Roles of human apolipoprotein E in the infectivity and replication of hepatitis C virus genotype 2a

Hepatitis C virus (HCV) infection is associated with lipoproteins, and apolipoprotein E (apoE) plays an essential role in infectious HCV particles. Although the role of apoE in HCV infection is well known, its role in the replication of HCV remains unclear. The aims of this study were to determine the role of apoE in the RNA replication of major HCV genotypes 1b and 2a, and to determine whether this role is HCVgenotype-dependent using HCV genotype 1b replicon cells and HCV genotype 2a producing (HP) cells. HCV infection was blocked in Huh7.5 cells treated with low-density lipoproteins, very low-density lipoproteins, or apoE3. An apoE3-specific monoclonal antibody also efficiently neutralized HCV infectivity, and HCV infection was dramatically suppressed by the knockdown of apoE expression with an apoE-specific small interfering RNA, suggesting a requirement for apoE in infectious HCV particles. HCV RNA replication was not affected in HP cells treated with each apoE isoform or transfected with apoE-specific siRNAs. However, the knockdown of apoE expression suppressed RNA replication of HCV genotype 1b. The siRNA-mediated knockdown of apoE, apoA1, and apoB expression also suppressed the RNA replication of HCV genotype 1b, but not that of HCV genotype 2a. Taken together, these findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a. These results provide important information for the future development of HCV-genotypespecific anti-HCV agents.     

Apolipoprotein E but Not B Is Required for the Formation of Infectious Hepatitis C Virus Particles

Our previous studies have found that hepatitis C virus (HCV) particles are enriched in apolipoprotein E (apoE) and that apoE is required for HCV infectivity and production. Studies by others, however, suggested that both microsomal transfer protein (MTP) and apoB are important for HCV production. To define the roles of apoB and apoE in the HCV life cycle, we developed a single-cycle HCV growth assay to determine the correlation of HCV assembly with apoB and apoE expression, as well as the influence of MTP inhibitors on the formation of HCV particles. The small interfering RNA (siRNA)-mediated knockdown of apoE expression remarkably suppressed the formation of HCV particles. However, apoE expressed ectopically could restore the defect of HCV production posed by the siRNA-mediated knockdown of endogenous apoE expression. In contrast, apoB-specific antibodies and siRNAs had no significant effect on HCV infectivity and production, respectively, suggesting that apoB does not play a significant role in the HCV life cycle. Additionally, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked secretion of apoB-containing lipoproteins but did not affect HCV production unless apoE expression and secretion were inhibited. At higher concentrations, however, MTP inhibitors blocked apoE expression and secretion and consequently suppressed the formation of HCV particles. Furthermore, apoE was found to be sensitive to trypsin digestion and to interact with NS5A in purified HCV particles and HCV-infected cells, as demonstrated by coimmunoprecipitation. Collectively, these findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.Hepatitis C virus (HCV) is the leading cause of chronic viral hepatitis, affecting approximately 170 million people worldwide (8, 40). HCV coinfection with human immunodeficiency virus (HIV) is also common, occurring overall in 25 to 30% of HIV-positive persons (1). Individuals with chronic HCV infection are at high risk for the development of cirrhosis and hepatocellular carcinoma. A pegylated interferon and ribavirin combination is the standard therapy to treat hepatitis C but suffers from limited efficacy (<50% antiviral response among patients infected with the dominant genotype 1 HCV) and severe side effects (18, 27). More efficacious and safer antiviral drugs for effective treatment of hepatitis C are urgently needed. A thorough understanding of the HCV life cycle will likely provide novel targets for antiviral drug discovery and development to control HCV infection.HCV is an enveloped RNA virus containing a single-stranded, positive-sense RNA genome and is classified as a Hepacivirus in the Flaviviridae family (11, 33). The viral RNA genome carries a single open reading frame flanked by untranslated regions (UTRs) at both the 5′ and 3′ ends. The 5′ and 3′ UTRs contain cis-acting RNA elements important for the initiation of HCV polyprotein translation and viral RNA replication (24). Upon translation, the HCV polyprotein precursor is proteolytically processed by cellular peptidases and viral proteases into at least 10 different viral proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Studies with subgenomic HCV RNAs demonstrated that the NS3 to NS5B proteins, in association with intracellular membranes and cellular proteins, are essential and sufficient for HCV RNA replication in the cell (5, 14, 25). The newly synthesized HCV proteins and RNA genome are assembled to form progeny HCV particles by undetermined mechanisms.Our earlier work found that infectious HCV particles are highly enriched in apolipoprotein E (apoE), which is a major determinant of HCV infectivity and production in cell culture (10). ApoE-specific monoclonal antibodies (MAbs) effectively neutralized HCV infectivity, in a dose-dependent manner. The knockdown of apoE expression by specific small interfering RNA (siRNA) remarkably suppressed HCV production, suggesting that apoE is also important for the formation of infectious particles and/or egression (10). However, studies by others suggested that HCV assembly and production are dependent on microsomal transfer protein (MTP) and apolipoprotein B (apoB), both of which are essential components required for the assembly and secretion of very-low-density lipoproteins (VLDLs) (19, 21). In those studies, both apoB-specific siRNAs and MTP inhibitors were found to suppress HCV production (19, 21). It was speculated that HCV shares the same assembly and secretion pathway with VLDLs.To define the roles of apoB and apoE in the formation of HCV particles and egression, we developed a single-cycle HCV growth assay. Using this assay system, we have demonstrated that apoE but not apoB is required for the infectivity and formation of infectious HCV particles. First of all, apoB-specific MAb and polyclonal antibodies did not affect HCV infection. Additionally, apoE-specific siRNA potently inhibited the formation of infectious HCV particles, whereas HCV production was unaffected by the siRNA-mediated knockdown of apoB expression. Furthermore, two MTP inhibitors, CP-346086 and BMS-2101038, efficiently blocked apoB secretion but did not significantly affect HCV production prior to the blockage of apoE expression/secretion. At higher concentrations, however, both MTP inhibitors blocked apoE secretion and consequently suppressed the formation of infectious HCV particles. To further understand the role of apoE in HCV assembly, we carried out coimmunoprecipitation (co-IP) experiments and found that apoE-specific MAb pulled down NS5A but not other HCV proteins from lysed HCV particles, suggesting a specific interaction between apoE and NS5A during the formation of infectious HCV particles. Collectively, our findings demonstrate that apoE but not apoB is required for HCV assembly, probably via a specific interaction with NS5A.     

Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor

Several cell surface molecules have been proposed as receptor candidates, mediating cell entry of hepatitis C virus (HCV) on the basis of their physical association with virions or with soluble HCV E2 glycoproteins. However, due to the lack of infectious HCV particles, evidence that these receptor candidates support infection was missing. Using our recently described infectious HCV pseudotype particles (HCVpp) that display functional E1E2 glycoprotein complexes, here we show that HCV is a pH-dependent virus, implying that its receptor component(s) mediate virion internalization by endocytosis. Expression of the CD81 tetraspanin in non-permissive CD81-negative hepato-carcinoma cells was sufficient to restore susceptibility to HCVpp infection, confirming its critical role as a cell attachment factor. As a cell surface molecule likely to mediate endosomal trafficking, we demonstrate that the human scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein-internalization molecule that we previously proposed as a novel HCV receptor candidate due to its affinity with E2 glycoproteins, is required for infection of CD81-expressing hepatic cells. By receptor competition assays, we found that SR-B1 antibodies that blocked binding of soluble E2 could prevent HCVpp infectivity. Furthermore, we establish that the hyper-variable region 1 of the HCV E2 glycoprotein is a critical determinant mediating entry in SR-B1-positive cells. Finally, by correlating expression of HCV receptors and infectivity, we suggest that, besides CD81 and SR-B1, additional hepatocyte-specific co-factor(s) are necessary for HCV entry.     

Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection

In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.     

Biochemical and morphological properties of hepatitis C virus particles and determination of their lipidome

A hallmark of hepatitis C virus (HCV) particles is their association with host cell lipids, most notably lipoprotein components. It is thought that this property accounts for the low density of virus particles and their large heterogeneity. However, the composition of infectious virions and their biochemical and morphological properties are largely unknown. We developed a system in which the envelope glycoprotein E2 was N-terminally tagged with a FLAG epitope. This virus, designated Jc1E2(FLAG), produced infectivity titers to wild type levels and allowed affinity purification of virus particles that were analyzed for their protein and lipid composition. By using mass spectrometry, we found the lipid composition of Jc1E2(FLAG) particles to resemble the one very low- and low density-lipoprotein with cholesteryl esters accounting for almost half of the total HCV lipids. Thus, HCV particles possess a unique lipid composition that is very distinct from all other viruses analyzed so far and from the human liver cells in which HCV was produced. By electron microscopy (EM), we found purified Jc1E2(FLAG) particles to be heterogeneous, mostly spherical structures, with an average diameter of about 73 nm. Importantly, the majority of E2-containing particles also contained apoE on their surface as assessed by immuno-EM. Taken together, we describe a rapid and efficient system for the production of large quantities of affinity-purified HCV allowing a comprehensive analysis of the infectious virion, including the determination of its lipid composition.     

Apolipoprotein E Codetermines Tissue Tropism of Hepatitis C Virus and Is Crucial for Viral Cell-to-Cell Transmission by Contributing to a Postenvelopment Step of Assembly

Hepatitis C virus (HCV) predominantly infects human hepatocytes, although extrahepatic virus reservoirs are being discussed. Infection of cells is initiated via cell-free and direct cell-to-cell transmission routes. Cell type-specific determinants of HCV entry and RNA replication have been reported. Moreover, several host factors required for synthesis and secretion of lipoproteins from liver cells, in part expressed in tissue-specific fashion, have been implicated in HCV assembly. However, the minimal cell type-specific requirements for HCV assembly have remained elusive. Here we report that production of HCV trans-complemented particles (HCV_TCP) from nonliver cells depends on ectopic expression of apolipoprotein E (ApoE). For efficient virus production by full-length HCV genomes, microRNA 122 (miR-122)-mediated enhancement of RNA replication is additionally required. Typical properties of cell culture-grown HCV (HCVcc) particles from ApoE-expressing nonliver cells are comparable to those of virions derived from human hepatoma cells, although specific infectivity of virions is modestly reduced. Thus, apolipoprotein B (ApoB), microsomal triglyceride transfer protein (MTTP), and apolipoprotein C1 (ApoC1), previously implicated in HCV assembly, are dispensable for production of infectious HCV. In the absence of ApoE, release of core protein from infected cells is reduced, and production of extracellular as well as intracellular infectivity is ablated. Since envelopment of capsids was not impaired, we conclude that ApoE acts after capsid envelopment but prior to secretion of infectious HCV. Remarkably, the lack of ApoE also abrogated direct HCV cell-to-cell transmission. These findings highlight ApoE as a host factor codetermining HCV tissue tropism due to its involvement in a late assembly step and viral cell-to-cell transmission.     

Characterization of functional hepatitis C virus envelope glycoproteins

Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2, that assemble as a noncovalent heterodimer which is mainly retained in the endoplasmic reticulum. Because assembly into particles and secretion from the cell lead to structural changes in viral envelope proteins, characterization of the proteins associated with the virion is necessary in order to better understand how they mature to be functional in virus entry. There is currently no efficient and reliable cell culture system to amplify HCV, and the envelope glycoproteins associated with the virion have therefore not been characterized yet. Recently, infectious pseudotype particles that are assembled by displaying unmodified HCV envelope glycoproteins on retroviral core particles have been successfully generated. Because HCV pseudotype particles contain fully functional envelope glycoproteins, these envelope proteins, or at least a fraction of them, should be in a mature conformation similar to that on the native HCV particles. In this study, we used conformation-dependent monoclonal antibodies to characterize the envelope glycoproteins associated with HCV pseudotype particles. We showed that the functional unit is a noncovalent E1E2 heterodimer containing complex or hybrid type glycans. We did not observe any evidence of maturation by a cellular endoprotease during the transport of these envelope glycoproteins through the secretory pathway. These envelope glycoproteins were recognized by a panel of conformation-dependent monoclonal antibodies as well as by CD81, a molecule involved in HCV entry. The functional envelope glycoproteins associated with HCV pseudotype particles were also shown to be sensitive to low-pH treatment. Such conformational changes are likely necessary to initiate fusion.     

C-type lectins L-SIGN and DC-SIGN capture and transmit infectious hepatitis C virus pseudotype particles

The molecular mechanisms involved in the hepatic tropism of hepatitis C virus (HCV) have not been identified. We have shown previously that liver-expressed C-type lectins L-SIGN and DC-SIGN bind the HCV E2 glycoprotein with high affinity (Lozach, P. Y., Lortat-Jacob, H., de Lacroix de Lavalette, A., Staropoli, I., Foung, S., Amara, A., Houles, C., Fieschi, F., Schwartz, O., Virelizier, J. L., Arenzana-Seisdedos, F., and Altmeyer, R. (2003) J. Biol. Chem. 278, 20358-20366). To analyze the functional relevance of this interaction, we generated pseudotyped lentivirus particles presenting HCV glycoproteins E1 and E2 at the virion surface (HCV-pp). High mannose N-glycans are present on E1 and, to a lesser extent, on E2 proteins of mature infectious HCV-pp. Such particles bind to both L-SIGN and DC-SIGN, but they cannot use these receptors for entry into cells. However, infectious virus is transmitted efficiently when permissive Huh-7 cells are cocultured with HCV-pp bound to L-SIGN or to DC-SIGN-positive cell lines. HCV-pp transmission via L-SIGN or DC-SIGN is inhibited by characteristic inhibitors such as the calcium chelator EGTA and monoclonal antibodies directed against lectin carbohydrate recognition domains of both lectins. In support of the biological relevance of this phenomenon, dendritic cells expressing endogenous DC-SIGN transmitted HCV-pp with high efficiency in a DC-SIGN-dependent manner. Our results support the hypothesis that C-type lectins such as the liver sinusoidal endothelial cell-expressed L-SIGN could act as a capture receptor for HCV in the liver and transmit infectious virions to neighboring hepatocytes.     

Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies

Virion capture assays, in which immobilized antibodies (Abs) capture virus particles, have been used to suggest that nonneutralizing Abs bind effectively to human immunodeficiency virus type 1 (HIV-1) primary viruses. Here, we show that virion capture assays, under conditions commonly reported in the literature, give a poor indication of epitope expression on the surface of infectious primary HIV-1. First, estimation of primary HIV-1 capture by p24 measurements shows a very poor correlation with an estimation based on infectivity measurements. Second, virion capture appears to require relatively low Ab affinity for the virion, as shown by the ability of a monoclonal Ab to capture a wild-type and a neutralization escape variant virus equally well. Nevertheless, in a more interpretable competition format, it is shown that nonneutralizing anti-CD4 binding site (CD4bs) Abs compete with a neutralizing anti-CD4bs Ab (b12) for virus capture, suggesting that the nonneutralizing anti-CD4bs Abs are able to bind to the envelope species that is involved in virion capture in these experiments. However, the nonneutralizing anti-CD4bs Abs do not inhibit neutralization by b12 even at considerable excess. This suggests that the nonneutralizing Abs are unable to bind effectively to the envelope species required for virus infectivity. The results were obtained for three different primary virus envelopes. The explanation that we favor is that infectious HIV-1 primary virions can express two forms of gp120, an accessible nonfunctional form and a functional form with limited access. Binding to the nonfunctional form, which needs only to be present at relatively low density on the virion, permits capture but does not lead to neutralization. The expression of a nonfunctional but accessible form of gp120 on virions may contribute to the general failure of HIV-1 infection to elicit cross-neutralizing Abs and may represent a significant problem for vaccines based on viruses or virus-like particles.     

High density lipoprotein inhibits hepatitis C virus-neutralizing antibodies by stimulating cell entry via activation of the scavenger receptor BI

Hepatitis C virus (HCV) exploits serum-dependent mechanisms that inhibit neutralizing antibodies. Here we demonstrate that high density lipoprotein (HDL) is a key serum factor that attenuates neutralization by monoclonal and HCV patient-derived polyclonal antibodies of infectious pseudo-particles (HCVpp) harboring authentic E1E2 glycoproteins and cell culture-grown genuine HCV (HCVcc). Over 10-fold higher antibody concentrations are required to neutralize either HCV-enveloped particles in the presence of HDL or human serum, and less than 3-5-fold reduction of infectious titers are obtained at saturating antibody concentrations, in contrast to complete inhibition in serum-free conditions. We show that HDL interaction with the scavenger receptor BI (SR-BI), a proposed cell entry co-factor of HCV and a receptor mediating lipid transfer with HDL, strongly reduces neutralization of HCVpp and HCVcc. We found that HDL activation of target cells strongly stimulates cell entry of viral particles by accelerating their endocytosis, thereby suppressing a 1-h time lag during which cell-bound virions are not internalized and can be targeted by antibodies. Compounds that inhibit lipid transfer functions of SR-BI fully restore neutralization by antibodies in human serum. We demonstrate that this functional HDL/SR-BI interaction only interferes with antibodies blocking HCV-E2 binding to CD81, a major HCV receptor, reflecting its prominent role during the cell entry process. Moreover, we identify monoclonal antibodies targeted to epitopes in the E1E2 complex that are not inhibited by HDL. Consistently, we show that antibodies targeted to HCV-E1 efficiently neutralize HCVpp and HCVcc in the presence of human serum.     

A conformational change in Sindbis virus glycoproteins E1 and E2 is detected at the plasma membrane as a consequence of early virus-cell interaction.

A conformational change in the structure of Sindbis (SB) virus was detected after virion attachment to baby hamster kidney cells but before internalization. The alteration was manifested as increased virion binding of certain glycoprotein E1 and E2 monoclonal antibodies (MAbs) that recognized transitional epitopes. These epitopes were inaccessible to MAb on native virions but became accessible to their cognate MAbs in the early stages of infection. Transit of virions through a low-pH compartment apparently was not required for the conformational change. Exposure of transitional epitopes was unaffected by treatment of BHK cells with NH4Cl and occurred normally in Chinese hamster ovary cells temperature sensitive for endosomal acidification. However, the rearrangement was correlated with both the time course and temperature dependence of SB virus penetration, and the rearrangement occurred earlier with an SB virus mutant having an accelerated penetration phenotype. In addition, MAb to a transitional epitope, a probe specific for rearranged particles, retarded penetration of infectious virions. These results suggested that the SB virus E1/E2 glycoprotein spike undergoes a structural rearrangement as a consequence of virion interaction with the cell surface and that this altered virion form may be an important early intermediate in an entry pathway leading to productive infection.     

How does hepatitis C virus enter cells?

Hepatitis C virus (HCV) exists in different forms in the circulation of infected people: lipoprotein bound and lipoprotein free, enveloped and nonenveloped. Viral particles with the highest infectivity are associated with lipoproteins, whereas lipoprotein-free virions are poorly infectious. The detection of HCV's envelope proteins E1 and E2 in lipoprotein-associated virions has been challenging. Because lipoproteins are readily endocytosed, some forms of HCV might utilize their association with lipoproteins rather than E1 and E2 for cell attachment and internalization. However, vaccination of chimpanzees with recombinant envelope proteins protected the animals from hepatitis C infection, suggesting an important role for E1 and E2 in cell entry. It seems possible that different forms of HCV use different receptors to attach to and enter cells. The putative receptors and the assays used for their validation are discussed in this review.     

Definition of a conserved immunodominant domain on hepatitis C virus E2 glycoprotein by neutralizing human monoclonal antibodies

Development of a successful hepatitis C virus (HCV) vaccine requires the definition of neutralization epitopes that are conserved among different HCV genotypes. Five human monoclonal antibodies (HMAbs) are described that cross-compete with other antibodies to a cluster of overlapping epitopes, previously designated domain B. Each HMAb broadly neutralizes retroviral pseudotype particles expressing HCV E1 and E2 glycoproteins, as well as the infectious chimeric genotype 1a and genotype 2a viruses. Alanine substitutions of residues within a region of E2 involved in binding to CD81 showed that critical E2 contact residues involved in the binding of representative antibodies are identical to those involved in the binding of E2 to CD81.     

Characteristics of Monoclonal Antibodies to Lucerne Transient Streak Sobemovirus and Their Use in Epitope Localization

Murine monoclonal antibodies (mAbs) to lucerne transient streak sobemovirus (LTSV) were used as probes for examining the virion capsid organization. A panel of nine mAbs from approximately 100 hybridomas were chosen for this study. All of these mAbs interacted effectively with sites on intact capsid and isolated coat protein sub-units (i.e. metatopes) in ELISA and Western blots. Only one of these mAbs reacted with virions in gel diffusion test producing a visible precipitin band. Competitive binding assays showed that these mAbs recognized the same or very similar metatopes. None of the mAbs neutralized LTSV virion infectivity but rabbit polyclonal antibodies drastically reduced infectivity. Treatment of virions with EDTA (swollen LTSV). protein crosslinking reagents or sodium dodecyl sulphate caused no detectable alterations in their reactivities with these mAbs, The binding sites for monoclonal and polyclonal antibodies were located on denatured 5.5 kDa fragments resulting from partial trypsinization of LTSV coat protein and 8 kDa fragments formed with chymopapain proteolysis. These results indicate that these LTSV epitopes are of linear (or sequential) configuration and are located on the exposed, structurally stable domain of the virion capsid.     

Characterization of Hepatitis C Virus Particle Subpopulations Reveals Multiple Usage of the Scavenger Receptor BI for Entry Steps

Hepatitis C virus (HCV) particles assemble along the very low density lipoprotein pathway and are released from hepatocytes as entities varying in their degree of lipid and apolipoprotein (apo) association as well as buoyant densities. Little is known about the cell entry pathway of these different HCV particle subpopulations, which likely occurs by regulated spatiotemporal processes involving several cell surface molecules. One of these molecules is the scavenger receptor BI (SR-BI), a receptor for high density lipoprotein that can bind to the HCV glycoprotein E2. By studying the entry properties of infectious virus subpopulations differing in their buoyant densities, we show that these HCV particles utilize SR-BI in a manifold manner. First, SR-BI mediates primary attachment of HCV particles of intermediate density to cells. These initial interactions involve apolipoproteins, such as apolipoprotein E, present on the surface of HCV particles, but not the E2 glycoprotein, suggesting that lipoprotein components in the virion act as host-derived ligands for important entry factors such as SR-BI. Second, we found that in contrast to this initial attachment, SR-BI mediates entry of HCV particles independent of their buoyant density. This function of SR-BI does not depend on E2/SR-BI interaction but relies on the lipid transfer activity of SR-BI, probably by facilitating entry steps along with other HCV entry co-factors. Finally, our results underscore a third function of SR-BI governed by specific residues in hypervariable region 1 of E2 leading to enhanced cell entry and depending on SR-BI ability to bind to E2.     

Characterization of low- and very-low-density hepatitis C virus RNA-containing particles

The presence of hepatitis C virus (HCV) RNA-containing particles in the low-density fractions of plasma has been associated with high infectivity. However, the nature of circulating HCV particles and their association with immunoglobulins or lipoproteins as well as the characterization of cell entry have all been subject to conflicting reports. For a better analysis of HCV RNA-containing particles, we quantified HCV RNA in the low-density fractions of plasma corresponding to the very-low-density lipoprotein (VLDL), intermediate-density lipoprotein, and low-density lipoprotein (LDL) fractions from untreated chronically HCV-infected patients. HCV RNA was always found in at least one of these fractions and represented 8 to 95% of the total plasma HCV RNA. Surprisingly, immunoglobulins G and M were also found in the low-density fractions and could be used to purify the HCV RNA-containing particles (lipo-viro-particles [LVP]). Purified LVP were rich in triglycerides; contained at least apolipoprotein B, HCV RNA, and core protein; and appeared as large spherical particles with a diameter of more than 100 nm and with internal structures. Delipidation of these particles resulted in capsid-like structures recognized by anti-HCV core protein antibody. Purified LVP efficiently bind and enter hepatocyte cell lines, while serum or whole-density fractions do not. Binding of these particles was competed out by VLDL and LDL from noninfected donors and was blocked by anti-apolipoprotein B and E antibodies, whereas upregulation of the LDL receptor increased their internalization. These results suggest that the infectivity of LVP is mediated by endogenous proteins rather than by viral components providing a mechanism of escape from the humoral immune response.     

Identification of a residue in hepatitis C virus E2 glycoprotein that determines scavenger receptor BI and CD81 receptor dependency and sensitivity to neutralizing antibodies

Hepatitis C virus (HCV) infection is dependent on at least three coreceptors: CD81, scavenger receptor BI (SR-BI), and claudin-1. The mechanism of how these molecules coordinate HCV entry is unknown. In this study we demonstrate that a cell culture-adapted JFH-1 mutant, with an amino acid change in E2 at position 451 (G451R), has a reduced dependency on SR-BI. This altered receptor dependency is accompanied by an increased sensitivity to neutralization by soluble CD81 and enhanced binding of recombinant E2 to cell surface-expressed and soluble CD81. Fractionation of HCV by density gradient centrifugation allows the analysis of particle-lipoprotein associations. The cell culture-adapted mutation alters the relationship between particle density and infectivity, with the peak infectivity occurring at higher density than the parental virus. No association was observed between particle density and SR-BI or CD81 coreceptor dependence. JFH-1 G451R is highly sensitive to neutralization by gp-specific antibodies, suggesting increased epitope exposure at the virion surface. Finally, an association was observed between JFH-1 particle density and sensitivity to neutralizing antibodies (NAbs), suggesting that lipoprotein association reduces the sensitivity of particles to NAbs. In summary, mutation of E2 at position 451 alters the relationship between particle density and infectivity, disrupts coreceptor dependence, and increases virion sensitivity to receptor mimics and NAbs. Our data suggest that a balanced interplay between HCV particles, lipoprotein components, and viral receptors allows the evasion of host immune responses.     

Partial inhibition of the human immunodeficiency virus type 1 protease results in aberrant virus assembly and the formation of noninfectious particles.

The production of infectious particles by human immunodeficiency virus type 1 is dependent on the accurate cleavage of its Gag and Gag/Pol precursors by a virally encoded protease. In the absence of protease activity, morphologically abnormal particles which are noninfectious are formed. Recently, inhibitors of the protease of human immunodeficiency virus type 1 have been developed as potential therapeutic agents. We have examined the basis for the loss of infectivity at the limiting inhibitor concentrations that are likely to be achieved in clinical settings. We found that subtle defects in processing are correlated with profound deficits in infectivity. Further, we correlated this partially disrupted processing with an altered virion morphology. These data suggest that accurate and complete processing is essential to the formation of infectious, morphologically normal virions and that the pathway by which these precursors are processed and assembled is sensitive to partial inhibition of the protease by an inhibitor disproportionate to the effect of the inhibitor on the viral protease itself.