125 μM of APY29. gD-1 protein expression was determined via In-cell Western, and normalized by DRAQ5 fluorescence at 24 h p.i. Data are mean values ± SD of triplicate determinations (** p < 0.01). Representative results are shown; (E) Inhibition of IRE1α kinase activity had an inhibitory effect on HSV-1 replication in HEC-1-A cells. Cells were presence or absence of APY29 (3.125 μM) or STF-083010 (60 μM) or both of two drugs and then infected with HSV-1 (HF) at moi = 1. Total cellular cells lysates were prepared at 24 h p.i. and subject to Western blot analysis against antibodies to total IRE1α, phosphorylated IRE1α and gD-1; (F) Inactivation of IRE1α kinase activity suppressed HSV-1 replication in Hela cells. Hela cells were transfected with vehicle or IRE1 alpha KA-pcDNA3.EGFP or IRE1 alpha-pcDNA3.EGFP plasmid for 48 h and then infected or mock-infected with HSV-1 (moi = 1). The total and phosphorylated IRE1α, 
 Title: Opposite Roles of RNase and Kinase Activities of Inositol-Requiring Enzyme 1 (IRE1) on HSV-1 Replication
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