Autophagy is an important cellular process by which ATG5 initiates the formation of double membrane vesicles (DMVs). Upon infection, DMVs have been shown to harbor the replicase complex of positive-strand RNA viruses such as MHV, poliovirus, and equine arteritis virus. Recently, it has been shown that autophagy proteins are proviral factors that favor initiation of hepatitis C virus (HCV) infection. Here, we identified ATG5 as an interacting protein for the HCV NS5B. ATG5/NS5B interaction was confirmed by co-IP and metabolic labeling studies. Furthermore, ATG5 protein colocalizes with NS4B, a constituent of the membranous web. Importantly, immunofluorescence staining demonstrated a strong colocalization of ATG5 and NS5B within perinuclear regions of infected cells at 2 days postinfection. However, colocalization was completely lacking at 5 DPI, suggesting that HCV utilizes ATG5 as a proviral factor during the onset of viral infection. Finally, inhibition of autophagy through ATG5 silencing blocks HCV replication.

Human PCSK9 is known to enhance the degradation of membrane-bound receptors such as the hepatocyte low-density lipoprotein receptor (LDLR), ApoER2, and very low-density lipoprotein receptor. Because the LDLR is suspected to be involved in hepatitis C virus (HCV) entry, we also tested whether PCSK9 can affect the levels of CD81, a major HCV receptor. Interestingly, stable expression of PCSK9 or a more active membrane-bound form of the protein (PCSK9-ACE2) resulted in a marked reduction in CD81 and LDLR expression. Therefore, we analyzed the antiviral effect of PCSK9 in vitro using the HCV genotype 2a ( H epatitis C virus (HCV) is a worldwide leading cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. 1 In the absence of a prophylactic vaccine or a specific antiviral agent, the best treatment currently available for HCV infection is the combination therapy of pegylated interferon and ribavirin. 2 HCV is a positive-strand RNA enveloped virus classified as a Hepacivirus within the Flaviviridae family. 3 Viral entry within target cells, primarily human hepatocytes, is not very well understood. Several cell surface proteins have been suggested to play a role in the binding of HCV to hepatocytes and/or be critical for viral entry. 4,5 Among the acknowledged receptors or coreceptors of HCV are: CD81, 6 scavenger receptor class B type I, 7 lowdensity lipoprotein receptor (LDLR), 8 and the recently identified claudin-1, claudin-6, claudin-9, 9 and occludin. 10 However, it is unclear which ones are required for viral propagation in vivo. Indeed, HCV particles recovered from infected plasma migrated at two distinct densities (1.25 g/mL and 1.06 g/mL). 11,12 It has been suggested that the most infectious virus is located in the low-density fraction that corresponds to lipoviroparticles of HCV coated with apolipoprotein B and E. 11 Because LDLR controls the uptake of circulating cholesterol principally through internalization of apolipoprotein B-and E-containing lipoproteins, such as LDL and very low-density lipoprotein, the implication of LDLR as a nonspecific receptor for HCV entry may be significant, at least in vivo.

1 Chronic renal failure (CRF) is associated with a decrease in liver cytochrome P450 (P450). The mechanism remains poorly understood. The present study aimed to investigate the e ects of the serum of rats with CRF on liver P450. 2 Normal rat hepatocytes were incubated for 24 h with serum (concentration of 10%) from rats with CRF and from control animals in order to measure (1) total P450 level, (2) protein expression and mRNA levels of major P450 isoforms, and (3) some of their speci®c metabolic activities (Ndemethylation of erythromycin). Time-course experiments (incubation time from 12 to 48 h) and dose-response curves (concentration of serum ranging from 1 to 30%) have been conducted. 3 In normal hepatocytes incubated for 24 h with serum (concentration of 10%) from rats with CRF, total P450 level, protein expression and mRNA levels of several P450 isoforms (CYP2C6, 2C11, 3A1 and 3A2) were decreased by more than 35% (P50.001) compared to serum from control animals. The protein expression as well as the mRNA levels of CYP2D were similar in hepatocytes incubated with serum from either control or CRF rats. The N-demethylation of erythromycin was decreased by more than 35% (P50.001) in hepatocytes incubated with serum from rats with CRF.The inhibitory e ect of serum from rats with CRF tended to peak at 48 h of incubation and was maximum at a concentration of 20%. 4 In conclusion, uremic serum contains mediator(s) that down-regulate the cytochrome P450 of normal hepatocytes secondary to reduced gene expression.

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