To ensure equal infection conditions in each experiment TCID50, MIO and HCV copy number were measured repeatedly as described in the methods (Figure S1a�Cb and data not shown). During culture, cells may display different levels of ER stress/UPR activation. In order to assess base line levels of ER stress we cultured product info non-infected cells in parallel to infected cells. We did not observe significant activation of ER stress in non-infected cells during days 1�C5 of the experiment (Figure S3a). Next, we monitored the time course of UPR induction. The three arms of the UPR were activated simultaneously in a wave-like pattern. Levels of phosphorylated IRE1, cleaved ATF6 and phosphorylated eIF2��, peaked on days 3�C5 by 4.13��0.6 fold, 8.3��1.8 fold and 13.6��1.

14 fold compared to non-infected cells (Figure 1a, b and c, respectively), in a manner that coincided with the peak viral replication. Phosphorylated eIF2�� leads to induction of CHOP and its translocation into the nucleus. Indeed, we observed accumulation of CHOP in the nucleus of HCV infected cells as compared to non-infected controls (Figure 1d). XBP-1 splicing followed a similar wave pattern, which reached a peak at day 3 post infection (Figure 2a). To determine if the IRE1-XBP-1 and PERK-eIF2�� arms are transcriptionally active we measured expression of their direct targets by real time PCR. mRNA levels of p58ipk and ERDJ4, ATF3 and ATF4 were up-regulated in a temporal pattern similar to XBP-1 splicing (3.8��1.54, 15.14��0.37, 31.96��15.44 and 2.86��1.01 fold increase compared to non-infected cells, respectively).

ATF4 was shown to up-regulate transcription of several UPR targets including that of the growth arrest and DNA damage (GADD)34 gene. GADD34 was shown to promote dephosphorylation of eIF2�� via protein phosphatase 1 (PP1c) thereby leading to recovery from translational inhibition in the UPR. We observed a marked increase in GADD34 transcription peaking on day 3 post-infection (8.1��1.2 fold increase compared to non-infected cells) (Figure 2b�Cf). Figure 1 HCV infection induces a wave like activation of the 3 arms of the UPR. Figure 2 Downstream target genes of the UPR are up-regulated following HCV infection. ER stress/UPR pathways are interrelated to inflammatory pathways via IRE1/TRAF2. We therefore looked at associated inflammatory pathways with regard to ER stress.

We observed a marked increase in the phosphorylation of JNK, peaking on days 3�C4 in parallel to the peak of viral replication and the activation of the three arms of the UPR (Figure S3b). Anacetrapib These results suggest that inflammatory pathways are activated in tandem to UPR pathways. We conclude that the IRE1-XBP-1-ERdj4-p58ipkand PERK-eIF2��-ATF4-ATF3-CHOP-GADD34 pathways are activated following HCV infection. ATF6 was cleaved, a marker of its activation. Downstream target genes of ATF6 were not investigated in our study.