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 ...