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HCV is a major cause of chronic liver disease worldwide. Much has been learned about HCV since its discovery 27 years ago by molecular cloning when standard virological methods failed to identify this important human pathogen. Elaboration of in vitro models enabled studies of the HCV life cycle and a search for inhibitors of viral replication, leading to the development of effective therapies of chronic HCV infection based on direct-acting antivirals with cure rates of more than 95%.
Despite of the tremendous success of HCV therapy and substantial progress in our knowledge of the virus life cycle, the structure, composition and morphogenesis of HCV particles circulating in the blood of infected patients still remain elusive and determination of the ultrastructure of HCV particles by electron microscopy is a significant challenge.
The putative infectious HCV particle consists of a nucleocapsid-containing the single-stranded RNA genome associated with the viral core protein and a lipid bilayer where the viral envelope proteins (E1 and E2) are assembled as heterodimers. In reality, the structure of HCV is more complex and the virus exhibits unusual and striking features. Indeed, a hallmark of HCV particles is their association with host-cell lipids and lipoproteins, mainly very low-density lipoproteins (VLDL) and low-density lipoproteins (LDL).1 ,2 Thus, virus particles in patients' sera have very low buoyant density and are extremely heterogeneous.2 Based on their composition, HCV virions have been called lipo-viro particles (LVPs).1
LVP is a hybrid particle composed of viral components and cell-derived triglyceride-rich lipoproteins including several apolipoproteins (ApoE, ApoB-100 (or ApoB-48), ApoCI, ApoCII, ApoCIII). The lipid constituents integrated in the virus particle define hepatotropism and infectivity and are required for virion stability, cell entry, morphogenesis and release of virus particles. Indeed, the entire HCV life cycle is linked to hepatic lipid metabolism and the production of HCV particles is related to VLDL assembly and release pathways. Moreover, virus-associated lipoproteins (especially ApoE) mask neutralising epitopes of the HCV envelope, thus participating in viral escape mechanisms from the host immune response.
The ultrastructure and composition of HCV particles produced in hepatoma cell lines (HCVcc) have been extensively investigated.3–5 These studies revealed that HCVcc has a unique composition, different from other viruses but similar to VLDL and LDL, with cholesteryl esters as a major component of HCV lipids.4 HCVcc particles are spherical with spike-like projections and sizes ranging from 40 to 100 nm.5 However, serum-derived HCV particles might have different ultrastructure from HCVcc due to higher lipid content. Studies of LVPs by electron microscopy have been limited because of their high lipid content and low viral load in the serum.
In this issue, Piver et al 6 report ultrastructural studies of serum-derived HCV. The authors take advantage of the immunocapture technique, based on coating microscopy grids with antibodies directed against the HCV envelope or virus-associated apolipoproteins, to bind LVPs directly from the serum for analysis by electron microscopy. Immunocapture has been previously used for studies of non-enveloped HCV nucleocapsids isolated from human sera and imaging by electron microscopy7 and for the binding of HCVcc particles to microscopy grids coated with anti-E2 antibodies for analysis by cryoelectron microscopy and tomography.5 Piver et al 6 have combined immunocapture with immunogold antibody labelling to investigate the properties of serum-derived HCV particles by transmission electron microscopy.
Two viral populations were identified: LVPs and lipoprotein-like subviral particles. The LVPs were composed of a nucleocapsid with an electron-dense centre enclosing the HCV RNA genome, and an external detergent-sensitive crescent composed of lipids. Strikingly, nucleocapsids were often detected at the periphery of the virus particle. Immune-electron microscopy analysis revealed the presence of E1 and E2 envelope proteins, and ApoE and ApoB100 at the surface of LVPs. The second viral population was composed of lipoprotein-like particles, with electron-light structure and a smooth surface, which contained the E1 and E2 envelope glycoproteins and ApoE and ApoB but apparently lacked the nucleocapsid. These lipoprotein-like particles correspond most probably to previously described nucleocapsid-free, subviral particles composed of triglyceride-rich lipoproteins and the E1E2 glycoprotein complex, which represent a predominant form of HCV in the blood largely outnumbering the HCV-RNA-positive LVPs.8
The association of HCV particles with lipoproteins renders them heterogeneous and subject to dynamic structural changes.9 Indeed, changes in dietary triglycerides can induce alterations in the physical properties, infectivity and clearance of HCV from the circulation.9 The present findings extend these observations suggesting that differences in blood triglyceride levels can also induce changes in the morphology of LVPs.
Several questions still remain to be answered concerning the various forms of HCV circulating in the blood (shown in figure 1) and their role in chronic infection and pathogenesis. Does the proportion of LVPs and lipoprotein-like particles change during chronic HCV infection? How do these changes influence the effectiveness of current therapies? Considering the relatively low levels of LVPs in the blood8 the question arises, what is the contribution of LVPs to the transmission of infection during chronic hepatitis C as compared with alternative routes of infection mediated by exosomes bearing HCV RNA or direct cell-to-cell virus spread, both independent of neutralising antibodies? What are the benefits for HCV from the production of subviral lipoprotein-like particles6 ,8 or non-enveloped nucleocapsids7 and how do these virus forms contribute to the physiopathology of chronic hepatitis C?
The study by Piver et al 6 provides further evidence of LVP as a putative HCV virion. The reported ultrastructure is compatible with currently proposed LVP models,10 even though infectivity of the characterised LVPs is not directly demonstrated. Nevertheless, this work represents a major step in understanding the ultrastructure of HCV—a still enigmatic causative agent of chronic hepatitis C and confirms the presence of diverse populations of HCV particles in the blood of infected patients. Better understanding the ultrastructural details of the circulating HCV particles would bring a new insight into HCV neutralisation mechanisms and aid the design of future HCV vaccine and therapy.
Competing interests None declared.
Provenance and peer review Commissioned; internally peer reviewed.
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