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 (JFH1) virus. The results clearly demonstrated that cells expressing PCSK9 or PCSK9-ACE2, but not the ACE2 control protein, were resistant to HCV infection. Furthermore, addition of purified soluble PCSK9 to cell culture supernatant impeded HCV infection in a dose-dependent manner. As expected, HuH7 cells expressing PCSK9-ACE2 were also resistant to infection by HCV pseudoparticles. In addition, we showed that CD81 cell surface expression is modulated by PCSK9 in an LDLR-independent manner. Finally, in the liver of single Pcsk9 and double (Pcsk9 + Ldlr) knockout mice, both LDLR and/or CD81 protein expression levels were significantly reduced, but not those of transferrin and scavenger receptor class B type 1. Conclusion: Our results demonstrate an antiviral effect of the circulating liver PCSK9 on HCV in cells and show that PCSK9 down-regulates the level of mouse liver CD81 expression in vivo. Therefore, we propose that the plasma level and/or activity of PCSK9 may modulate HCV infectivity in humans. (Hepatology 2009.)

Worldwide there are approximately 240million individuals chronically infected with the hepatitis B virus (HBV), including 15-20million coinfected with the hepatitis delta virus (HDV). Treatments available today are not fully efficient and often associated to important side effects and development of drug resistance. Targeting the HBV/HDV entry step using preS1-specific lipopeptides appears as a promising strategy to block viral entry for both HBV and HDV (Gripon et al., 2005; Petersen et al., 2008). Recently, the human Sodium Taurocholate Cotransporting Polypeptide (hNTCP) has been identified as a functional, preS1-specific receptor for HBV and HDV. This groundbreaking discovery has opened a very promising avenue for the treatment of chronic HBV and HDV infections. Here we investigated the ability of FDA approved therapeutics with documented inhibitory effect on hNTCP cellular function to impair viral entry using a HDV in vitro infection model based on a hNTCP-expressing Huh7 cell line. We demonstrate the potential of three FDA approved molecules, irbesartan, ezetimibe, and ritonavir, to alter HDV infection in vitro.

Hepatitis C virus (HCV) infection induces intracellular membrane rearrangements, thus forming a membranous web (MW) in which HCV replication and assembly occur. The HCV-induced MW is primarily composed of double membrane vesicles (DMVs) transfused by multi-membrane vesicles. The autophagy machinery has been proposed to participate in the formation of such vesicles. However, no clear evidence has been found linking autophagy to the formation of these DMVs. In this study, we evaluated the role of the autophagy elongation complex (ATG5-12/16L1) in HCV replication and MW formation. Using a dominant negative form of ATG12 and an siRNA approach, we demonstrated that the ATG5-12 conjugate, but not LC3-II formation, is crucial for efficient viral replication. Furthermore, purification of HCV MW revealed the presence of ATG5-12 and ATG16L1 along with HCV nonstructural proteins. Interestingly, LC3 was not recruited along with the elongation complex to the site of viral replication. Finally, inhibition of the elongation complex, but not LC3, greatly impaired the formation of the wild-type MW phenotype. To our knowledge, this study provides the first evidence of the involvement of autophagy proteins in the formation of wild-type MWs.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an important factor in plasma cholesterol regulation through modulation of low-density lipoprotein receptor (LDLR) levels. Naturally occurring mutation can lead to hyper- or hypo-cholesterolemia in human. Recently, we reported that PCSK9 was also able to modulate CD81 in Huh7 cells. In the present study, several gain-of-function (GOF) and loss-of-function (LOF) as well as engineered mutants of PCSK9 were compared for their ability to modulate the cell surface expression of LDLR and CD81. While PCSK9 GOF D374Y enhances the degradation both receptors, D374H and D129N seem to only reduce LDLR levels. In contrast, mutations in the C-terminal hinge-CHRD segment primarily affect the PCSK9-induced CD81 degradation. Furthermore, when C-terminally fused to an ACE2-transmembrane anchor, the secretory N-terminal catalytic or hinge-CHRD domains of PCSK9 were able to reduce CD81 and LDLR levels. These data confirm that PCSK9 reduces CD81 levels via an intracellular pathway, as reported for LDLR. Using immunocytochemistry, a proximity ligation assay and coimmunoprecipitation, we found that the cell surface level of PCSK9 was enhanced upon overexpression of CD81, and that both PCSK9 and LDLR interact with this tetraspanin protein. Interestingly, using CHO-A7 cells lacking LDLR expression, we revealed that LDLR was not required for the degradation of CD81 by PCSK9, but its presence strengthened the PCSK9 effect.