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Keywords:

  • hepatitis C virus (HCV);
  • lipid droplet;
  • lipoprotein;
  • virus assembly;
  • virus entry

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

HCV (hepatitis C virus) represents a major global health problem. A consistent body of evidence has been accumulating, suggesting a peculiar overlap between the HCV life cycle and lipid metabolism. This association becomes evident both for the clinical symptoms of HCV infection and the molecular mechanisms underlying the morphogenesis and entry process of this virus. The HCV core–lipid droplets association seems to be central to the HCV morphogenesis process. Moreover, the biogenesis pathway of very-low-density lipoproteins has been shown to be involved in HCV morphogenesis with MTP (microsomal triacylglycerol transfer protein), ApoB (apolipoprotein B) and ApoE (apolipoprotein E) as essential elements in the production of infectious HCV particles. HCV infectivity also correlates with the lipidation status of the particles. Furthermore, some HCV cellular receptors and the regulation of the entry process are also connected to lipoproteins and lipid metabolism. Specifically, lipoproteins modulate the entry process and the cholesterol transporter SR-BI (scavenger receptor class B type I) is a cellular entry factor for HCV. The present review aims to summarize the advances in our understanding of the HCV–lipid metabolism association, which may open new therapeutic avenues.


Abbreviations used:
ApoB

apolipoprotein B

ApoCI

apolipoprotein CI

ApoE

apolipoprotein E

ER

endoplasmic reticulum

HCV

hepatitis C virus

HCVcc

HCV produced in cell culture

HCVpp

HCV pseudoparticles

HDL

high-density lipoprotein

LDL

low-density lipoprotein

LDL-R

LDL receptor

MTP

microsomal triacylglycerol transfer protein

ORF

open reading frame

SAA

serum amyloid A

SR-BI

scavenger receptor class B type I

VLDL

very-LDL

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

HCV (hepatitis C virus) represents a serious health problem worldwide as approx. 130 million people (2% of the world population) are infected (Shepard et al., 2005). HCV infection is mainly restricted to hepatocytes, and since most of the infected individuals fail to spontaneously clear the virus from the liver, this leads to a chronic infection that can evolve towards liver fibrosis, cirrhosis and hepatocellular carcinoma over a period of decades (Lemon et al., 2007).

HCV is a positive-stranded RNA virus that belongs to the Hepacivirus genus in the Flaviviridae family (reviewed in Lindenbach et al., 2007). A single ORF (open reading frame) encoding a polyprotein precursor is co- and post-translationally processed by endogenous and virally encoded proteases to produce ten mature proteins (Figure 1). The structural proteins (core and envelope proteins E1 and E2) are located in the N-terminal part of the polyprotein and the non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) are located towards the C-terminus. The non-structural proteins NS3 to NS5B are involved in the replication of the viral genome, whereas the structural proteins are the components of the viral particle (reviewed in Moradpour et al., 2007).

image

Figure 1. Genomic organization of HCV

The genome of HCV is a positive stranded RNA. It contains 5′ and 3′ non-coding regions and a single ORF, which is translated into a polyprotein. This polyprotein is co- and post-translationally processed by cellular (signal peptidase) and viral proteases (NS2 autoprotease and NS3 protease) to generate ten viral proteins. An additional cleavage, mediated by a signal peptide peptidase, takes place in the C-terminal region of the core protein. The structural proteins, core and envelope proteins E1 and E2 (light grey), constitute the virus make up. The non-structural proteins NS3 to NS5B (dark grey) are responsible for HCV RNA replication. The non-structural proteins p7 and NS2 (medium grey) are involved in the assembly of the viral particle. Besides p7, NS2 and the structural proteins, some non-structural proteins are also involved in virus morphogenesis, which represents a peculiarity of the Flaviviridae family (reviewed in Murray et al., 2008).

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For reasons still unknown, HCV clinical isolates do not propagate in cell culture. However, a few years ago, a cell culture system that enables relatively efficient amplification of HCV [HCVcc (HCV produced in cell culture)] has finally been developed (Lindenbach et al., 2005; Wakita et al., 2005; Zhong et al., 2005). This system is based on the transfection of the human hepatoma cell line Huh-7 with genomic HCV RNA derived from a cloned viral genome of an HCV isolate from a Japanese patient with fulminant hepatitis. Data accumulated with the HCVcc system begin to shed light on the life cycle of this virus (Figure 2).

image

Figure 2. HCV life cycle

HCV particles are made of viral components (green spheres) associated with lipoproteins (yellow spheres). Viral particles enter hepatocytes using at least four entry factors including the cholesterol transporter SR-BI. HCV entry takes place by receptordependent endocytosis. Upon arrival in early endosomes, the envelope proteins enable the fusion of the viral envelope with the endosome and release of the genome into the cytosol. The viral RNA is then translated into a polyprotein that is processed to generate the viral proteins. The non-structural proteins NS3 to NS5B form the replication complex in association with ER-derived membranes called membranous web (Gosert et al., 2003), and they replicate the genome. After accumulation of neosynthesized genomic RNA and viral proteins, the HCV particle is assembled in an ER-related compartment in close connection with the VLDL biogenesis pathway. This process seems to occur in the proximity of lipid droplets (LD) (Miyanari et al., 2007). Then, HCV particles, associated with lipoproteins, are exported through the secretory pathway. Note that the polarization of the cell can potentially affect HCV entry and/or release (Mee et al., 2009). However, in the absence of detailed information on HCV infection of polarized hepatocytes, the life cycle of HCV is presented in the context of a non-polarized cell.

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HCV infection of the hepatocyte begins with a complex interaction of the virion with a series of cellular entry factors (for recent reviews, see Dubuisson et al., 2008; Helle and Dubuisson, 2008; Timpe and McKeating, 2008; von Hahn and Rice, 2008; Burlone and Budkowska, 2009). The viral particle is then internalized by clathrin-mediated endocytosis (Blanchard et al., 2006; Meertens et al., 2006). It is believed that after fusion between the viral envelope and the membrane of an early endosome (Meertens et al., 2006), the viral RNA is released into the cytosol. The genome is then translated and processed to generate the viral proteins mentioned above. The non-structural proteins assemble the replication complex, which is tightly linked to ER (endoplasmic reticulum)-derived membranes (reviewed in Moradpour et al., 2007). With the progressive accumulation of new genomic RNA and structural proteins, progeny viral particles are formed in an intracellular compartment and they are released from the cell through the secretory pathway.

Today, an increasing body of data indicates that HCV exploits the lipid metabolism of the hepatocyte for the assembly and entry steps of its life cycle. In the present review, we discuss the molecular mechanisms of this close association.

HCV and lipid droplets

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

HCV core protein has been suggested to be a major determinant of HCV-induced liver damage (reviewed in Roingeard and Hourioux, 2008; McLauchlan, 2009). The HCV core was shown to accumulate around the lipid droplets, which are intracellular stores of triacylglycerols and cholesteryl esters surrounded by a single layer of phospholipids (Moradpour et al., 1996; Barba et al., 1997). Moreover, HCV core expression in transgenic mice inhibits the activity of MTP (microsomal triacylglycerol transfer protein) and the subsequent secretion of VLDLs [very-LDLs (low-density lipoproteins)] (Perlemuter et al., 2002).

Based on amino acid distribution and hydrophobicity plots, the mature form of HCV core protein can be divided into two domains, D1 and D2 (McLauchlan, 2000; Hope et al., 2002; Boulant et al., 2005). D1 is rich in basic residues and is located at the N-terminal two-thirds of the core, whereas D2 encompasses the C-terminus and it is more hydrophobic. In its immature form, the HCV core also contains a signal peptide at its C-terminus that is responsible for the translocation of the ectodomain of the envelope glycoprotein E1 in the ER lumen (Santolini et al., 1994). After cleavage by a signal peptidase between core and E1, this signal peptide is removed from the core protein by an additional cleavage mediated by a signal peptide peptidase (McLauchlan et al., 2002). This latter processing is essential for the localization of the core around lipid droplets (McLauchlan et al., 2002) as well as for viral infectivity (Targett-Adams et al., 2008).

The association between HCV core protein and lipid droplets is a peculiarity of the Hepaciviruses in the Flaviviridae family (Hope and McLauchlan, 2000). This is due to the presence of domain D2 which has only been observed in the Hepacivirus genus and is absent from the other genera (McLauchlan, 2000). The structural peculiarity of the HCV core might partly explain some physiopathological differences between HCV and the other viruses within the Flaviviridae family. Thus the D2 domain of the HCV core gained special attention and was thoroughly characterized both structurally and functionally. NMR analyses of two overlapping peptides spanning this domain showed that it consists of two amphipathic α-helices connected by a hydrophobic loop. Mutation analyses showed the importance of the hydrophobic residues of the helices in the association with lipid droplets (Boulant et al., 2006). Despite its apparent lack of secondary structure, the hydrophobic loop is also essential for lipid droplet association. Notably, mutation of two prolines within the hydrophobic loop abolished the targeting of the core protein to lipid droplets (Hope et al., 2002; Boulant et al., 2006).

Functional studies of the relevance of D2 and core–lipid droplet association became possible when the HCV life cycle could be reproduced in cell culture with the HCVcc system. Interestingly, chimaeric viruses with domains exchanged in the core region showed variations in the efficiency of virus formation and kinetics of virus production (Shavinskaya et al., 2007). In this work, the D2 domain was identified to be responsible for virus production efficiency. Further analyses also revealed an inverse correlation between the extent of lipid droplets–core association and virus production (Shavinskaya et al., 2007). Thus the HCV assembly process appears to be spatially associated with lipid droplets.

Recently, it has also been shown that besides the core protein, viral non-structural proteins like NS5A and NS3 and double-stranded viral RNA are also present around lipid droplets (Miyanari et al., 2007). The association between the core and lipid droplets is essential for the recruitment of the other viral proteins and for virus production. Thus core mutations that abolish the lipid droplet association impair virus production (Boulant et al., 2007; Miyanari et al., 2007). The current assembly model assumes that the core protein associates with lipid droplets via its D2 domain and recruits NS5A to lipid droplets through direct interactions (Masaki et al., 2008). Furthermore, NS5A plays a double role in both replication and assembly processes as a potential switch between the two steps (Evans et al., 2004; Appel et al., 2008; Tellinghuisen et al., 2008). Unfortunately, our view on HCV assembly currently lacks data concerning the envelopment step and the role of non-structural proteins in this process. In conclusion, lipid droplets are a major actor in the HCV assembly process, but at the same time are also central to lipoprotein secretion by hepatocytes (Olofsson et al., 2008). Recently, the two assembly pathways were shown to intersect (Huang et al., 2007; Gastaminza et al., 2008).

HCV and the VLDL assembly pathway

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

Hepatocytes play an essential role in maintaining the lipid homoeostasis in the body by assembly and secretion of VLDLs. The main protein component of VLDL is ApoB100 (apolipoprotein B100), a very large glycoprotein (4536 amino acids) capable of binding lipids due to its amphipathic nature. VLDL assembly begins with the co-translocational lipidation of ApoB100 by MTP (Hussain et al., 2003). MTP interacts with ApoB to transfer the triacylglycerols and generate a poorly lipidated form of VLDL called pre-VLDL. Subsequently, pre-VLDL fuses with bulk lipids from luminal lipid droplets present in the secretory pathway to become mature VLDL of lower density (Shelness and Sellers, 2001). The picture of mature VLDL formation is still unclear regarding the location, the origin of triacylglycerols and the role of MTP in the process (Fisher and Ginsberg, 2002). ApoB100 levels are regulated at different stages in VLDL assembly. While the lack of triacylglycerols induces co-translational degradation of ApoB by the proteasome pathway, there are data suggesting a late non-proteasomal degradation of assembled VLDL lipoproteins (Fisher and Ginsberg, 2002).

The parallel between VLDL and HCV assembly pathways was established recently by two groups using different approaches. Huang et al. (2007) purified NS5A-positive membranes and characterized their composition (Huang et al., 2007). They found that besides the viral non-structural proteins involved in HCV replication (NS3, NS4B and NS5B) and viral RNA, the membranes contained numerous proteins involved in lipid metabolism. Pharmacological inhibition of MTP activity and siRNA (small interfering RNA) down-regulation of ApoB showed the requirement of a functional VLDL secretion pathway for the production of infectious HCV particles (Huang et al., 2007). The similarity between HCV and VLDL production pathways is highlighted further by Gastaminza et al. (2008), who used different pharmacological treatments to follow the maturation of the high-density intracellular HCV particles to mature secreted particles (Gastaminza et al., 2008). Using brefeldin A, which stops the transport between ER and Golgi compartments and different pharmacological inhibitors, they showed that the intracellular high-density HCV particles are degraded in a post-ER compartment and in a non-proteasomal manner. MTP activity inhibition and ApoB down-regulation prevented the assembly of infectious particles at an early stage and the effect was not genotype specific (Gastaminza et al., 2008).

Besides ApoB and MTP, ApoE (apolipoprotein E) is also an essential player in the synthesis of mature VLDLs (Fazio et al., 2000). Interestingly, it has also been shown that down-regulation of ApoE reduces considerably the production of infectious particles (Chang et al., 2007). Furthermore, HCV infectivity is also highly neutralized by anti-ApoE antibodies (Chang et al., 2007).

The overlap between HCV and VLDL assembly processes was further illustrated by the finding that recombinant HCV envelope glycoproteins, expressed in the absence of other viral components, can be secreted along with triacylglycerol-rich lipoproteins by differentiated intestinal Caco-2 cells. HCV envelope glycoproteins were shown to be associated with lipoprotein containing ApoB and their secretion was dependent on the activity of MTP (Icard et al., 2009).

As summarized in Figure 3, our understanding of the HCV assembly process is still in its infancy, but the characterization of the physical and biochemical properties of the end product, the HCV particles, has strengthened and guided our current picture of HCV assembly.

image

Figure 3. A model of the HCV assembly process

The association of core protein (green) with lipid droplets (LD) is essential for the HCV assembly process. The replication complex (RC) is also recruited to the LD potentially through direct interaction with the core. Core interaction with viral RNA is supposed to trigger nucleocapsid formation, which leads to the budding process involving the envelope proteins (red). Virion formation is paralleled by VLDL assembly with MTP transferring triacylglycerols (TG) to the nascent ApoB chain. A high-density particle may appear through the association between the HCV virion and the poorly lipidated ApoB. The ApoB part of the hybrid particle is lipidated further to yield the mature low-density HCV virion. The process assumes a second TG transfer and the involvement of ApoE and MTP as suggested for the VLDL maturation process.

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HCV particles

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

Despite the recent advances in the HCV cell culture system, we are still missing a detailed characterization of HCV particles. HCV particles isolated from patient sera were found to be heterogeneous in terms of their density and composition. The disease stage, patient health status and the purification protocol can add further variability in the reported characterizations of HCV particles. Density gradient analyses indicate that HCV RNA is found in particles of low and higher density (Andre et al., 2005). The low-density fraction contained lipoproteins with ApoB and ApoE, triacylglycerols, core and the envelope glycoproteins (Thomssen et al., 1992; Prince et al., 1996; Andre et al., 2002; Maillard et al., 2006; Nielsen et al., 2006). Furthermore, only the low-density population of HCV isolated from sera (≤1.09 g/ml) is highly infectious in chimpanzee (Bradley et al., 1991; Beach et al., 1992). Recently, ApoCI (apolipoprotein CI) was also reported to be associated with infectious HCV particles (Meunier et al., 2008).

The HCV cell culture system allowed the production of HCV particles (HCVcc) at higher titres and their biophysical properties were characterized. In density gradients, most of the secreted RNA-containing particles are poorly infectious and fractionate at high densities (1.14 g/ml), whereas HCVcc particles of high infectivity were shown to mainly fractionate at low densities (1.10 g/ml) (Gastaminza et al., 2006; Lindenbach et al., 2006). Since viral particles produced in cell culture can infect chimpanzees and mice transplanted with human hepatocytes, the biophysical properties of viral particles produced in animals were also characterized (Wakita et al., 2005; Lindenbach et al., 2006). Interestingly, the specific infectivity of virus recovered from these animals was much higher than virus produced in cell culture and it was also associated with a lower density than cell culture-derived virus (Lindenbach et al., 2006). These observations suggest that the composition or maturation of the lipoproteins associated with HCV particles can modulate their infectivity. This is in agreement with the observation that depletion of cholesterol and sphingomyelin from the particle inhibits HCVcc infectivity (Aizaki et al., 2008).

The participation of different apolipoproteins in the make up of cell culture-derived virus was further proved by the neutralization of HCV infectivity with antibodies against ApoB, ApoE or ApoCI (Andreo et al., 2007; Chang et al., 2007; Meunier et al., 2008). The nature of the association between HCV particle, lipids and lipoproteins is currently not known, but more hints are coming from the involvement of lipid metabolism in the HCV entry process.

HCV entry

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

For a long time, it has been difficult to study HCV entry because of the difficulties in propagating the virus in cell culture. However, surrogate models have been extensively used to decipher the early steps of the HCV life cycle. HCVpp (HCV pseudoparticles) have been the most robust system used to study the full process of HCV entry into host cells (Bartosch et al., 2003a; Drummer et al., 2003; Hsu et al., 2003). These particles consist of retroviral particles that have HCV envelope glycoproteins E1 and E2 instead of retroviral glycoproteins anchored in their lipid envelope. However, HCVpp do not seem to be associated with VLDLs (Bartosch et al., 2003a), which is due to differences in the assembly process between HCVpp and HCVcc. Indeed, HCVcc assemble in an ER-derived compartment in association with VLDLs (Huang et al., 2007), whereas HCVpp are assembled in a post-Golgi compartment (Sandrin et al., 2005). Therefore the HCVcc system is a more appropriate tool to study the potential role of the lipoproteins associated with the particle in the entry process.

Virus entry is often initiated by the binding of particles to attachment factors, which helps to concentrate viruses on the cell surface. As for many other viruses, glycosaminoglycans seem to be an initial docking site for HCV attachment (Germi et al., 2002; Barth et al., 2003, 2006; Koutsoudakis et al., 2006; Basu et al., 2007; Morikawa et al., 2007). After the initial attachment to the host cell, a virus generally binds to specific entry factors, which are responsible for initiating a series of events that eventually lead to the release of the viral genome into the cytosol. Several cell surface proteins have been described as specific entry factors for HCV.

The first identified and best characterized HCV entry factor is the tetraspanin CD81, which was initially shown to interact with HCV envelope glycoprotein E2 (Pileri et al., 1998). Since its discovery, the involvement of CD81 in HCV entry has been extensively confirmed using the HCVpp and HCVcc systems (for recent reviews, see Dubuisson et al., 2008; Helle and Dubuisson, 2008; Timpe and McKeating, 2008; von Hahn and Rice, 2008; Burlone and Budkowska, 2009). Although CD81 is essential for HCV infection of hepatocytes with cell-free virus, CD81-independent cell-to-cell transmission has also been reported (Timpe et al., 2008; Witteveldt et al., 2009).

Besides CD81, SR-BI (scavenger receptor class B type I; also called CLA-1) has also been shown to interact with HCV glycoprotein E2 and has therefore been proposed to be another potential entry factor for this virus (Scarselli et al., 2002). The involvement of SR-BI in HCV entry has also been confirmedusing the HCVpp and HCVcc systems (Bartosch et al., 2003b; Catanese et al., 2007; Kapadia et al., 2007; Zeisel et al., 2007; Dreux et al., 2009). Interestingly, kinetics of inhibition with anti-SR-BI and anti-CD81 antibodies suggest that SR-BI might act concomitantly with CD81 (Zeisel et al., 2007).

Recently, the tight junction proteins Claudin-1 and Occludin have also been identified as new additional entry factors for HCV (Evans et al., 2007; Ploss et al., 2009). No direct interaction between Claudin-1 and HCV envelope glycoproteins associated with HCV particles has been reported yet (Evans et al., 2007; Zheng et al., 2007). In contrast, an interaction between HCV envelope glycoproteins and Occludin has been shown (Benedicto et al., 2008; Liu et al., 2009).

Together with the identification of CD81 and SR-BI, the recent identification of the tight junction proteins Claudin-1 and Occludin suggests some level of complexity in HCV entry. The current characterization of HCV entry factors indicates that HCV entry into the hepatocyte is a slow and potentially multistep process. However, the precise role of HCV entry factors in the early steps of the HCV life cycle remains to be determined. Furthermore, as discussed above, native HCV particles are associated with host lipoproteins, supporting the hypothesis that HCV entry is connected to the host lipid metabolism (Burlone and Budkowska, 2009). This latter observation provides an additional level of complexity to the understanding of HCV entry into its host cells.

Due to the association of lipoproteins with HCV particles, the LDL-R (LDL receptor) has rapidly been proposed to be involved in HCV entry (Agnello et al., 1999; Monazahian et al., 1999). The most important ligand for this receptor is LDL, which is responsible for the transport of most of the plasma cholesterol. It has been shown that cell surface adsorption of HCV particles isolated from patients and accumulation of viral RNA in cells can be inhibited by antibodies directed against the LDL-R as well as purified LDLs and VLDLs. Furthermore, a correlation has been shown between the accumulation of HCV RNA into primary hepatocytes and the expression of LDL-R mRNA and LDL entry (Molina et al., 2007). Finally, the inhibition of HCVcc entry by anti-ApoE or -ApoB antibodies is another potential argument in favour of a role for the LDL-R in HCV entry (Andreo et al., 2007; Chang et al., 2007). However, we cannot exclude that the LDL-R leads to non-productive entry, since lipoprotein lipase treatment of Huh-7 cells leads to HCV uptake but inhibits HCVcc infection (Andreo et al., 2007). Lipoprotein lipase is a key enzyme in the metabolism of lipoproteins, which hydrolyses triacylglycerols in the core of lipoprotein particles and targets them to the liver for their clearance from the blood circulation (Mead et al., 2002). Since secreted HCV particles are potentially associated with VLDLs, one can imagine that after digestion with lipoprotein lipase, a large proportion of these particles will interact with the LDL-R on the hepatocytes and be targeted for degradation (Merkel et al., 2002). From the data that have been accumulated, the LDL-R is likely to be an attachment factor for the lipoproteins associated with HCV. However, further investigations with the HCVcc system are needed to determine whether this interaction leads to a productive or a non-productive entry pathway.

Among HCV entry factors, SR-BI is another receptor involved in lipoprotein metabolism. This protein was in fact initially identified as the major physiological receptor for HDLs (high-density lipoproteins) in the liver, and it was later shown to be a multiligand receptor (Connelly and Williams, 2004). Although HCV envelope glycoprotein E2 interacts with SR-BI, the lipoproteins associated with HCV particles can also potentially play a role in HCV–SR-BI interaction (Maillard et al., 2006). Thus SR-BI is also a receptor for VLDLs and LDLs (Connelly and Williams, 2004).

Interestingly, several SR-BI ligands have been found to modulate HCV infectivity. First, HDL has been shown to enhance HCV entry in a process that depends on the lipid transfer function of SR-BI and the presence of ApoCI (Bartosch et al., 2005; Meunier et al., 2005; Voisset et al., 2005; Catanese et al., 2007; Dreux et al., 2007; Zeisel et al., 2007). It seems therefore that HCV exploits the physiological functions of SR-BI during the entry process. Furthermore, the HDL-enhancing effect on HCV entry reduces the sensitivity of HCV to neutralizing antibodies (Dreux et al., 2006; Voisset et al., 2006). In contrast with HDLs, oxidized LDLs, another ligand of SR-BI, have been reported to inhibit HCV entry (von Hahn et al., 2006). In addition, VLDLs also block the binding of HCV particles isolated from HCV-infected patients to SR-BI (Maillard et al., 2006). Finally, SAA (serum amyloid A), an acute phase apolipoprotein produced by the liver, also inhibits HCV entry (Lavie et al., 2006; Cai et al., 2007). In this case, by interacting with the viral particle, SAA blocks HCV binding to target cells (Lavie et al., 2006). Details of the early steps of the HCV life cycle are summarized in Figure 4.

image

Figure 4. A model of HCV entry

Initial host-cell attachment may involve glycosaminoglycans (GAGs). In addition, due to its association with VLDL, the LDL-R can also potentially play a role in the initial step of entry. After initial binding to the cell, the particle appears to interact with four entry factors: SR-BI, the tetraspanin CD81 and the tight junction proteins Claudin-1 (CLDN1) and Occludin (OCLN). After binding to several components of the host cell, the HCV particle is internalized by clathrin-mediated endocytosis. Several serum components may modulate HCV entry. Indeed, SAA, oxidized LDL (LDLox) and lipoprotein lipase (LPL) have been shown to inhibit HCV entry. In contrast, HDL has been shown to increase HCV entry.

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Besides the effects of some lipoproteins and apolipoproteins, the lipid composition of the plasma membrane can also modulate HCV entry into host cells. For instance, depletion of cholesterol from the plasma membrane affects HCV entry by reducing the cell surface expression of CD81 (Kapadia et al., 2007). Furthermore, altering the sphingomyelin/ceramide ratio of the plasma membrane can also affect HCV entry by also decreasing the cell surface expression of CD81 (Voisset et al., 2008).

Conclusions and perspectives

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

The molecular mechanisms that govern the connection between HCV life cycle and lipid metabolism as summarized in Table 1 are just beginning to be understood. There are still essential questions to be answered. (i) What is the mechanism of HCV association to lipoproteins? In order to address the issue, it will be necessary to characterize the different intermediates of HCV assembly in terms of composition and lipidation status. Furthermore, the common mechanisms between VLDL and HCV assembly pathways need to be determined. (ii) What is the architecture of the HCV particle? Better biochemical and electron microscopy studies will have to be developed to characterize HCV particles released from cell culture preparations. Knowledge of HCV particle architecture is crucial in addressing the next question. (iii) How does the HCV particle lipid composition determine the receptor association and the entry process? We need to address the role of lipids in virus–receptor interaction, receptor complex dynamics and the activation of signalling pathways that determine particle internalization. Moreover, the different cues involved in the HCV fusion process with vesicles of the endocytic pathway remain to be unravelled.

Table 1.  Interaction between the HCV life cycle and lipid metabolism
HCVLipid metabolismInteraction
HCV morphogenesis  
 HCV coreLipid dropletsHCV core localizes around lipid droplets, a crucial event for HCV assembly
 NS5A (replication complex)Lipid dropletsNS5A and other replication complex components are recruited in a core-dependent fashion to lipid droplets. This recruitment is important for HCV morphogenesis
 ?VLDL assembly pathway (ApoB, ApoE and MTP)A functional VLDL assembly pathway was shown to be essential for assembly of infectious particles
HCV particleApoB, ApoE, ApoCI and lipidsApolipoproteins have been shown to be associated with the viral particle. Furthermore, HCV infectivity correlates with the lipid content (density) of the particle
HCV entrySR-BISR-BI is an entry factor for HCV
 LDL-RThe V(LDL) moiety of the HCV particle can interact with the LDL-R
 SAA, an acute phase lipoproteinSAA inhibits HCV entry by interacting with the viral particle
 Plasma membrane lipid compositionAltering the plasma membrane lipid composition prevents HCV entry

Interestingly, the connection between HCV and lipid metabolism is also illustrated by the physiopathology of the infection. Chronic hepatitis C is indeed often associated with accumulation of triacylglycerols in the liver, which is called steatosis (Negro, 2006). HCV may induce steatosis in three ways: impaired lipoprotein secretion, impaired lipid degradation and increased lipogenesis. It has also been shown that infected patients have decreased levels of ApoB and cholesterol in their serum, but they recover upon treatment (Negro, 2006). Moreover, triacylglycerol accumulation in the liver has also been shown to be associated with insulin resistance and diabetes in HCV-infected patients (Serfaty and Capeau, 2009). Therefore a better understanding of the interactions between HCV and lipid metabolism should also help us to understand some of the physiopathological mechanisms that take place during chronic HCV infection. Finally, investigation of the different aspects of the HCV–lipid metabolism connection will also indicate new therapy strategies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

We thank Sophana Ung for his assistance in drawing the Figures, and Dr Birke Tews and Gabrielle Vieyres for a critical reading of this paper.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References

This work was supported by the Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) [grant number 2009-139]; and by the Marie Curie Research Training Network [grant number MRTN-CT-2006-035599]. J.D. is an international scholar of the Howard Hughes Medical Institute (HHMI).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. HCV and lipid droplets
  5. HCV and the VLDL assembly pathway
  6. HCV particles
  7. HCV entry
  8. Conclusions and perspectives
  9. Acknowledgements
  10. Funding
  11. References