Protein fragments were analyzed by N-terminal peptide sequencing

Protein fragments were analyzed by N-terminal peptide sequencing. == Results == HCV NS3/4A protease cleaved C4 in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4 cleavage. NS3/4A proteasemediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4 fragments. Caftaric acid Such C4 processing, with a concomitant reduction in levels of full-length C4, was also observed in HCV-infected cells expressing C4. == Conclusions == C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection. == Introduction == Hepatitis C virus Caftaric acid (HCV) is a single-stranded positive-strand RNA virus of the Flaviviridae family. The viral genome encodes four structural proteins and six non-structural (NS) proteins [1]. NS3/4A, a complex consisting of NS3 with serine protease activity and cofactor NS4A, plays an essential role in processing of HCV proteins. NS3/4A is a target of direct-acting antiviral agents (DAA) [2,3], and use of an NS3/4A protease inhibitor as a DAA markedly increases the therapeutic effect of other anti-HCV agents. Thus, NS3/4A protease may play an important role in interfering with the antiviral response. HCV has been hypothesized to Caftaric acid block the host immune response against persistent infection [4]. Furthermore, the time required for HCV-infected patients to develop hepatic cirrhosis varies among individuals; in Caftaric acid particular, the progression of hepatic fibrosis seems to be slower in HCV carriers with persistent normal alanine aminotransferase (ALT) levels than in chronic hepatitis patients with elevated ALT levels [5]. These differences in clinical features might be caused by variations in the host immune response, but the underling mechanism is unclear. In the course of proteomic analyses aimed at identifying proteins potentially involved in the pathophysiology of hepatic diseases, we found that a specific peptide fragment of complement component 4 (C4) was significantly more abundant in HCV carriers with persistent normal ALT than in patients with chronic hepatitis [6], as well as more abundant in HCV carriers, Caftaric acid regardless of ALT levels, compared to healthy controls. Assuming that C4 expression levels are similar among these groups, this C4 fragment may be generated by post-translational processing in HCV-infected individuals. The complement system is part of the innate immune system, which can be activated through three pathways: the classical pathway, the mannose-binding lectin pathway, and the alternative pathway. C4, which is involved in the classical- and mannose-binding lectin pathways, can be cleaved by certain cellular protease(s), leading to a cascade of C4 activation [7]. In this study, we provide the first evidence that HCV NS3/4A cleaves C4, and that this cleavage attenuates activation of the classical pathway of complement system. == Materials and Methods == == Materials == HCV NS3/4A protease (217 amino acid [aa] fusion protein with NS4A co-factor fused to the N-terminus of NS3 protease domain) with His-tag, HCV core (aa 1102) with GST-tag, and HCV NS5 (aa 20612302) with GST-tag were purchased from AnaSpec (Fremont, CA) or ProSpec (Rehovot, Israel). Isolated human-derived complement components (C1, C2) were obtained from Hycult Biotech (Uden, Netherlands), and C4 Rabbit polyclonal to AnnexinA10 and C4-deficient guinea pig serum (C4d-GPS) were purchased from Sigma-Aldrich (St. Louis, MO). VX950, a HCV NS3/4A serine protease inhibitor, was obtained from Selleck Chemicals (Houston, TX). Veronal buffer, sheep erythrocytes, and hemolysin were purchased from Wako (Osaka, Japan), Nippon Biotest Laboratories Inc. (Tokyo, Japan), and Denka Seiken Co. (Tokyo, Japan),.