A human claudin-1-derived peptide inhibits hepatitis C virus entry

Hepatitis C virus (HCV) entry is a complicated process that requires multiple host factors, such as CD81, scavenger receptor BI, claudin-1 (CLDN1), and occludin. The interaction of virus and cellular entry factors represents a promising target for novel anti-HCV drug development. In this study, we sought to identify peptide inhibitors for HCV entry by screening a library of overlapping peptides covering the four above-mentioned entry factors. An 18-amino acid peptide (designated as CL58) that was derived from the CLDN1 intracellular and first transmembrane region inhibited both de novo and established HCV infection in vitro. Unlike previously reported peptides corresponding to CLDN1 extracellular loops, CL58 did not alter the normal distribution of CLDN1 and was not cytotoxic in vitro at concentrations nearly 100-fold higher than the effective antiviral dose. The inhibitory effect of CL58 appeared to occur at a late step during viral entry, presumably after initial binding. Finally, overexpressed CL58 was able to interact with HCV envelope proteins. CONCLUSION: We identified a novel CLDN1-derived peptide that inhibits HCV entry at a postbinding step. The findings expand our knowledge of the roles that CLDN1 play in HCV entry and highlight the potential for developing a new class of inhibitors targeting the viral entry process.     

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture-derived HCV (HCVcc). Anti-SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI-specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81-HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti-SR-BI antibodies and SR-BI-specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV-CD81 interaction.     

GB virus type C interactions with HIV: the role of envelope glycoproteins

Summary.  GB virus C/hepatitis G virus (GBV-C/HGV) is the most closely related human virus to hepatitis C virus (HCV). GBV-C is lymphotropic and not associated with any known disease, although it is associated with improved survival in HIV-infected individuals. In peripheral blood mononuclear cells, GBV-C induces the release of soluble ligands for HIV entry receptors (RANTES, MIP-1a, MIP-1b and SDF-1), suggesting that GBV-C may interact with lymphocytes to induce a chemokine and/or cytokine milieu that is inhibitory to HIV infection. Expression of GBV-C envelope glycoprotein E2 in CD4+ T cells or addition of recombinant E2 to CD4 cells recapitulates the HIV inhibition seen with GBV-C infection. Like HCV E2, GBV-C E2 is predicted to be post-translationally processed in the endoplasmic reticulum and is involved with cell binding. The C-termini of GBV-C E1 and E2 proteins contain predicted transmembrane domains sharing features with HCV TM domains. To date, cellular receptor(s) for GBV-C E2 have not been identified. GBV-C E2-mediated HIV inhibition is dose-dependent and HIV replication is blocked at the binding and/or entry step. In addition, a putative GBV-C E2 fusion peptide interferes with HIV gp41 peptide oligomerization required for HIV-1 fusion, further suggesting that GBV-C E2 may inhibit HIV entry. Additional work is needed to identify the GBV-C E2 cellular receptor, characterize GBV-C E2 domains responsible for HIV inhibition, and to examine GBV-C E2-mediated fusion in the context of the entire envelope protein or viral-particles. Understanding the mechanisms of action may identify novel approaches to HIV therapy.     

The receptor-binding region of human apolipoprotein E has direct anti-infective activity

BACKGROUND: The APOE genotype has a uniquely strong influence on the outcome of viral infection. The mechanism is unknown, although one possibility is direct inhibition of viral entry into cells. METHODS: We have examined the direct anti-infective activity of a peptide analogue of the receptor-binding region of apolipoprotein E (apoE) that is known as "apoE dimer tandem repeat peptide" (apoEdp) and has previously been shown to mimic some of the biological effects of apoE and that recently was shown to bind low-density lipoprotein receptor-related protein. RESULTS: apoEdp has activity against herpes simplex virus types 1 and 2, human immunodeficiency virus, Pseudomonas aeruginosa, and Staphylococcus aureus; concentrations in the range of 1-20 micromol/L inhibit infection by 50%. These biological actions depend on adoption of an alpha -helical structure, as has been found for other biological effects of apoE peptides. The peptide interferes with the earliest stages of viral infection, preventing viral attachment and exerting a mild virucidal action. In addition, an N-terminal fragment of apoE that also contains this binding domain has antiviral activity. CONCLUSIONS: These data suggest that human apoE or fragments containing the receptor-binding domain may contribute to innate immunity to viral infection by direct disruption of viral particles and/or inhibition of viral attachment, thus reducing viral entry.     

Effect of human immunodeficiency virus infection on hepatitis C virus infection in hemophiliacs

Chronic liver disease due to hepatitis C virus (HCV) infection is a major problem in hemophiliacs. Recent reports suggested that hemophiliacs coinfected with hepatitis C virus and human immunodeficiency virus (HIV) have an increased incidence of liver failure but the mechanism of accelerated liver injury is not clear. We tested plasma from 100 hemophiliacs for anti-HCV by second generation ELISA, anti-HIV by EIA, and HCV RNA and HIV RNA by branched DNA and polymerase chain reaction assays to determine if hemophiliacs coinfected with HCV and HIV have higher HCV RNA levels and more active liver disease. Seventy-nine (79%) patients were anti-HCV positive, of whom 85% were HCV RNA positive. None of the anti-HCV-negative patients had detectable HCV RNA in plasma. Forty-two (42%) patients were anti-HIV positive, of whom 47% had detectable HIV RNA. All the anti-HIV-positive patients were also anti-HCV positive. The prevalence of both anti-HCV and anti-HIV increased significantly with age. There was no difference in HCV RNA levels between anti-HIV-positive and anti-HIV-negative patients (mean: 21±4 vs 18±5 Meq/ml), although HCV RNA levels were significantly higher in anti-HIV-positive patients with CD4 counts<200/mm³ (P=0.008). There was an inverse correlation between HCV RNA levels and CD4 counts but no correlation was found between HCV RNA and serum aminotransferase levels. We found a high prevalence of HCV and HIV coinfection in our hemophiliacs. Hepatitis C virus replication appears to be increased in patients with severe immunodeficiency secondary to progressive HIV infection. However, there was no correlation between HCV RNA and serum ALT level, suggesting that HCV is not directly cytopathic.     

CD4 T lymphocyte proliferative responses to hepatitis C virus (HCV) antigens in patients coinfected with HCV and human immunodeficiency virus who responded to anti-HCV treatment

CD4 T lymphocyte proliferative responses to hepatitis C virus (HCV) antigens were evaluated before and during an anti-HCV regimen (interferon-alpha2a and ribavirin) in 36 patients coinfected with HCV and human immunodeficiency virus (HIV), to determine whether immune responses against HCV antigens are present in such patients, whether these responses are modified by anti-HCV treatment, and whether they are correlated with treatment efficacy. The CD4 responses against HCV antigens (primarily core antigens) detected at study entry in one-half of the patients did not correlate with anti-HCV treatment efficacy. Of 36 patients, 8 had patterns of persistent immune response to infection by genotypes 3 or 4 that were significantly correlated with sustained virologic response. Persistent immunologic reactivity and sustained virologic response coexisted only in patients infected with genotype 3. These findings suggest that HCV genotype may influence specific immune response, which, in turn, is implicated in virologic control.     

The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry

Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. Current antiviral therapy fails to clear infection in a substantial proportion of cases. Drug development is focused on nonstructural proteins required for RNA replication. Individuals undergoing orthotopic liver transplantation face rapid, universal reinfection of the graft. Therefore, antiviral strategies targeting the early stages of infection are urgently needed for the prevention of HCV infection. In this study, we identified the polyphenol, epigallocatechin-3-gallate (EGCG), as an inhibitor of HCV entry. Green tea catechins, such as EGCG and its derivatives, epigallocatechin (EGC), epicatechin gallate (ECG), and epicatechin (EC), have been previously found to exert antiviral and antioncogenic properties. EGCG had no effect on HCV RNA replication, assembly, or release of progeny virions. However, it potently inhibited Cell-culture-derived HCV (HCVcc) entry into hepatoma cell lines as well as primary human hepatocytes. The effect was independent of the HCV genotype, and both infection of cells by extracellular virions and cell-to-cell spread were blocked. Pretreatment of cells with EGCG before HCV inoculation did not reduce HCV infection, whereas the application of EGCG during inoculation strongly inhibited HCV infectivity. Moreover, treatment with EGCG directly during inoculation strongly inhibited HCV infectivity. Expression levels of all known HCV (co-)receptors were unaltered by EGCG. Finally, we showed that EGCG inhibits viral attachment to the cell, thus disrupting the initial step of HCV cell entry. Conclusion: The green tea molecule, EGCG, potently inhibits HCV entry and could be part of an antiviral strategy aimed at the prevention of HCV reinfection after liver transplantation.     

Mother-to-infant transmission of hepatitis C virus.

OBJECTIVE: To describe the rate of perinatal transmission of hepatitis C virus (HCV). DESIGN: Follow-up study of newborn children of mothers with chronic HCV infection. SETTING: A university hospital in Sweden. PARTICIPANTS: Fourteen women with chronic HCV infection and their 21 newly born children. MAIN OUTCOME MEASURES: Detection of HCV RNA in serum by the polymerase chain reaction and detection of anti-HCV antibody by second generation assays. RESULTS: All mothers were found to be positive for anti-HCV antibody both by second-generation enzyme-linked immunosorbent assay (ELISA) and by second-generation recombinant immunoblot assay (RIBA-2); all also had detectable serum HCV RNA. Two children had long-lasting alanine aminotransferase (ALT) elevations, and one of them became HCV RNA positive. None of the other children developed biochemical hepatitis. However, two additional children had temporary viremia. Only the child with biochemical and biopsy-proven hepatitis and detectable HCV RNA in multiple blood samples actively produced anti-HCV antibody. CONCLUSIONS: Mother-to-infant transmission of HCV infection from chronically infected women without human immunodeficiency virus (HIV) infection seems to be uncommon.     

Oxidized low-density lipoprotein inhibits hepatitis C virus cell entry in human hepatoma cells

Cell entry of hepatitis C virus, pseudoparticles (HCVpp) and cell culture grown virus (HCVcc), requires the interaction of viral glycoproteins with CD81 and other as yet unknown cellular factors. One of these is likely to be the scavenger receptor class B type I (SR-BI). To further understand the role of SR-BI, we examined the effect of SR-BI ligands on HCVpp and HCVcc infectivity. Oxidized low-density lipoprotein (oxLDL), but not native LDL, potently inhibited HCVpp and HCVcc cell entry. Pseudoparticles bearing unrelated viral glycoproteins or bovine viral diarrhea virus were not affected. A dose-dependent inhibition was observed for HCVpp bearing diverse viral glycoproteins with an approximate IC50 of 1.5 microg/mL apolipoprotein content, which is within the range of oxLDL reported to be present in human plasma. The ability of lipoprotein components to bind to target cells associated with their antiviral activity, suggesting a mechanism of action which targets a cell surface receptor critical for HCV infection of the host cell. However, binding of soluble E2 to SR-BI or CD81 was not affected by oxLDL, suggesting that oxLDL does not act as a simple receptor blocker. At the same time, oxLDL incubation altered the biophysical properties of HCVpp, suggesting a ternary interaction of oxLDL with both virus and target cells. In conclusion, the SR-BI ligand oxLDL is a potent cell entry inhibitor for a broad range of HCV strains in vitro. These findings suggest that SR-BI is an essential component of the cellular HCV receptor complex.     

经血液(非静脉吸毒者)和性途径传播的HIV感染者合并HBV和HCV感染的现状

目的 探讨我国经血液(非静脉吸毒者)和性途径传播的HIV感染者合并乙型肝炎和丙型肝炎的状况.方法 回顾性分析2005年1至9月在全国13个研究中心就诊的362例HIV/AIDS患者(静脉吸毒者除外),应用酶联免疫试剂盒分别测定其HBsAg、抗-HBs,HBeAg、抗-Hbe、抗-HBc和抗-HCV.采用t检验和X2检验分别对计量和计数结果进行统计学分析.结果 315例检测血HBV和HCV的患者中,HBsAg阳性14例,占4.4%;抗-HCV阳性158例,占50.2%,抗-HCV阴性157例,占49.8%;HIV、HBV、HCV共感染2例,占0.6%.抗-HCV阳性组中经血液和性传播的比例分别占92%和4%,以血液传播为主;抗-HCV阴性组中经血液和性传播的比例分别占11%和66%,以性传播为主.抗-HCV阳性组的HIV确诊时间、CD4+T淋巴细胞绝对计数、ALT和AST均高于抗-HCV阴性组.两组患者的HBV标志物表达也存在差异,其中抗-HCV阳性组中HBsAg阳性2例,占1.3%,抗-HCV阴性组中HBsAg阳性12例,占7.6%,两组比较差异有统计学意义(X2=7.542,P<0.01).10例HBsAg阳性者进行HBV DNA检测,其中4例阳性,抗-HCV均为阴性.57例抗-HCV阳性患者进行HCV RNA检测,阳性者占63.2%.结论 我国输血和性传播途径的HIV感染合并HBV或HCV感染,以合并HCV感染为主,并多见于经输血感染者.合并HCV感染可加重HIV患者的肝脏损伤,同时也可能存在干扰HBV复制的情况.     

From the Cover: Identification of transferrin receptor 1 as a hepatitis C virus entry factor

Hepatitis C virus (HCV) is a liver tropic pathogen that affects ∼170 million people worldwide and causes liver pathologies including fibrosis, cirrhosis, steatosis, iron overload, and hepatocellular carcinoma. As part of a project initially directed at understanding how HCV may disrupt cellular iron homeostasis, we found that HCV alters expression of the iron uptake receptor transferrin receptor 1 (TfR1). After further investigation, we found that TfR1 mediates HCV entry. Specifically, functional studies showed that TfR1 knockdown and antibody blocking inhibit HCV cell culture (HCVcc) infection. Blocking cell surface TfR1 also inhibited HCV pseudoparticle (HCVpp) infection, demonstrating that TfR1 acts at the level of HCV glycoprotein-dependent entry. Likewise, a TfR1 small-molecule inhibitor that causes internalization of surface TfR1 resulted in a decrease in HCVcc and HCVpp infection. In kinetic studies, TfR1 antibody blocking lost its inhibitory activity after anti-CD81 blocking, suggesting that TfR1 acts during HCV entry at a postbinding step after CD81. In contrast, viral spread assays indicated that HCV cell-to-cell spread is less dependent on TfR1. Interestingly, silencing of the TfR1 trafficking protein, a TfR-1 specific adaptor protein required for TfR1 internalization, also inhibited HCVcc infection. On the basis of these results, we conclude that TfR1 plays a role in HCV infection at the level of glycoprotein-mediated entry, acts after CD81, and possibly is involved in HCV particle internalization.     

Hepatitis C virus antigens enzyme immunoassay for one-step diagnosis of hepatitis C virus coinfection in human immunodeficiency virus infected individuals

BACKGROUND Current diagnosis of hepatitis C virus(HCV) infection requires two sequential steps: testing for anti-HCV followed by HCV RNA PCR to confirm viremia. We have developed a highly sensitive and specific HCV-antigens enzyme immunoassay(HCV-Ags EIA) for one-step diagnosis of viremic HCV infection.AIM To assess the clinical application of the HCV-Ags EIA in one-step diagnosis of viremic HCV infection in human immunodeficiency virus(HIV)-coinfected individuals.METHODS The study blindly tested HCV-Ags EIA for its performance in one-step diagnosing viremic HCV infection in 147 sera: 10 without HCV or HIV infection;54 with viremic HCV monoinfection; 38 with viremic HCV/HIV coinfection; and45 with viremic HCV and non-viremic HIV coinfection.RESULTS Upon decoding, it was 100% accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR test. In five sera with HCV infection, HCV RNA was as low as50-59 IU/mL, and four out of five tested positive for HCV-Ags EIA. Likewise, it was also 100% accordance of HCV-Ags EIA to HCV infection status by HCV RNA PCR in 83 sera with HCV and HIV coinfection, regardless if HIV infection was active or not.CONCLUSION The modified HCV-Ags EIA has a lower detection limit equivalent to serum HCV RNA levels of approximately 100 IU/mL. It is highly sensitive and specific in the setting of HIV coinfection, regardless of HIV infection status and CD4 count.These data support the clinical application of the HCV-Ags EIA in one-step diagnosis of HCV infection in HIV-infected individuals.     

Prevalence of coinfection with human immunodeficiency virus and hepatitis C virus in Japan

People with human immunodeficiency virus (HIV) infection are frequently infected with hepatitis C virus (HCV), because of the common transmission routes. Since the dissemination of hyperactive antiretrovirus therapy (HAART), the morbidity and mortality associated with HIV infection have declined. However, the reduction in mortality due to opportunistic infection has made HCV-associated liver diseases the leading cause of mortality in Western countries. A similar situation is assumed in Japan, but the status of coinfection with HIV and HCV is unclear. We conducted a nationwide survey to determine the prevalence of coinfection with HIV and HCV by distributing a questionnaire to the hospitals in the HIV/AIDS Network of Japan. Among 4877 patients reported to be HIV-positive, 935 (19.2%) were also positive for the anti-HCV antibody. Most (84.1%) of the patients coinfected with HIV and HCV were recipients of blood products. These data, for the first time, show the current status of coinfection with HIV and HCV in Japan. A detailed analysis of the progression and severity of liver diseases in the coinfected patients is expected.     

Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

Summary.  Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262–290) of hepatitis C virus (HCV) may function as a fusion peptide. Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid substitutions appeared to disrupt necessary interactions between E1 and E2. For some mutants, reductions in HCVpp-associated E1 were associated with the incorporation of a high molecular weight, hyperglycosylated E2 that displayed decreased CD81-binding. Other entry-deficient mutants displayed normal E1E2 incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production. However, the S283P mutation had a different effect in the two systems as it did not increase production of infectious HCVcc. This comprehensive mutational analysis of the putative HCV fusion peptide provides insight into the role of E1 in its interaction with E2 and in HCV entry.     

L-SIGN (CD 209L) is a liver-specific capture receptor for hepatitis C virus

Hepatitis C virus (HCV) infects nearly 3% of the population of the world and is a major cause of liver disease. However, the mechanism whereby the virus targets the liver for infection remains unknown, because none of the putative cellular receptors for HCV are both expressed specifically in the liver and capable of binding HCV envelope glycoproteins. Liver/lymph node-specific intercellular adhesion molecule-3-grabbing integrin (L-SIGN) is a calcium-dependent lectin expressed on endothelial cells of liver and lymph nodes. Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a homologous molecule expressed on dendritic cells, binds HIV and promotes infection. By using a virus-binding assay, we demonstrate that L-SIGN and DC-SIGN specifically bind naturally occurring HCV present in the sera of infected individuals. Further studies demonstrate that binding is mediated by the HCV envelope glycoprotein E2 and is blocked by specific inhibitors, including mannan, calcium chelators, and Abs to the lectin domain of the SIGN molecules. Thus, L-SIGN represents a liver-specific receptor for HCV, and L-SIGN and DC-SIGN may play important roles in HCV infection and immunity.     

Heterosexual transmission of hepatitis C virus and the possible role of coexistent human immunodeficiency virus infection in the index case: A multicentre study of 423 pairings

Abstract. Objectives. To define the role that sexual transmission plays in the spread of hepatitis C virus (HCV) infection, and to examine the influence of coexistent human immunodeficiency virus (HIV) infection on this mode of transmission. Design. A multicentre, seroprevalence study of anti-HCV performed in the stable heterosexual partners (SHP) of index cases reactive for anti-HCV. Setting. Department of Internal Medicine and Section of Gastroenterology of three University Hospitals, Spain. Subjects. A total of 423 stable heterosexual partners of index cases reactive for anti-HCV. This included a group of 142 intravenous drug users (IVDU), 120 of whom were coinfected with HIV. Additionally, 2886 first-time voluntary blood donors selected at random were included to compare the prevalence of anti-HCV. Main outcome measures. Serum samples were screened for anti-HCV by a commercially available, second-generation enzyme-linked immunoassay. Tests repeatedly reactive for anti-HCV were analysed by a four-antigen, recombinant immunoblot assay. Anti-HIV was tested by enzyme immunoassay and Western blot was used for confirmation of positive cases. Results. The prevalence of anti-HCV, was 7.1% in SHP and 1.2% in random donors (P < 0.001). This prevalence was higher in SHP of index cases co-infected with HIV in comparison with that shown in the SHP of index cases only reactive for the anti-HCV (9.1 vs. 6.3%; P = 0.2), particularly when a younger and more homogeneous group such as the SHP of IVDU index cases was considered alone (9.2 vs. 0%; P = 0.1). However, the SHP of IVDU index cases coinfected with HIV were almost three times more likely to be infected with HIV than HCV (24.2 vs. 9.2%). Conclusions. These data suggest that HCV infection may be sexually transmitted but with low efficiency, and this could be increased in the presence of coexistent HIV infection in the index case.     

Apolipoprotein E-epsilon 4 protects against severe liver disease caused by hepatitis C virus

The outcome of infection with hepatitis C virus (HCV) varies greatly. The virus associates with serum lipoproteins, including those containing apolipoprotein E (apoE) and apolipoprotein B (apoB), and may enter cells via the low-density lipoprotein receptor (LDLR). ApoE genotypes can affect the extent of damage in diseases caused by 2 other viruses--herpes simplex virus type 1 (HSV1; in Alzheimer's disease and herpes labialis) and human immunodeficiency virus (HIV). We therefore investigated whether specific apoE and apoB alleles were associated with different outcomes of HCV infection. A total of 156 anti-HCV-positive patients and 104 non-HCV-infected patients were studied. Liver biopsy specimens from patients with chronic HCV infection (n = 111) were assessed for disease severity by the Knodell system. ApoE and apoB genotypes were determined by standard polymerase chain reaction (PCR) methods. There was no significant difference among the apoE genotypes of HCV-infected subjects compared with previously published population data, or between HCV-RNA positive or negative patients. However, chronically HCV-infected subjects with mild liver disease (n = 65) had a significantly higher apoE-epsilon 4 allele frequency (20.0%) than those (n = 46) with severe disease (6.5%). ApoB alleles alone or in combination with apoE were not associated with mild or severe disease. The overall apoE allele frequencies of patients with liver disease not caused by HCV were similar to those of the total HCV group and in contrast to the HCV patients, the apoE allele frequencies were similar in those patients with no or mild fibrosis as compared with those with bridging fibrosis or cirrhosis. In conclusion, carriage of an apoE-epsilon 4 allele may be protective against liver damage caused by HCV, but not against damage due to various nonviral causes. This is yet another case in which apoE may determine the severity of a viral disease.     

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

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.