Due to difficulties in cell culture propagation, the mechanisms of hepatitis C virus (HCV) entry are poorly understood. Here, postbinding cellular mechanisms of HCV entry were studied using both retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the HCV clone JFH-1 propagated in cell culture (HCVcc). HCVpp entry was measured by quantitative real-time PCR after 3 h of contact with target cells, and HCVcc infection was quantified by immunoblot analysis and immunofluorescence detection of HCV proteins expressed in infected cells. The functional role of clathrin-mediated endocytosis in HCV entry was assessed by small interfering RNA-mediated clathrin heavy chain depletion and with chlorpromazine, an inhibitor of clathrin-coated pit formation at the plasma membrane. In both conditions, HCVpp entry and HCVcc infection were inhibited. HCVcc infection was also inhibited by pretreating target cells with bafilomycin A1 or chloroquine, two drugs known to interfere with endosome acidification. These data indicate that HCV enters target cells by clathrin-mediated endocytosis, followed by a fusion step from within an acidic endosomal compartment.Hepatitis C virus (HCV) infects about 170 million people around the world. Despite the importance of HCV as a human pathogen, little is known about its cell biology. The virus was identified and cloned more than 15 years ago (7), but the lack of a robust system allowing for the production of HCV in cell culture has hampered for many years functional studies on HCV infection.In recent years, two major advances have made it possible to investigate HCV entry. A first advance has been the production of infectious retroviral particles pseudotyped with HCV envelope glycoproteins (3,14,24). Using this system of HCV pseudoparticles (HCVpp), observations on receptor usage and the facilitating role of high-density lipoprotein during entry were reported (3, 24, 55). A second major advance has been the recent development of a cell culture model for HCV (30,56,59). This system allows for the production of virus that can be efficiently propagated in cell culture (HCVcc). Therefore, the cell entry of HCV can now be investigated in the context of an infectious cycle.HCV belongs to the Hepacivirus genus in the Flaviviridae family, which also includes the Flavivirus and Pestivirus genera (31). The HCV genome encodes three structural proteins, capsid protein C and envelope glycoproteins E1 and E2, which are associated in the form of a heterodimer (13). Several cellular proteins were reported to interact in vitro with isolated E2. These putative receptors include the tetraspanin CD81 (42), the scavenger receptor class B type I (SR-BI) (50), the lectins L-SIGN and DC-SIGN (18, 32), the asialoglycoprotein receptor (49), and heparan-sulfate proteoglycans (2). The low-density lipoprotein receptor was also proposed as a candidate receptor (1). The importance of CD81 and SR-BI in HCV entry was confirmed with HCVpp (3, 4, 24) as well as with HCVcc for CD81 (30,56). Beyond receptor bind...

The scavenger receptor class B type I (SR-BI) has recently been shown to interact with hepatitis C virus (HCV) envelope glycoprotein E2, suggesting that it might be involved at some step of HCV entry into host cells. However, due to the absence of a cell culture system to efficiently amplify HCV, it is not clear how SR-BI contributes to HCV entry. Here, we sought to determine how high density lipoproteins (HDLs), the natural ligand of SR-BI, affect HCV entry. By using the recently described infectious HCV pseudotyped particles (HCVpps) that display functional E1E2 glycoprotein complexes, we showed that HDLs are able to markedly enhance HCVpp entry. We did not find any evidence of HDL association with HCVpps, suggesting that HCVpps do not enter into target cells using HDL as a carrier to bind to its receptor. Interestingly, lipid-free apoA-I and apoA-II, the major HDL apolipoproteins, were unable to enhance HCVpp infectivity. In addition, drugs inhibit- Approximately 170 million individuals are infected worldwide by hepatitis C virus (HCV).1 HCV infection causes acute hepatitis, which has a high probability of becoming chronic and in the long term can lead to cirrhosis and hepatocellular carcinoma (1). Because of the serious consequences of its infection in humans, much effort is being made to understand the basic mechanisms of HCV lifecycle. Because of the absence of an efficient cell culture system to replicate HCV, several laboratories have tried to develop surrogate models to study HCV entry (2). These models are based on the expression of HCV envelope glycoproteins E1 and E2, which form a noncovalent heterodimer. Several difficulties have been encountered in these approaches because HCV envelope glycoproteins are located in the endoplasmic reticulum (3), and their folding and assembly is very sensitive to mutations or deletions affecting the endoplasmic reticulum retention domains (4). Recently, infectious pseudotyped particles (HCVpps) that are assembled by displaying unmodified HCV envelope glycoproteins onto retroviral core particles have successfully been generated and now enable studies of HCV entry (5, 6).The cellular tropism of enveloped viruses is largely determined by selective interactions of viral envelope glycoproteins with specific cell-surface receptors. Several candidate receptors for HCV have recently been proposed. Molecules like the CD81 tetraspanin (7), the scavenger receptor class B type I (SR-BI) (8), the LDL receptor (9, 10), and the asialoglycoprotein receptor (11) are potential candidate receptors for HCV, whereas the mannose binding lectins DC-SIGN and L-SIGN have been shown to function as HCV capture receptors but do not mediate viral entry into target cells (12-16). Among these molecules there is mounting evidence that CD81 and SR-BI are necessary for HCV entry (5,6,17). Whereas the role of CD81 in HCV entry is now well documented (5,6,(17)(18)(19), the contribution of SR-BI in HCV entry needs to be further characterized. SR-BI is a 509-amino acid glycoprotein with two cytoplasmic ...

Due to the recent development of a cell culture model, hepatitis C virus (HCV) can be efficiently propagated in cell culture. This allowed us to reinvestigate the subcellular localization of HCV structural proteins in the context of an infectious cycle. In agreement with previous reports, confocal immunofluorescence analysis of the subcellular localization of HCV structural proteins indicated that, in infected cells, the glycoprotein heterodimer is retained in the endoplasmic reticulum. However, in contrast to other studies, the glycoprotein heterodimer did not accumulate in other intracellular compartments or at the plasma membrane. As previously reported, an association between the capsid protein and lipid droplets was also observed. In addition, a fraction of labeling was consistent with the capsid protein being localized in a membranous compartment that is associated with the lipid droplets. However, in contrast to previous reports, the capsid protein was not found in the nucleus or in association with mitochondria or other well-defined intracellular compartments. Surprisingly, no colocalization was observed between the glycoprotein heterodimer and the capsid protein in infected cells. Electron microscopy analyses allowed us to identify a membrane alteration similar to the previously reported "membranous web." However, no virus-like particles were found in this type of structure. In addition, dense elements compatible with the size and shape of a viral particle were seldom observed in infected cells. In conclusion, the cell culture system for HCV allowed us for the first time to characterize the subcellular localization of HCV structural proteins in the context an infectious cycle.

The modulation of canonical macroautophagy/autophagy for therapeutic benefit is an emerging strategy of medical and pharmaceutical interest. Many drugs act to inhibit autophagic flux by targeting lysosome function, while others were developed to activate the pathway. Here, we report the surprising finding that many therapeutically relevant autophagy modulators with lysosomotropic and ionophore properties, classified as inhibitors of canonical autophagy, are also capable of activating a parallel noncanonical autophagy pathway that drives MAP1LC3/LC3 lipidation on endolysosomal membranes. Further, we provide the first evidence supporting drug-induced noncanonical autophagy in vivo using the local anesthetic lidocaine and human skin biopsies. In addition, we find that several published inducers of autophagy and mitophagy are also potent activators of noncanonical autophagy. Together, our data raise important issues regarding the interpretation of LC3 lipidation data and the use of autophagy modulators, and highlight the need for a greater understanding of the functional consequences of noncanonical autophagy.

INSERM U966, Université Franç ois Rabelais and CHRU de Tours, FranceLike most other positive-strand RNA viruses, hepatitis C virus (HCV) induces changes in the host cell's membranes, resulting in a membranous web. The non-structural proteins of the viral replication complex are thought to be associated with these newly synthesized membranes. We studied this phenomenon, using a Huh7.5 cell clone displaying high levels of replication of a subgenomic replicon of the JFH-1 strain. Electron microscopy of ultrathin sections of these cells revealed the presence of numerous double membrane vesicles (DMVs), resembling those observed for other RNA viruses such as poliovirus and coronavirus. Some sections had more discrete multivesicular units consisting of circular concentric membranes organized into clusters surrounded by a wrapping membrane. These structures were highly specific to HCV as they were not detected in naive Huh7.5 cells. Preparations enriched in these structures were separated from other endoplasmic reticulum-derived membranes by cell cytoplasm homogenization and ultracentrifugation on a sucrose gradient. They were found to contain the non-structural NS3 and NS5A HCV proteins, HCV RNA and LC3-II, a specific marker of autophagic membranes. By analogy to other viral models, HCV may induce DMVs by activating the autophagy pathway. This could represent a strategy to conceal the viral RNA and help the virus to evade double-stranded RNA-triggered host antiviral responses. More detailed characterization of these virus-cell interactions may facilitate the development of new treatments active against HCV and other RNA viruses that are dependent on newly synthesized cellular membranes for replication. INTRODUCTIONThe formation of a membrane-associated replication complex, consisting of viral proteins, replicating RNA, altered cellular membranes and other host factors, is a hallmark of all positive-strand RNA viruses including those infecting mammalian, insect or plant cells (Miller & Krijnse-Locker, 2008;Mackenzie, 2005). Depending on the virus, replication may occur on rearranged convoluted membranes, single or double membrane vesicles of various sizes derived from the endoplasmic reticulum (ER), the intermediate compartment between the ER and the Golgi apparatus, the trans-Golgi network, mitochondria or even lysosomes (Miller & KrijnseLocker, 2008). These membrane structures induced by positive-strand RNA viruses probably serve as a scaffold for the assembly of viral replication complexes by providing an organization and environment facilitating viral replication (Lyle et al., 2002; Schwartz et al., 2002). Some positivestranded RNA viruses including some strains of poliovirus (Jackson et al., 2005), coxsackievirus B3 (Wong et al., 2008), dengue virus (Lee et al., 2008) and mouse hepatitis coronavirus (Prentice et al., 2004) may take over the host autophagy machinery to facilitate their own replication. These positive-stranded RNA viruses trigger autophagosome-like formation without triggering the ultimate degradati...

The data provide a molecular explanation for HCV genotype 3-specific lipid accumulation. This difference between genotypes may be due to phenylalanine having a higher affinity for lipids than tyrosine (Y). These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis.

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and cirrhosis and may lead to hepatocellular carcinoma. With an estimated 170 million people worldwide chronically infected with HCV, this disease has emerged as a serious global health problem since the cloning of the viral genome in 1989 (6). HCV is an enveloped RNA virus belonging to the genus Hepacivirus of the Flaviviridae family. Its genome is a 9.6-kb single-stranded RNA of positive polarity with a 5Ј untranslated region (UTR) that functions as an internal ribosome entry site, a single long open reading frame encoding a polyprotein of approximately 3,000 amino acids (aa) and a 3Ј UTR (1). This polypeptide is posttranslationally cleaved by host cell peptidases to yield three structural proteins and by viral proteases, which generate the five nonstructural proteins. The structural proteins, which are located in the amino-terminal region of the polyprotein, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins (NS) 2 to 5B are separated from the structural proteins by the short hydrophobic polypeptide p7, the function of which is unknown (1). By analogy to related positive-strand RNA viruses, replication occurs by means of a negative-strand RNA intermediate and is catalyzed by the NS proteins, which form a cytoplasmic membrane-associated replicase complex.Several entire cloned HCV genomes are able to initiate infection when introduced directly into chimpanzee livers (11). However, the transfection of cell lines with constructs containing these genomes does not result in HCV replication in vitro (1). Similarly, the use of infected sera to infect cell lines or primary cell cultures has yielded disappointing results because infection and replication are very inefficient (2). Molecular studies of the HCV infectious cycle in the host cell and the development of specific anti-HCV agents have been considerably hampered by the inability to achieve propagation of the virus in cultured cells in vitro. A significant advance in HCV research was recently made with the development of subgenomic HCV RNAs consisting of sequences encoding nonstructural proteins flanked by the 5Ј and 3Ј UTRs, which selfreplicate in hepatoma cells (4,18). However, this model cannot be used to address the structural features of the virion or its assembly pathway. In addition, viral particles are difficult to observe by electron microscopy in the plasma or liver tissues of infected patients. A number of attempts have bee...

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.