Purified 20S proteasome complexes were subsequently incubated with the precursor substrate HBV core 131C162 polypeptide for the indicated time periods. lymphocyte epitope was impaired in processing experiments using isolated 20S proteasomes from HCV-infected cells and was restored by the silencing of PKR expression. In conclusion, our data point to a novel mechanism of immune regulation by HCV that affects the antigen-processing machinery through the PKR-mediated suppression of immunoproteasome induction in infected cells. Introduction The clearance of viral infection is dependent on vigorous CD8+ cytotoxic T lymphocyte (CTL) responses, which must be tightly regulated to prevent immune-mediated host tissue damage. Virus-infected cells are recognized and destroyed by specific CTLs that bind to virus-derived peptide epitopes associated with cell surface major histocompatibility complex (MHC) class I molecules.1, 2 Most of these antigenic peptides, which are usually 8C10 amino-acid residues in length, are generated by the 30S proteasome complex, which is the central proteolytic machinery of the ubiquitin-proteasome-system.3, 4 The 30S complex is composed of the 20S proteasome proteolytic core complex and two associated 19S regulatory particles.4, 5 The 20S complex is arranged as four staggered rings, each containing seven non-identical subunits. The outer rings contain the subunits (1C7), which form the gates’ through which substrates enter and products are released.5 Each of the two inner rings contains the subunits (1C7), three of which (1, 2 and 5) harbor the six active sites.5 Type I and II interferons (IFNs), which are major cytokines in viral infection, induce the expression of the immunosubunits (i-subunits) 1i/LMP2, 2i/MECL-1 and 5i/LMP7 in non-immune cells and the Manidipine (Manyper) assembly of the so called immunoproteasomes (i-proteasomes).5, 6 In addition, i-proteasomes are constitutively expressed in hematopoietic/immune cells, such as dendritic cells.7, 8 Because of the altered proteolytic activity, i-proteasomes have been shown to exhibit altered frequencies in cleavage site usage. This affects the relative abundance of the generated antigenic peptides, which in turn can influence the quality of the peptide-specific CD8+ Manidipine (Manyper) CTL response.9 For example, the generation of the hepatitis B virus (HBV) core 141C151 epitope has been shown to be strongly influenced by the structural presence of the i-subunit 5i/LMP7.10 Additionally, it has been shown that 1i/LMP2- or 5i/LMP7-deficient mice are unable to efficiently generate and present some CD8+ CTL epitopes11, 12, 13 while the CD8+ CTL response was barely affected in 1i/LMP2- or 5i/LMP7-deficient mice infected with lymphocytic choriomeningitis virus.14, 15 Recent reports demonstrated that quantitative changes in the epitope generation of i-subunit-deficient mice result in alterations of the immunodominance hierarchy and the T-cell TIL4 repertoire in a murine influenza infection model.16 Another study using mice completely lacking i-proteasomes indicated that the peptide repertoire presented by dendritic cells in the lymphoid organs differed from that presented by wild-type dendritic cells by 50%.17 In addition to affecting the outcome of the CTL response, i-proteasomes also possess an important proteostatic function in preserving cell viability under conditions of IFN-induced oxidative stress.18, 19 For example, in a murine model of coxsackievirus infection, i-proteasomes were shown to protect mice against oxidant protein damage in the injured myocardium.20 Hepatitis C virus (HCV) is one of the most common causes of chronic liver disease. Although some patients successfully clear the virus after acute HCV infection, most patients fail to eliminate the virus and develop chronic persistent infection accompanied by inflammatory liver injury.21 The outcome of HCV infection is determined by virus-specific cellular immune responses.22, 23, 24, 25 Indeed, patients who control their HCV infection have broad CD8+ T-cell responses with higher functional avidity, whereas CD8+ T-cell responses are impaired in patients with persistent HCV infection.23, 24, 25, 26 HCV Manidipine (Manyper) evades host immune responses through various mechanisms, leading to chronic persistent infection.27 However, little is known regarding the effects of HCV infection on the epitope-processing machinery, which is essential for the recognition of infected cells by CTLs. In the present study, we investigated the i-proteasome induction in HCV-infected cells..