Strands Of Hepatitis C Virus RNA Research Articles

Ribosome Pausing at Inefficient Codons at the End of the Replicase Coding Region Is Important for Hepatitis C Virus Genome Replication.

Hepatitis C virus (HCV) infects liver cells and often causes chronic infection, also leading to liver cirrhosis and cancer. In the cytoplasm, the viral structural and non-structural (NS) proteins are directly translated from the plus strand HCV RNA genome. The viral proteins NS3 to NS5B proteins constitute the replication complex that is required for RNA genome replication via a minus strand antigenome. The most C-terminal protein in the genome is the NS5B replicase, which needs to initiate antigenome RNA synthesis at the very 3′-end of the plus strand. Using ribosome profiling of cells replicating full-length infectious HCV genomes, we uncovered that ribosomes accumulate at the HCV stop codon and about 30 nucleotides upstream of it. This pausing is due to the presence of conserved rare, inefficient Wobble codons upstream of the termination site. Synonymous substitution of these inefficient codons to efficient codons has negative consequences for viral RNA replication but not for viral protein synthesis. This pausing may allow the enzymatically active replicase core to find its genuine RNA template in cis, while the protein is still held in place by being stuck with its C-terminus in the exit tunnel of the paused ribosome.

A New Age-Structured Multiscale Model of the Hepatitis C Virus Life-Cycle During Infection and Therapy With Direct-Acting Antiviral Agents

The dynamics of hepatitis C virus (HCV) RNA during translation and replication within infected cells were added to a previous age-structured multiscale mathematical model of HCV infection and treatment. The model allows the study of the dynamics of HCV RNA inside infected cells as well as the release of virus from infected cells and the dynamics of subsequent new cell infections. The model was used to fit in vitro data and estimate parameters characterizing HCV replication. This is the first model to our knowledge to consider both positive and negative strands of HCV RNA with an age-structured multiscale modeling approach. Using this model we also studied the effects of direct-acting antiviral agents (DAAs) in blocking HCV RNA intracellular replication and the release of new virions and fit the model to in vivo data obtained from HCV-infected subjects under therapy.

Does the mosquito Culex pipiens represent a potential vector of hepatitis C virus?

Infection with hepatitis C virus (HCV) is a public health burden in several countries. Although transmission through blood is the most likely potential route of HCV infection, other sources warrant exploration. This study was designed to examine the possibility that the mosquito Culex pipiens (Diptera: Culicidae) might serve as a vector of HCV. A series of laboratory experiments were conducted in female Cx.pipiens that were fed on blood taken from HCV patients and tested for the presence of HCV RNAs using a reverse transcription polymerase chain reaction technique. In addition, the ability of the female mosquito to transmit HCV to human blood through membrane feeding or to its offspring (larvae) was tested. Although positive strand RNA was detected on days 1,7 and 14, negative strand HCV RNA was detected in mosquito body homogenate on days 7 and 14. Positive strands were also detected in the head, alimentary canal and salivary glands of mosquito adults at 1 week post-feeding, as well as in their offspring (larvae). An ex vivo assay demonstrated that HCV-infected mosquitoes were able to transmit the virus RNA into naive human blood samples via a membrane feeder. The present data indicate that the mosquito Cx.pipiens may be a potential vector of HCV.

Synthesis of Oxadiazolyl, Pyrazolyl and Thiazolyl Derivatives of Thiophene-2-Carboxamide as Antimicrobial and Anti-HCV Agents.

Introduction:Three series of pyrazole, thiazole and 1,3,4-oxadiazole, derivatives were synthesized starting from 5-amino-4-(hydrazinocarbonyl)-3-methylthiophene-2-carboxamide (2).Methods:All compounds were investigated for their preliminary antimicrobial activity. They were proved to exhibit remarkable antimicrobial activity against Pseudomonas aeruginosa with insignificant activity towards Gram positive bacterial strains and fungi.Results: In-vitro testing of the new compounds on hepatitis-C virus (HCV) replication in hepatocellular carcinoma cell line HepG2 infected with the virus utilizing the reverse transcription polymerase chain reaction technique (RT-PCR) generally showed inhibition of the replication of HCV RNA (–) strands at low concentration, while, eight compounds; 3a, 6, 7a, 7b, 9a, 9b, 10a and 11b proved to inhibit the replication of HCV RNA (+) and (–) strands at very low concentration range 0.08-0.36 μg/mL.Conclusion:Compounds 7b and 11b displayed the highest anti-HCV and antimicrobial activities in this study.

Hepatitis C virus addiction to liver miR-122 takes its Toll on the host.

Hepatitis C virus addiction to liver miR-122 takes its Toll on the host.

Defining the spatial relationship between hepatitis C virus infection and interferon-stimulated gene induction in the human liver

Defining the spatial relationship between hepatitis C virus infection and interferon-stimulated gene induction in the human liver

Detection of hepatitis C virus (HCV) negative strand RNA and NS3 protein in peripheral blood mononuclear cells (PBMC): CD3+, CD14+ and CD19+

BackgroundAlthough hepatitis C virus (HCV) is primarily hepatotropic, markers of HCV replication were detected in peripheral blood mononuclear cells (PBMC) as well as in ex vivo collected tissues and organs. Specific strains of HCV were found to be capable to infect cells of the immune system: T and B cells and monocytes/macrophages as well as cell lines in vitro. The direct invasion of cells of the immune system by the virus may be responsible for extrahepatic consequences of HCV infection: cryoglobulinemia and non-Hodgkin’s lymphoma.The aim of the present study was to determine the prevalence of markers of HCV infection: negative strand HCV RNA and non-structural NS3 protein in PBMC subpopulations: CD3+, CD14+ and CD19+. The presence of virus and the proportion of affected cells within a particular PBMC fraction could indicate a principal target cell susceptible for HCV.MethodsPBMC samples were collected from 26 treatment-free patients chronically infected with HCV. PBMC subpopulations: CD3+, CD14+, CD19+ were obtained using positive magnetic separation. The presence of negative strand RNA HCV and viral NS3 protein were analyzed by strand-specific RT-PCR and NS3 immunocytochemistry staining.ResultsNegative strand HCV RNA was detectable in 7/26 (27%), whereas NS3 protein in 15/26 (57.6%) of PBMC samples. At least one replication marker was found in 13/26 (50%) of CD3+ cells then in 8/26 (30.8%) of CD14+ and CD19+ cells. The highest percentage of cells harboring viral markers in single specimen was also observed in CD3+ (2.4%), then in CD19+ (1.2%), and much lower in CD14+ (0.4%) cells.ConclusionsOur results indicate that CD3+ cells are a dominant site for extrahepatic HCV replication, although other PBMC subpopulations may also support virus replication.

Decreased level of intracellular cholesterol in peripheral blood mononuclear cells is associated with chronic hepatitis C virus infection

Peripheral blood mononuclear cells (PBMCs) constitute the main extrahepatic place of, hepatitis C virus (HCV) replication. We aimed to determine the impact of CHC infection and microRNA-, 122 expression on cholesterol expression in PBMCs. HCV RNA strand, intracellular cholesterol, HMGCoA, reductase and miR-122 expression in PBMC were determined in 54 CHC patients. The study shows that significant decrease of intracellular cholesterol level in PBMC (p=0.000000), accompanied by serum hypocholesterolemia is the characteristic feature of chronic hepatitis C infection. Although, microRNA-122 expression was detectable in PBMCs of CHC patients (52.5%), the alteration of intracellular cholesterol level was independent of miR-122 expression.

Hepatitis C virus (HCV) infection of peripheral blood mononuclear cells in patients with type II cryoglobulinemia

ObjectivesType II cryoglobulinemia is a common extrahepatic manifestation of chronic hepatitis C virus (HCV) infection. The mechanisms behind its development are unclear, but could be related to direct infection of the immune cells. MethodsPeripheral blood mononuclear cells from 18 patients with type II cryoglobulinemia were separated into CD3+ (T cells), CD19+ (B cells) and CD14+ (monocytes) and analyzed for the presence of negative strand HCV RNA, which is a viral replicative intermediate, and for the presence of HCV non-structural protein 3 (NS3). Control group consisted of 182 consecutive chronic hepatitis C patients prior to initiation of antiviral therapy. ResultsNegative strand HCV RNA was detected in PBMC from six (33.3%), patients and in 15 (8.2%) controls (p<0.01). Negative strand was most frequently detected in B cells (3 patients), followed by T cells (2 patients), and monocytes (2 patients). One patient was positive both in CD3+ and CD14+ cells. NS3 protein was detected in six (33.3%) patients; five were positive in T cells, three in B cells, and another three were positive in monocytes. Two patients were positive in all analyzed cell subpopulation and one patient was positive in CD14+ and CD19+ cells, but not in CD3+ cells. Altogether, 11 patients (61.1%) were positive either for the negative strand HCV RNA or NS3 protein in at least one of the analyzed cell compartments. ConclusionOur findings of common presence of viral replication in cells of the immune system suggest that direct HCV infection could play a role in the etiology of cryoglobulinemia.

Replication of hepatitis C virus in peripheral blood mononuclear cells in patients with chronic hepatitis C treated with pegylated interferon alpha and ribavirin

Hepatitis C virus (HCV) is a primarily hepatotropic virus, but hepatocytes are not the only localization of its replication. It is still unclear if extrahepatic HCV replication, measured as the detection of HCV RNA negative strand in peripheral blood mononuclear cells (PBMCs) before initiation of treatment, has an influence on therapy response. Detection of HCV RNA in extrahepatic sites for assessment of therapy efficacy is not routinely used in clinical practice. The aim of the study was to evaluate whether the replication of HCV in PBMCs affects the rate of sustained virological response (SVR). The study group comprised 55 patients with chronic hepatitis C, originally treatment naive. They were treated with pegylated interferon (PEG-IFN) alpha 2a and ribavirin, with the standard dosing schedule. Parallel serum samples for HCV RNA and PBMC samples for HCV RNA negative strand were obtained at baseline, at the end of treatment and 24 weeks after finishing therapy. Undetectable HCV RNA in serum at the end of therapy was found in 48 patients (87.3%), while 33 patients (60.0%) achieved sustained virological response (SVR) (51% for HCV genotype 1 and 78% for genotype 3, respectively). Fifteen individuals (31.3%) were relapsers. Factors associated with significantly higher rate of SVR were young age, mild or no fibrosis and infection with HCV genotype 3. HCV RNA negative strand in PBMCs before treatment was found in 21.8% (12 out of 55 patients). HCV RNA negative strand was detected at baseline more frequently in patients who later achieved SVR. Relapse appeared significantly more often in patients with negative strand at the end of therapy: in 2 out of 15 individuals compared to 0 out of 33 patients (p=0.03). Presence of negative HCV RNA strand in PBMCs before treatment may be suggested as a potential marker of good treatment response. Detection of negative strand at the end of therapy is a predictor of relapse.

Comparison of HCV Plus-and Minus-Strand RNA in PBMCs of Responders and non-Responders of Chronically Infected Patients Receiving Ribavirin and Interferon Therapy

Background and Aims: Hepatitis C virus (HCV) can cause hepatocellular carcinoma (HCC) in a significant proportion (≈ 20 %) of individuals with chronic HCV infection (CHC). Currently, CHC is treated with peginterferon and ribavirin, which depending on genotype approximately 50 to 70% of patients are cured. The so-called “extrahepatic HCV infection” or viral replication in regions of the body other than liver, e.g. peripheral mononuclear cells (PBMCs), is likely to contribute to the lack of response to treatment in non-responders. In this study, HCV infection of PBMCs was compared between responders- and non-responders HCV-infected patients. Materials and Methods: RT-nested PCR was utilized to detect the plus- and minus- strands of HCV RNA in PBMCs using type-specific primers specific for the HCV core region. Results: Both the plus- and the minus strands of HCV genome were significantly lower in PBMCs of responder- than non-responder patients. Conclusion: The presence of both plus and minus HCV strands in PBMCs was associated with the treatment outcome, such that HCV infection of PBMCs was identified in higher proportion of non-responders relative to responders.

Coordinated increase of miRNA-155 and miRNA-196b expression correlates with the detection of the antigenomic strand of hepatitis C virus in peripheral blood mononuclear cells

A tight relationship has been revealed between cellular microRNAs (miRNAs) and the course of hepatitis C virus (HCV) replication in human hepatoma cells. Although the detection of the antigenomic HCV RNA strand in peripheral blood mononuclear cells (PBMCs) has provided evidence for viral replication in PBMCs, no reports have shown how miRNAs are affected upon HCV RNA synthesis in PBMCs. The aim of the present study was to assess if and how the expression levels of miRNA-155 and miRNA-196b in PBMCs are related to HCV replication in PBMCs of chronic hepatitis C (CHC) patients. Supporting analyses were performed to evaluate the expression of precursor pri-miR-155 (BIC) and Dicer protein. The genomic and antigenomic HCV RNA strands in PBMCs were detected by strand-specific qRT-PCR. The expression levels of miRNAs, BIC RNA and Dicer protein were assayed on PBMCs by qRT-PCR and Western blotting, respectively. miRNA-155 and miRNA-196b were detected in all studied PBMC samples, but their levels varied according to the presence of the antigenomic HCV RNA strand in PBMCs. Increased expression levels of miRNA-155 and miRNA-196b were associated with the presence of the antigenomic HCV RNA strand in PBMCs. In this group of patients higher frequency of BIC RNA and Dicer protein detection was also found. This study demonstrates that HCV RNA replication in PBMCs of CHC patients is connected with the increased and coordinated expression of miRNA-155 and miRNA-196b.

Human cell types important for Hepatitis C Virus replication in vivo and in vitro. Old assertions and current evidence

Hepatitis C Virus (HCV) is a single stranded RNA virus which produces negative strand RNA as a replicative intermediate. We analyzed 75 RT-PCR studies that tested for negative strand HCV RNA in liver and other human tissues. 85% of the studies that investigated extrahepatic replication of HCV found one or more samples positive for replicative RNA. Studies using in situ hybridization, immunofluorescence, immunohistochemistry, and quasispecies analysis also demonstrated the presence of replicating HCV in various extrahepatic human tissues, and provide evidence that HCV replicates in macrophages, B cells, T cells, and other extrahepatic tissues. We also analyzed both short term and long term in vitro systems used to culture HCV. These systems vary in their purposes and methods, but long term culturing of HCV in B cells, T cells, and other cell types has been used to analyze replication. It is therefore now possible to study HIV-HCV co-infections and HCV replication in vitro.

Lymphotropic HCV strain can infect human primary naïve CD4+ cells and affect their proliferation and IFN-γ secretion activity.

Lymphotropic hepatitis C virus (HCV) infection of B and T cells might play an important role in the pathogenesis of hepatitis C. Recently, we showed that a lymphotropic HCV (SB strain) could infect established T-cell lines and B-cell lines. However, whether HCV replication interferes with cell proliferation and function in primary T lymphocytes is still unclear. The aim of this study was to analyze whether HCV replication in primary T lymphocytes affected their development, proliferation, and Th1 commitment. SB strain cell culture supernatant (2 × 10(4) copies/ml HCV) was used to infect several kinds of primary lymphocyte subsets. Mock, UV-irradiated SB-HCV, JFH-1 strain, and JFH-1 NS5B mutant, which could not replicate in T cells, were included as negative controls. Carboxyfluorescein succinimidyl ester (CFSE) and CD45RA double staining was used to evaluate the proliferative activity of CD4(+)CD45RA(+)CD45RO(-) naïve CD4(+) cells. Interferon (IFN)-γ and interleukin (IL)-10 secretion assays magnetic cell sorting (MACS) were carried out. Negative strand HCV RNA was detected in CD4(+), CD14(+), and CD19(+) cells. Among CD4(+) cells, CD4(+)CD45RA(+)RO(-) cells (naïve CD4(+) cells) were most susceptible to replication of the SB strain. The levels of CFSE and CD45RA expression gradually declined during cell division in uninfected cells, while HCV-infected naïve CD4(+) cells expressed higher levels of CFSE and CD45RA than Mock or UV-SB infected naïve CD4(+) cells. Moreover, the production of IFN-γ was significantly suppressed in SB-infected naïve CD4(+) cells. Lymphotropic HCV replication suppressed proliferation and development, including that towards Th1 commitment, in human primary naïve CD4(+) cells.

Investigating a New Generation of Ribozymes in Order to Target HCV

For a long time nucleic acid-based approaches directed towards controlling the propagation of Hepatitis C Virus (HCV) have been considered to possess high potential. Towards this end, ribozymes (i.e. RNA enzymes) that specifically recognize and subsequently catalyze the cleavage of their RNA substrate present an attractive molecular tool. Here, the unique properties of a new generation of ribozymes are taken advantage of in order to develop an efficient and durable ribozyme-based technology with which to target HCV (+) RNA strands. These ribozymes resulted from the coupling of a specific on/off adaptor (SOFA) to the ribozyme domain derived from the Hepatitis Delta Virus (HDV). The former switches cleavage activity “on” solely in the presence of the desired RNA substrate, while the latter was the first catalytic RNA reported to function naturally in human cells, specifically in hepatocytes. In order to maximize the chances for success, a step-by-step approach was used for both the design and the selection of the ribozymes. This approach included the use of both bioinformatics and biochemical methods for the identification of the sites possessing the greatest potential for targeting, and the subsequent in vitro testing of the cleavage activities of the corresponding SOFA-HDV ribozymes. These efforts led to a significant improvement in the ribozymes' designs. The ability of the resulting SOFA-HDV ribozymes to inhibit HCV replication was further examined using a luciferase-based replicon. Although some of the ribozymes exhibited high levels of cleavage activity in vitro, none appears to be a potential long term inhibitor in cellulo. Analysis of recent discoveries in the cellular biology of HCV might explain this failure, as well as provide some ideas on the potential limits of using nucleic acid-based drugs to control the propagation of HCV. Finally, the above conclusions received support from experiments performed using a collection of SOFA-HDV ribozymes directed against HCV (−) strands.

Hepatitis C virus replication in patients with occult hepatitis C virus infection†

We read with interest the review by Welker and Zeuzem1 on occult hepatitis C virus (HCV) infection. In this review, the authors claim that viral replication has not been proven in occult HCV infection. However, the presence of the antigenomic HCV RNA strand has been demonstrated in liver and in peripheral blood mononuclear cells (PBMCs) of patients with occult HCV who are negative for anti-HCV and serum HCV RNA2 (and references in Carreño et al.2). There is no doubt that detection of the antigenomic strand is an indicator of viral replication as recognized by Welker and Zeuzem in their review.1 Furthermore, ultracentrifugation of serum samples before performance of real-time reverse transcription polymerase chain reaction (RT-PCR) detects very low levels of viremia in patients with occult HCV infection, and the density of the circulating HCV particles was identical to those found in the serum of patients with chronic hepatitis C.2 In addition, family members of patients with occult HCV infection show serological markers of HCV infection even more frequently than those of patients with chronic hepatitis C.3 Other authors have reported that low-level persisting HCV remains infectious.4 Finally, HCV-specific CD4+ T cell proliferative responses to nonstructural 3 and/or 4 proteins have been detected among patients infected with occult HCV,2 which may not have occurred unless there was ongoing HCV replication, either within the liver or in immune-privileged sites. So, taking all these data into account, one may convincingly assume that HCV replication takes place in patients with occult HCV infection who no doubt are potentially infectious. However, Welker and Zeuzem1 review two studies that unconvincingly failed to detect occult HCV infection in cryptogenic hepatitis,5, 6 because of several concerns with the methodology employed. Thus, one study5 used whole blood to assess occult HCV infection, but it was demonstrated in patients with occult hepatitis C that the sensitivity of HCV RNA detection substantially decreased when using whole blood instead of PBMCs.2 Another study6 included a very small and heterogeneous population, and these authors did not perform detection of HCV RNA either in liver or in serum by PCR after ultracentrifugation. Thus, their results do not exclude the existence of occult HCV infection. On the other hand, an anti-HCV core antibody assay has been developed recently, which adds another diagnostic tool because it has been proposed as a potential new marker of the serologically silent form of occult HCV infection.7 In contrast, other studies have confirmed the existence of occult HCV infection by detection of HCV RNA in liver.8, 9 Furthermore, contrary to what is concluded by Welker and Zeuzem, HCV proteins have been detected in liver10 and in lymph nodes11 of patients who are negative for anti-HCV and serum HCV RNA. In summary, considering the data available, there is cumulative evidence strongly supporting the idea that HCV replication takes place in patients with occult HCV infection. Therefore, in order to diagnose occult HCV infection among these patients, it is mandatory to perform appropriate molecular and immunoserological tests in serum, PBMCs, and liver biopsies. Vicente Carreño*, Javier Bartolomé*, Inmaculada Castillo*, Juan Antonio Quiroga*, * Fundación para el Estudio de las Hepatitis Virales, Madrid, Spain.

HCV RNA in peripheral blood cell subsets in HCV–HIV coinfected patients at the end of PegIFN/RBV treatment is associated with virologic relapse

Extrahepatic replication may have important implications for the treatment of hepatitis C virus (HCV). Our aim was to analyse the association between the presence of positive/negative strand HCV RNA in different peripheral blood cell subsets at the end of PegIFN/RBV treatment, and treatment response in HIV-coinfected patients. Thirty-four HCV-HIV coinfected patients who concluded 48 weeks of PegIFN/RBV treatment were included in the present study. Positive/negative strand HCV RNA was detected by amplification of the 5' untranslated region (5' UTR) using high-temperature RT-PCR in immunomagnetic-isolated cell subsets. Twenty-three patients (67.6%) had sustained virologic response (SVR) while 11 patients (32.4%) relapsed. The frequency of positive/negative strand HCV RNA in any cell subsets was significantly lower in patients with SVR (8.6%) compared to relapsers (63.6%) (P = 0.002). Baseline HCV viral load was statistically higher among patients who relapsed (P = 0.008), while patients with SVR had very early virologic response more frequently (P = 0.003). Multivariate analysis showed, among these three variables, that only the presence of positive/negative strand HCV RNA was independently associated with relapse [P = 0.024; OR 14 (14-137)]. In conclusion, the presence of positive/negative strand HCV RNA at the end of treatment is associated with relapse among HCV-HIV coinfected patients and might have important implications in the clinical practice.

Desperately seeking hepatitis C virus

Spanish investigators described recently the so-called occult hepatitis C virus (HCV) infection, emphasizing the detection of genomic and antigenomic HCV RNA strands in liver and peripheral blood mononuclear cells. Therefore, the persistence of viral replication in occult HCV infection should be considered as a putative source of infection among family members and patients undergoing invasive procedures, transfusion or transplantation. Additionally, the most worrisome finding is that an occult HCV infection may persist in patients with sustained virological response.

HCV infection facilitates asymptomatic carotid atherosclerosis: preliminary report of HCV RNA localization in human carotid plaques

Background: Clinical and experimental evidence suggests that hepatitis C virus (HCV) infection shows peculiar characteristics that strongly support a role in the development of atherosclerosis. We aimed to investigate whether (a) HCV infection can facilitate asymptomatic carotid lesions and (b) the presence of HCV RNA sequences can be shown in plaque tissues. Methods: The status of carotid arteries, studied as intima-media thickness (IMT) in carotid bifurcation and prevalence and severity of plaques in internal carotid artery, was investigated by high-resolution B-mode ultrasonography in 31 HCV seropositive (HCV+) and in 120 age-matched HCV seronegative (HCV-) subjects evaluated for cardiovascular risk factors. The atherosclerotic risk profile, inflammation markers and main liver function tests were also studied in all patients. HCV RNA sequences were investigated by highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) in plaque tissues and serum of 2 HCV+ patients who underwent carotid revascularization. Results: Genomic and antigenomic HCV RNA strands were evidenced within both the carotid plaque tissues examined. The prevalence of an IMT > 1 mm, but not the prevalence and severity of internal carotid plaques, was significantly higher (P < 0.001) in HCV+ than in HCV patients. The atherosclerotic risk profile for traditional and inflammatory factors did not differ between the HCV+ and HCV- groups. Main liver function tests did not differ between the two groups. HCV positivity was significantly associated with >1 mm IMT (P < 0.01) according to univariate analysis, and this association remained significant in multivariate regression analysis. Conclusions: The novel finding of HCV RNA sequences within carotid plaques suggests a local pro-atherogenetic action of the virus inside the plaque. On the whole our data strongly support that HCV infection facilitates the occurrence of carotid atherosclerotic lesions.

The low-density lipoprotein receptor plays a role in the infection of primary human hepatocytes by hepatitis C virus

The direct implication of low-density lipoprotein receptor (LDLR) in hepatitis C virus (HCV) infection of human hepatocyte has not been demonstrated. Normal primary human hepatocytes infected by serum HCV were used to document this point. Expression and activity of LDLR were assessed by RT-PCR and LDL entry, in the absence or presence of squalestatin or 25-hydroxycholesterol that up- or down-regulates LDLR expression, respectively. Infection was performed in the absence or presence of LDL, HDL, recombinant soluble LDLR peptides encompassing full-length (r-shLDLR4-292) or truncated (r-shLDLR4-166) LDL-binding domain, monoclonal antibodies against r-shLDLR4-292, squalestatin or 25-hydroxycholesterol. Intracellular amounts of replicative and genomic HCV RNA strands used as end point of infection were assessed by RT-PCR. r-shLDLR4-292, antibodies against r-shLDLR4-292 and LDL inhibited viral RNA accumulation, irrespective of genotype, viral load or liver donor. Inhibition was greatest when r-shLDLR4-292 was present at the time of inoculation and gradually decreased as the delay between inoculation and r-shLDLR4-292 treatment increased. In hepatocytes pre-treated with squalestatin or 25-hydroxycholesterol before infection, viral RNA accumulation increased or decreased in parallel with LDLR mRNA expression and LDL entry. LDLR is involved at an early stage in infection of normal human hepatocytes by serum-derived HCV virions.