Several clinical observations point to an intricate crosstalk between iron (Fe) metabolism and chronic hepatitis C virus (HCV) infection. In this study, we wanted to investigate the molecular control that Fe levels exert on HCV replication at the hepatocyte level. In keeping with previous observations we confirmed that supra-physiological intracellular Fe induced by haemin treatment down-modulated HCV replication from subgenomic replicons. We also found that RNAi-mediated knockdown of the key Fe modulator hepcidin increased intracellular ferritin and inhibited HCV replication. Conversely, HCV replication did not modulate ferritin content in hepatocytes. Finally, we demonstrated that hepcidin is modulated at the mRNA level by alpha interferon through STAT3. We propose that in Huh7 cells hepcidin modulation leads to an unfavourable intracellular environment for HCV replication. These data may therefore contribute to a better understanding of the complex interplay between HCV and cellular physiology during infection. INTRODUCTIONHepatitis C is a blood-borne infectious disease that is caused by the hepatitis C virus (HCV) (Choo et al., 1989). The estimated prevalence of HCV infection is 2.2 % worldwide (Alter, 2007). In most cases, the infection causes hepatic inflammation (hepatitis) that is often asymptomatic, but about 80 % of infected individuals develop chronic hepatitis that may ultimately lead to cirrhosis and hepatocellular carcinoma (HCC) (Lauer & Walker, 2001). HCV is able to establish a persistent infection also because of its efficiency in evading the immune response of the host (Sklan et al., 2009). HCV is a serious health care problem that requires careful understanding of the molecular mechanisms that regulate its replication in the host cells in order to develop efficient antiviral strategies. The outcome of an HCV infection depends both on the host genetic background and on the viral subtype as well as from external factors. Among these, iron (Fe) overload has been shown to be common among patients with chronic hepatitis C (Bonkovsky, 2002; Boucher et al., 1997;Di Bisceglie et al., 1992) and to correlate with a poor response to antiviral therapy (Franchini et al., 2008;Sherrington & Olynyk, 2002).Fe is subject to a tightly coordinated regulation by cellular factors involved in Fe absorption, transport, uptake and storage (Hentze et al., 2004). Circulating transferrin (Tf) bound to Fe 3+ is endocytosed into cells by the transferrin receptor 1 (TfR1) through clathrin-coated pits into endosomes. The divalent metal transporter 1 (DMT-1, also known as DCT1 or Nramp2) transfers Fe 2+ across the endosomal membrane into the cytoplasm, but is also able to mediate direct absorption of non-Tf-bound Fe from the plasma in the intestine (Fleming et al., 1997). Fe is stored in the cells in a complex with ferritin (Torti & Torti, 2002) and is exported from cells by the Fe exporter ferroportin 1 (FPN1, also known as Ireg1) (Abboud & Haile, 2000;Donovan et al., 2000;McKie et al., 2000). Regulation of Fe homeostasis ...

(HCV) is a single-stranded positive-sense RNA hepatotropic virus. Despite cellular defenses, HCV is able to replicate in hepatocytes and to establish a chronic infection that could lead to severe complications and hepatocellular carcinoma. An important player in subverting the host response to HCV infection is the viral nonstructural protein NS5A, which, in addition to its role in replication and assembly, targets several pathways involved in the cellular response to viral infection. Several unbiased screens identified nucleosome assembly protein 1-like 1 (NAP1L1) as an interaction partner of HCV NS5A. Here we confirmed this interaction and mapped it to the C terminus of NS5A of both genotype 1 and 2. NS5A sequesters NAP1L1 in the cytoplasm, blocking its nuclear translocation. However, only NS5A from genotype 2 HCV, and not that from genotype 1, targets NAP1L1 for proteosome-mediated degradation. NAP1L1 is a nuclear chaperone involved in chromatin remodeling, and we demonstrated the NAP1L1-dependent regulation of specific pathways involved in cellular responses to viral infection and cell survival. Among those, we showed that lack of NAP1L1 leads to a decrease of RELA protein levels and a strong defect of IRF3 TBK1/IKKε-mediated phosphorylation, leading to inefficient RIG-I and Toll-like receptor 3 (TLR3) responses. Hence, HCV is able to modulate the host cell environment by targeting NAP1L1 through NS5A. Viruses have evolved to replicate and to overcome antiviral countermeasures of the infected cell. Hepatitis C virus is capable of establishing a lifelong chronic infection in the liver, which could develop into cirrhosis and cancer. Chronic viruses are particularly able to interfere with the cellular antiviral pathways by several different mechanisms. In this study, we identified a novel cellular target of the viral nonstructural protein NS5A and demonstrated its role in antiviral signaling. This factor, called nucleosome assembly protein 1-like 1 (NAP1L1), is a nuclear chaperone involved in the remodeling of chromatin during transcription. When it is depleted, specific signaling pathways leading to antiviral effectors are affected. Therefore, we provide evidence for both a novel strategy of virus evasion from cellular immunity and a novel role for a cellular protein, which has not been described to date.