Human low-density lipoprotein receptor plays an important role in hepatitis B virus infection.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, resulting in chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HBV vaccine is effective to prevent new HBV infection but does not offer therapeutic benefit to hepatitis B patients. Neither are current antiviral drugs curative of chronic hepatitis B. A more thorough understanding of HBV infection and replication holds a great promise for identification of novel antiviral drugs and design of optimal strategies towards the ultimate elimination of chronic hepatitis B. Recently, we have developed a robust HBV cell culture system and discovered that human apolipoprotein E (apoE) is enriched on the HBV envelope and promotes HBV infection and production. In the present study, we have determined the role of the low-density lipoprotein receptor (LDLR) in HBV infection. A LDLR-blocking monoclonal antibody potently inhibited HBV infection in HepG2 cells expressing the sodium taurocholate cotransporting polypeptide (NTCP) as well as in primary human hepatocytes. More importantly, small interfering RNAs (siRNAs)-mediated knockdown of LDLR expression and the CRISPR/Cas9-induced knockout of the LDLR gene markedly reduced HBV infection. A recombinant LDLR protein could block heparin-mediated apoE pulldown, suggesting that LDLR may act as an HBV cell attachment receptor via binding to the HBV-associated apoE. Collectively, these findings demonstrate that LDLR plays an important role in HBV infection probably by serving as a virus attachment receptor.

Human apolipoprotein E promotes hepatitis B virus infection and production.

Hepatitis B virus (HBV) is a common cause of liver diseases, including chronic hepatitis, steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). HBV chronically infects about 240 million people worldwide, posing a major global health problem. The current standard antiviral therapy effectively inhibits HBV replication but does not eliminate the virus unlike direct-acting antivirals (DAA) for curing hepatitis C. Our previous studies have demonstrated that human apolipoprotein E (apoE) plays important roles in hepatitis C virus infection and morphogenesis. In the present study, we have found that apoE is also associated with HBV and is required for efficient HBV infection. An apoE-specific monoclonal antibody was able to capture HBV similar to anti-HBs. More importantly, apoE monoclonal antibody could effectively block HBV infection, resulting in a greater than 90% reduction of HBV infectivity. Likewise, silencing of apoE expression or knockout of apoE gene by CRISPR/Cas9 resulted in a greater than 90% reduction of HBV infection and more than 80% decrease of HBV production, which could be fully restored by ectopic apoE expression. However, apoE silencing or knockout did not significantly affect HBV DNA replication or the production of nonenveloped (naked) nucleocapsids. These findings demonstrate that human apoE promotes HBV infection and production. We speculate that apoE may also play a role in persistent HBV infection by evading host immune response similar to its role in the HCV life cycle and pathogenesis. Inhibitors interfering with apoE biogenesis, secretion, and/or binding to receptors may serve as antivirals for elimination of chronic HBV infection.

Functional Characterization of Apolipoproteins in the HCV Life Cycle.

Apolipoprotein E (apoE) plays dual functions in the HCV life cycle by promoting HCV infection and virion assembly and production. ApoE is a structural component on the HCV envelope. It mediates HCV cell attachment through specific interactions with the cell surface receptors such as syndecan-1 (SDC-1) and SDC-2 heparan sulfate proteoglycans (HSPGs). It also interacts with NS5A and E2, resulting in an enhancement of HCV morphogenesis. It can bind HCV extracellularly and promotes HCV infection. It is critical for HCV cell-to-cell transmission and may also play a role in HCV persistence by interfering with the action of HCV-neutralizing antibodies. Other apolipoproteins particularly apoB and apoC1 were also found on the HCV envelope, but their roles in the HCV life cycle remain unclear. In the last decade, a number of genomic, immunological, structural, and cell biology methodologies have been developed and used for determining the importance of apoE in the HCV life cycle. These methods and protocols will continue to be valuable to further understand the importance and the underlying molecular mechanism of various apolipoproteins in HCV infection and pathogenesis.

Robust Human and Murine Hepatocyte Culture Models of Hepatitis B Virus Infection and Replication.

Hepatitis B virus (HBV) is a major cause of chronic liver diseases, including hepatitis, cirrhosis, and hepatocellular carcinoma. HBV research has been hampered by the lack of robust cell culture and small animal models of HBV infection. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV receptor has been a landmark advance in HBV research in recent years. Ectopic expression of NTCP in nonpermissive HepG2, Huh7, and AML12 cell lines confers HBV susceptibility. However, HBV replication in these human and murine hepatocyte cell lines appeared suboptimal. In the present study, we constructed stable NTCP-expressing HepG2 and AML12 cell lines and found that HBV permissiveness is correlated with NTCP expression. More significantly, we developed robust HBV cell culture models by treating the HBV-infected cells with dimethyl sulfoxide (DMSO) and hydrocortisone, which significantly promoted HBV replication and production. Mechanistic studies suggested that hydrocortisone significantly enhanced the transcription and expression of PGC1α and HNF4α, which are known to promote HBV transcription and replication. These new human and murine hepatocyte culture systems of HBV infection and replication will accelerate the determination of molecular aspects underlying HBV infection, replication, and morphogenesis in human and murine hepatocytes. We anticipate that our HBV cell culture models will also facilitate the discovery and development of antiviral drugs towards the ultimate eradication of chronic hepatitis B virus infection.IMPORTANCE HBV research has been greatly hampered by the lack of robust cell culture and small animal models of HBV infection and propagation. The discovery of NTCP as an HBV receptor has greatly impacted the field of HBV research. Although HBV infection of NTCP-expressing human and murine hepatocyte cell lines has been demonstrated, its replication in cell culture appeared inefficient. To further improve cell culture systems of HBV infection and replication, we constructed NTCP-expressing HepG2 and AML12 cell lines that are highly permissive to HBV infection. More significantly, we found that DMSO and hydrocortisone markedly enhanced HBV transcription and replication in human and murine hepatocytes when added to the cell culture medium. These new cell culture models of HBV infection and replication will facilitate HBV research and antiviral drug discovery towards the ultimate elimination of chronic hepatitis B virus infection.

Identification of cellular genes and pathways important for tumorigenicity of hepatocellular carcinoma cell lines by proteomic profiling.

Hepatocellular carcinoma (HCC) is the most commonly diagnosed malignancy of the liver. A more thorough understanding of HCC pathogenesis will provide novel targets for development of cancer drugs to effectively treat HCC. To further this goal, we carried out a proteomic profiling of HCC cell lines Huh-7.4 and Huh-7.5. These two cell lines were derived from subgenomic HCV RNA-replicating Huh-7 cells upon clearance of HCV RNA by antiviral drug treatment. Initially, the tumorigenicity of each cell line was determined and compared in parallel in the same immunedeficient mice. Strikingly, the Huh-7.4 cell line was able to induce tumors, whereas the Huh-7.5 cell line failed to do so, providing unique model systems for identifying cellular genes and pathways important for HCC development and progression. Subsequently, one-dimensional LC-MS/MS proteomic and bioinformatics analyses were performed in the hope of identifying unique cellular genes and pathways responsible for HCC tumorigenicity. Interestingly, a total of 130 cellular genes were found to be significantly up- or downregulated between these two cell lines (r>3 fold, P<0.001). Also, EIF (EIF2&4), mTOR/p70S6K, ERK5, and EGFR signaling pathways were significantly different. Overall, these results provide significant new information to shed light on the underlying biological processes involved in HCC development and progression.

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.

The Serum Very-Low-Density Lipoprotein Serves as a Restriction Factor against Hepatitis C Virus Infection.

UNLABELLED: Recent studies demonstrated that transgenic mice expressing key human hepatitis C virus (HCV) receptors are susceptible to HCV infection, albeit at very low efficiency. Robust mouse models of HCV infection and replication are needed to determine the importance of host factors in HCV replication, pathogenesis, and carcinogenesis as well as to facilitate the development of antiviral agents and vaccines. The low efficiency of HCV replication in the humanized mouse models is likely due to either the lack of essential host factors or the presence of restriction factors for HCV infection and/or replication in mouse hepatocytes. To determine whether HCV infection is affected by restriction factors present in serum, we examined the effects of mouse and human sera on HCV infectivity. Strikingly, we found that mouse and human sera potently inhibited HCV infection. Mechanistic studies demonstrated that mouse serum blocked HCV cell attachment without significant effect on HCV replication. Fractionation analysis of mouse serum in conjunction with targeted mass spectrometric analysis suggested that serum very-low-density lipoprotein (VLDL) was responsible for the blockade of HCV cell attachment, as VLDL-depleted mouse serum lost HCV-inhibitory activity. Both purified mouse and human VLDL could efficiently inhibit HCV infection. Collectively, these findings suggest that serum VLDL serves as a major restriction factor of HCV infection in vivo. The results also imply that reduction or elimination of VLDL production will likely enhance HCV infection in the humanized mouse model of HCV infection and replication. IMPORTANCE: HCV is a major cause of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Recently, several studies suggested that humanized mouse or transgenic mouse expressing key HCV human receptors became susceptible to HCV infection. However, HCV infection and replication in the humanized animals were very inefficient, suggesting either the lack of cellular genes important for HCV replication or the presence of restriction factors inhibiting HCV infection and replication in the mouse. In this study, we found that both mouse and human sera effectively inhibited HCV infection. Mechanistic studies demonstrated that VLDL is the major restriction factor that blocks HCV infection. These findings suggest that VLDL is beneficial to patients by restricting HCV infection. More importantly, our findings suggest that elimination of VLDL will lead to the development of more robust mouse models for the study of HCV pathogenesis, host response to HCV infection, and evaluation of HCV vaccines.

A Dual-reporter system for real-time monitoring and high-throughput CRISPR/Cas9 library screening of the hepatitis C virus.

The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections.

Characterization of hepatitis C virus interaction with heparan sulfate proteoglycans.

UNLABELLED: Hepatitis C virus (HCV) entry involves binding to cell surface heparan sulfate (HS) structures. However, due to the lipoprotein-like structure of HCV, the exact contribution of virion components to this interaction remains controversial. Here, we investigated the relative contribution of HCV envelope proteins and apolipoprotein E in the HS-binding step. Deletion of hypervariable region 1, a region previously proposed to be involved in HS binding, did not alter HCV virion binding to HS, indicating that this region is not involved in this interaction in the context of a viral infection. Patient sera and monoclonal antibodies recognizing different regions of HCV envelope glycoproteins were also used in a pulldown assay with beads coated with heparin, a close HS structural homologue. Although isolated HCV envelope glycoproteins could interact with heparin, none of these antibodies was able to interfere with the virion-heparin interaction, strongly suggesting that at the virion surface, HCV envelope glycoproteins are not accessible for HS binding. In contrast, results from kinetic studies, heparin pulldown experiments, and inhibition experiments with anti-apolipoprotein E antibodies indicated that this apolipoprotein plays a major role in HCV-HS interaction. Finally, characterization of the HS structural determinants required for HCV infection by silencing of the enzymes involved in the HS biosynthesis pathway and by competition with modified heparin indicated that N- and 6-O-sulfation but not 2-O-sulfation is required for HCV infection and that the minimum HS oligosaccharide length required for HCV infection is a decasaccharide. Together, these data indicate that HCV hijacks apolipoprotein E to initiate its interaction with specific HS structures. IMPORTANCE: Hepatitis C is a global health problem. Hepatitis C virus (HCV) infects approximately 130 million individuals worldwide, with the majority of cases remaining undiagnosed and untreated. In most infected individuals, the virus evades the immune system and establishes a chronic infection. As a consequence, hepatitis C is the leading cause of cirrhosis, end-stage liver disease, hepatocellular carcinoma, and liver transplantation. Virus infection is initiated by entry of the virus into the host cell. In this study, we provide new insights into the viral and cellular determinants involved in the first step of HCV entry, the binding of the virus to host cells. We show that apolipoprotein E is likely responsible for virus binding to heparan sulfate and that N- and 6-O-sulfation of the heparan sulfate proteoglycans is required for HCV infection. In addition, the minimal HS length unit required for HCV infection is a decasaccharide.

Apolipoprotein E mediates attachment of clinical hepatitis C virus to hepatocytes by binding to cell surface heparan sulfate proteoglycan receptors.

Our previous studies demonstrated that the cell culture-grown hepatitis C virus of genotype 2a (HCVcc) uses apolipoprotein E (apoE) to mediate its attachment to the surface of human hepatoma Huh-7.5 cells. ApoE mediates HCV attachment by binding to the cell surface heparan sulfate (HS) which is covalently attached to the core proteins of proteoglycans (HSPGs). In the present study, we further determined the physiological importance of apoE and HSPGs in the HCV attachment using a clinical HCV of genotype 1b (HCV1b) obtained from hepatitis C patients and human embryonic stem cell-differentiated hepatocyte-like cells (DHHs). DHHs were found to resemble primary human hepatocytes. Similar to HCVcc, HCV1b was found to attach to the surface of DHHs by the apoE-mediated binding to the cell surface HSPGs. The apoE-specific monoclonal antibody, purified HSPGs, and heparin were all able to efficiently block HCV1b attachment to DHHs. Similarly, the removal of heparan sulfate from cell surface by treatment with heparinase suppressed HCV1b attachment to DHHs. More significantly, HCV1b attachment was potently inhibited by a synthetic peptide derived from the apoE receptor-binding region as well as by an HSPG-binding peptide. Likewise, the HSPG-binding peptide prevented apoE from binding to heparin in a dose-dependent manner, as determined by an in vitro heparin pull-down assay. Collectively, these findings demonstrate that HSPGs serve as major HCV attachment receptors on the surface of human hepatocytes to which the apoE protein ligand on the HCV envelope binds.

Syndecan-1 serves as the major receptor for attachment of hepatitis C virus to the surfaces of hepatocytes.

Our recent studies demonstrated that apolipoprotein E mediates cell attachment of hepatitis C virus (HCV) through interactions with the cell surface heparan sulfate (HS). HS is known to covalently attach to core proteins to form heparan sulfate proteoglycans (HSPGs) on the cell surface. The HSPG core proteins include the membrane-spanning syndecans (SDCs), the lycosylphosphatidylinositol-linked glypicans (GPCs), the basement membrane proteoglycan perlecan (HSPG2), and agrin. In the present study, we have profiled each of the HSPG core proteins in HCV attachment. Substantial evidence derived from our studies demonstrates that SDC1 is the major receptor protein for HCV attachment. The knockdown of SDC1 expression by small interfering RNA (siRNA)-induced gene silence resulted in a significant reduction of HCV attachment to Huh-7.5 cells and stem cell-differentiated human hepatocytes. The silence of SDC2 expression also caused a modest decrease of HCV attachment. In contrast, the siRNA-mediated knockdown of other SDCs, GPCs, HSPG2, and agrin had no effect on HCV attachment. More importantly, ectopic expression of SDC1 was able to completely restore HCV attachment to Huh-7.5 cells in which the endogenous SDC1 expression was silenced by specific siRNAs. Interestingly, mouse SDC1 is also fully functional in mediating HCV attachment when expressed in the SDC1-deficient cells, consistent with recent reports that mouse hepatocytes are also susceptible to HCV infection when expressing other key HCV receptors. Collectively, our findings demonstrate that SDC1 serves as the major receptor protein for HCV attachment to cells, providing another potential target for discovery and development of antiviral drugs against HCV.

Ribosomal protein S25 dependency reveals a common mechanism for diverse internal ribosome entry sites and ribosome shunting.

During viral infection or cellular stress, cap-dependent translation is shut down. Proteins that are synthesized under these conditions use alternative mechanisms to initiate translation. This study demonstrates that at least two alternative translation initiation routes, internal ribosome entry site (IRES) initiation and ribosome shunting, rely on ribosomal protein S25 (RPS25). This suggests that they share a mechanism for initiation that is not employed by cap-dependent translation, since cap-dependent translation is not affected by the loss of RPS25. Furthermore, we demonstrate that viruses that utilize an IRES or a ribosome shunt, such as hepatitis C virus, poliovirus, or adenovirus, have impaired amplification in cells depleted of RPS25. In contrast, viral amplification of a virus that relies solely on cap-dependent translation, herpes simplex virus, is not hindered. We present a model that explains how RPS25 can be a nexus for multiple alternative translation initiation pathways.

Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules.

Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm.

Hepatitis C virus inhibits AKT-tuberous sclerosis complex (TSC), the mechanistic target of rapamycin (MTOR) pathway, through endoplasmic reticulum stress to induce autophagy.

Hepatitis C virus (HCV) is able to induce autophagy via endoplasmic reticulum (ER) stress, but the exact molecular signaling pathway is not well understood. We found that the activity of the mechanistic target of rapamycin complex 1 (MTORC1) was inhibited in Huh7 cells either harboring HCV-N (genotype 1b) full-genomic replicon or infected with JFH1 (genotype 2a) virus, which led to the activation of UNC-51-like kinase 1 (ULK1) and thus to autophagy. We then analyzed activity upstream of MTORC1, and found that both protein kinase, AMP-activated, α (PRKAA, including PRKAA1 and PRKAA2, also known as AMP-activated protein kinase, AMPKα) and AKT (refers to pan AKT, including three isoforms of AKT1-3, also known as protein kinase B, PKB) were inhibited by HCV infection. The inhibition of the AKT-TSC-MTORC1 pathway contributed to upregulating autophagy, but inhibition of PRKAA downregulated autophagy. The net effect on autophagy was from AKT, which overrode the inhibition effect from PRKAA. It was further found that HCV-induced ER stress was responsible for the inhibition of the AKT pathway. Metformin, a PRKAA agonist, inhibited HCV replication not only by activating PRKAA as previously reported, but also by activating AKT independently of the autophagy pathway. Taken together, our data suggested HCV inhibited the AKT-TSC-MTORC1 pathway via ER stress, resulting in autophagy, which may contribute to the establishment of the HCV-induced autophagy.

Transient activation of the PI3K-AKT pathway by hepatitis C virus to enhance viral entry.

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed against CD81 and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction between HCV E2 and its two co-receptors CD81 and claudin-1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibited HCV infection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3K-AKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients.

Cell culture-adaptive mutations promote viral protein-protein interactions and morphogenesis of infectious hepatitis C virus.

Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demonstrated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCV morphogenesis is poorly understood. We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 orders of magnitude higher than that of wild-type virus. Sequence analysis identified a total of 16 amino acid mutations in core (C), E1, NS2, NS3, NS5A, and NS5B, with the majority of mutations clustered in NS5A. Reverse genetic analysis of these mutations individually or in different combinations demonstrated that amino acid mutations in NS2 and NS5A markedly enhanced HCV production. Additionally, mutations in C, E1, NS3, and NS5B synergistically promoted HCV production in the background of NS2 and NS5A mutations. Adaptive mutations in NS5A domains I, II, and III independently enhanced HCV production, suggesting that all three domains of NS5A are important for HCV morphogenesis. More importantly, adaptive mutations greatly enhanced physical interactions among HCV structural and NS proteins, as determined by studies with coimmunoprecipitation and mammalian two-hybrid assays. Collectively, these findings demonstrate that adaptive mutations can enhance specific protein-protein interactions among viral structural and NS proteins and therefore promote the assembly of infectious HCV particles.

Hepatitis C virus attachment mediated by apolipoprotein E binding to cell surface heparan sulfate.

Viruses are known to use virally encoded envelope proteins for cell attachment, which is the very first step of virus infection. In the present study, we have obtained substantial evidence demonstrating that hepatitis C virus (HCV) uses the cellular protein apolipoprotein E (apoE) for its attachment to cells. An apoE-specific monoclonal antibody was able to efficiently block HCV attachment to the hepatoma cell line Huh-7.5 as well as primary human hepatocytes. After HCV bound to cells, however, anti-apoE antibody was unable to inhibit virus infection. Conversely, the HCV E2-specific monoclonal antibody CBH5 did not affect HCV attachment but potently inhibited HCV entry. Similarly, small interfering RNA-mediated knockdown of the key HCV receptor/coreceptor molecules CD81, claudin-1, low-density lipoprotein receptor (LDLr), occludin, and SR-BI did not affect HCV attachment but efficiently suppressed HCV infection, suggesting their important roles in HCV infection at postattachment steps. Strikingly, removal of heparan sulfate from the cell surface by treatment with heparinase blocked HCV attachment. Likewise, substitutions of the positively charged amino acids with neutral or negatively charged residues in the receptor-binding region of apoE resulted in a reduction of apoE-mediating HCV infection. More importantly, mutations of the arginine and lysine to alanine or glutamic acid in the receptor-binding region ablated the heparin-binding activity of apoE, as determined by an in vitro heparin pulldown assay. HCV attachment could also be inhibited by a synthetic peptide derived from the apoE receptor-binding region. Collectively, these findings demonstrate that apoE mediates HCV attachment through specific interactions with cell surface heparan sulfate.

Hepatitis C virus infects the endothelial cells of the blood-brain barrier.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to progressive liver disease and is associated with a variety of extrahepatic syndromes, including central nervous system (CNS) abnormalities. However, it is unclear whether such cognitive abnormalities are a function of systemic disease, impaired hepatic function, or virus infection of the CNS. METHODS: We measured levels of HCV RNA and expression of the viral entry receptor in brain tissue samples from 10 infected individuals (and 3 uninfected individuals, as controls) and human brain microvascular endothelial cells by using quantitative polymerase chain reaction and immunochemical and confocal imaging analyses. HCV pseudoparticles and cell culture-derived HCV were used to study the ability of endothelial cells to support viral entry and replication. RESULTS: Using quantitative polymerase chain reaction, we detected HCV RNA in brain tissue of infected individuals at significantly lower levels than in liver samples. Brain microvascular endothelia and brain endothelial cells expressed all of the recognized HCV entry receptors. Two independently derived brain endothelial cell lines, hCMEC/D3 and HBMEC, supported HCV entry and replication. These processes were inhibited by antibodies against the entry factors CD81, scavenger receptor BI, and claudin-1; by interferon; and by reagents that inhibit NS3 protease and NS5B polymerase. HCV infection promotes endothelial permeability and cellular apoptosis. CONCLUSIONS: Human brain endothelial cells express functional receptors that support HCV entry and replication. Virus infection of the CNS might lead to HCV-associated neuropathologies.

Inhibition of hepatitis C virus replication using adeno-associated virus vector delivery of an exogenous anti-hepatitis C virus microRNA cluster.

UNLABELLED: RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 × 10(11) vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. CONCLUSION: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens.