It is noteworthy that most of these studies were performed not in the context of viral infection, but rather by using transfections of sub-genomic or full-length replicons, as well as cell lines (hepatocytes and non-hepatocyes) over-expressing specific viral proteins. As readouts, these studies used mostly luciferase reporters and were limited to single arms of the UPR. read me Furthermore, most did not explore downstream targets of UPR activation or stated that disrupted activation of UPR exists [16]�C[18], [20]�C[22]. More recently, induction of ER stress was also demonstrated in the context of the HuH7.5.1 model, a cell culture system which supports a full replication cycle of HCV [23], [24]. All three pathways of the UPR were activated: PERK underwent phosphorylation, ATF6 was cleaved and XBP-1 mRNA was spliced.

However, it was not conclusively shown whether the target genes of the various pathways were correspondingly activated. In contrast, Lerat et al, showed in a transgenic mouse model expressing the full replicon of HCV that ER stress genes are not activated [25]. In addition, Joyce et al, showed that infection of human hepatocytes by HCV in-vivo in the chimeric SCID/Alb-uPA mice resulted in enhanced Bip/Grp78 expression, but no significant activation of UPR target genes [26]. These data may suggest that the UPR was either partially activated or that its downstream signaling was suppressed or kept at a minimal level probably to avoid ER stress-mediated apoptosis. All of the studies were limited to the acute infection soon after virus introduction.

However, chronic infection by HCV, rather than the acute phase is the major component of HCV pathology in humans. In this study we explored whether HCV infection causes chronic long lasting ER stress conditions, and if so, is adaptation to continuous ER stress, which was demonstrated to occur in response to pharmacological ER agents develops. Using the HuH7.5.1 system we demonstrate that HCV induces the UPR in a wave-like fashion, which peaks on days 3�C5 after infection. The UPR then subsides but remains active at Brefeldin_A a low level. This chronic ER stress conferred adaptation and resistance to further drug-induced ER stress. Suppression of viral replication using interferon-�� 2a treatment restored UPR responsiveness, indicating that continues presence of the virus is required for adaptation. To address this phenomenon in vivo, we used mice expressing the HCV replicon in their liver (HCV-Tg). These mice displayed basal low level chronic ER stress and adaptation of UPR when administered tunicamycin, an ER stressor. These findings represent a novel mechanism by which chronic HCV infection may disrupt cellular homeostasis. Materials and Methods Cell Culture Huh 7.5.1 cells, a kind gift from Dr.