No abstract

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.

No abstract

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNAdependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 Å contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the ⌳1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive preinitiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template. Hepatitis C virus (HCV)1 is the major etiological agent of non-A non-B hepatitis and infects an estimated 3% of the world's population (1). Approximately 80% of the infected individuals will remain chronically infected for decades and may eventually develop severe liver cirrhosis and hepatocellular carcinoma (2). 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 interferon and ribavirin (3).HCV is an enveloped virus belonging to the hepacivirus gender in the Flaviviridae family, which also includes the flavivirus and pestivirus (4). The single-stranded positive-sense RNA genome is ϳ9.5 kb in length and produces a single polyprotein of 3010 -3040 amino acids (5). The polyprotein is processed by a combination of viral and cellular proteases, giving rise to at least 10 individual proteins (6). In the absence of a permissive cell culture replication system, the knowledge of ...

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.

In the context of viral infections, autophagy induction can be beneficial or inhibitory. Within the Paramyxoviridae family, only morbilliviruses have been investigated and are reported to induce autophagy. Here we show that morbilliviruses rapidly induce autophagy and require this induction for efficient cell-to-cell spread. Coexpression of both glycoproteins in cells expressing one of the cellular receptors was required for autophagy induction, and LC3 punctum formation, indicative of autophagy, was mainly observed in syncytia. A similar correlation between syncytium formation and autophagy induction was also observed for other paramyxovirus glycoproteins, suggesting that membrane fusion-mediated autophagy may be common among paramyxoviruses and possibly other enveloped viruses.A s obligate intracellular parasites, viruses have evolved to exploit cellular functions, while cells have in turn developed a broad spectrum of antiviral mechanisms. In addition to the wellcharacterized sensors of pathogen-associated molecular patterns (PAMPs) (18), alteration of autophagy activity is increasingly recognized as an early inducer of innate immune activation (36,45,46). Autophagy is a ubiquitous mechanism involved in the maintenance of homeostasis in response to cellular stress by recycling of long-lived proteins and cytoplasmic organelles. Briefly, damaged cellular constituents are sequestered into the autophagosomes, which fuse with lysosomes to transform into autolysosomes (9). Autophagosome formation involves the conjugation of autophagy-related gene 5 (Atg5) and Atg12 by the ubiquitin E1-like enzyme Atg7 and the E2-like enzyme Atg10 (31, 38), followed by the cleavage of the microtubule-associated protein light chain 3 (LC3) in LC3-I and LC3-II (37). LC3 is a major constituent of the autophagosome, and its intracellular localization and conversion from the cytoplasmic form, LC3-I, to its membrane-bound cleavage product, LC3-II, are frequently used to monitor autophagic activity (15,25). In the context of virus infections, autophagy may be either inhibited to interfere with innate immune activation or induced to promote virus replication (22).Morbilliviruses constitute a genus in the Paramyxoviridae family within the order Mononegavirales (5). These viruses initially infect a broad range of immune cells and then spread to epithelial cells. Attachment to the target cell is mediated by the hemagglutinin (H) protein, upon which the fusion (F) protein induces fusion of the viral and cellular membranes (19). The signaling lymphocyte activation molecule (SLAM), which is expressed on the surface of activated T and B lymphocytes, macrophages, and dendritic cells (43), serves as the immune cell receptor for all morbilliviruses (39), while CD46, a regulator of complement activation, is used only by attenuated and certain wild-type measles viruses (MeVs) (47). Recent reports demonstrate that both SLAM and CD46 recruit the vps34/beclin1 autophagic complex, suggesting that morbilliviruses might induce autophagy pathways upon recepto...

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.