Hepatitis C virus cell-cell transmission and resistance to direct-acting antiviral agents

Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs

Syndecan 4 Is Involved in Mediating HCV Entry through Interaction with Lipoviral Particle-Associated Apolipoprotein E

Hepatitis C virus (HCV) is a major cause of liver disease worldwide and HCV infection represents a major health problem. HCV associates with host lipoproteins forming host/viral hybrid complexes termed lipoviral particles. Apolipoprotein E (apoE) is a lipoprotein component that interacts with heparan sulfate proteoglycans (HSPG) to mediate hepatic lipoprotein uptake, and may likewise mediate HCV entry. We sought to define the functional regions of apoE with an aim to identify critical apoE binding partners involved in HCV infection. Using adenoviral vectors and siRNA to modulate apoE expression we show a direct correlation of apoE expression and HCV infectivity, whereas no correlation exists with viral protein expression. Mutating the HSPG binding domain (HSPG-BD) of apoE revealed key residues that are critical for mediating HCV infection. Furthermore, a novel synthetic peptide that mimics apoE's HSPG-BD directly and competitively inhibits HCV infection. Genetic knockdown of the HSPG proteins syndecan (SDC) 1 and 4 revealed that SDC4 principally mediates HCV entry. Our data demonstrate that HCV uses apoE-SDC4 interactions to enter hepatoma cells and establish infection. Targeting apoE-SDC interactions could be an alternative strategy for blocking HCV entry, a critical step in maintaining chronic HCV infection

Hepatitis C Virus Infection in Phenotypically Distinct Huh7 Cell Lines

In 2005, the first robust hepatitis C virus (HCV) infectious cell culture system was developed based on the HCV genotype 2a JFH-1 molecular clone and the human-derived hepatoma cell line Huh7. Although much effort has been made to dissect and expand the repertoire of JFH-1-derived clones, less attention has been given to the host cell despite the intriguing facts that thus far only Huh7 cells have been found to be highly permissive for HCV infection and furthermore only a limited number of Huh7 cell lines/stocks appear to be fully permissive. As such, we compiled a panel of Huh7 lines from disparate sources and evaluated their permissiveness for HCV infection. We found that although Huh7 lines from different laboratories do vary in morphology and cell growth, the majority (8 out of 9) were highly permissive for infection, as demonstrated by robust HCV RNA and de novo infectious virion production following infection. While HCV RNA levels achieved in the 8 permissive cell lines were relatively equivalent, three Huh7 lines demonstrated higher infectious virion production suggesting these cell lines more efficiently support post-replication event(s) in the viral life cycle. Consistent with previous studies, the single Huh7 line found to be relatively resistant to infection demonstrated a block in HCV entry. These studies not only suggest that the majority of Huh7 cell lines in different laboratories are in fact highly permissive for HCV infection, but also identify phenotypically distinct Huh7 lines, which may facilitate studies investigating the cellular determinants of HCV infection

Viral Kinetics Suggests a Reconciliation of the Disparate Observations of the Modulation of Claudin-1 Expression on Cells Exposed to Hepatitis C Virus

The tight junction protein claudin-1 (CLDN1) is necessary for hepatitis C virus (HCV) entry into target cells. Recent studies have made disparate observations of the modulation of the expression of CLDN1 on cells following infection by HCV. In one study, the mean CLDN1 expression on cells exposed to HCV declined, whereas in another study HCV infected cells showed increased CLDN1 expression compared to uninfected cells. Consequently, the role of HCV in modulating CLDN1 expression, and hence the frequency of cellular superinfection, remains unclear. Here, we present a possible reconciliation of these disparate observations. We hypothesized that viral kinetics and not necessarily HCV-induced receptor modulation underlies these disparate observations. To test this hypothesis, we constructed a mathematical model of viral kinetics in vitro that mimicked the above experiments. Model predictions provided good fits to the observed evolution of the distribution of CLDN1 expression on cells following exposure to HCV. Cells with higher CLDN1 expression were preferentially infected and outgrown by cells with lower CLDN1 expression, resulting in a decline of the mean CLDN1 expression with time. At the same time, because the susceptibility of cells to infection increased with CLDN1 expression, infected cells tended to have higher CLDN1 expression on average than uninfected cells. Our study thus presents an explanation of the disparate observations of CLDN1 expression following HCV infection and points to the importance of considering viral kinetics in future studies of receptor expression on cells exposed to HCV

Soluble Serum CD81 Is Elevated in Patients with Chronic Hepatitis C and Correlates with Alanine Aminotransferase Serum Activity

Aim: Cellular CD81 is a well characterized hepatitis C virus (HCV) entry factor, while the relevance of soluble exosomal CD81 in HCV pathogenesis is poorly defined. We performed a case-control study to investigate whether soluble CD81 in the exosomal serum fraction is associated with HCV replication and inflammatory activity. Patients and Methods: Four cohorts were investigated, patients with chronic hepatitis C (n = 37), patients with chronic HCV infection and persistently normal ALT levels (n = 24), patients with long term sustained virologic response (SVR, n = 7), and healthy volunteers (n = 23). Concentration of soluble CD81 was assessed semi-quantitatively after differential centrifugation ranging from 200 g to 100,000 g in the fifth centrifugation fraction by immunoblotting and densitometry. Results: Soluble CD81 was increased in patients with chronic hepatitis C compared to healthy subjects (p = 0.03) and cured patients (p = 0.017). Patients with chronic HCV infection and persistently normal ALT levels and patients with long term SVR had similar soluble CD81 levels as healthy controls (p>0.2). Overall, soluble CD81 levels were associated with ALT levels (r = 0.334, p = 0.016) and severe liver fibrosis (p = 0.027). Conclusion: CD81 is increased in the exosomal serum fraction in patients with chronic hepatitis C and appears to be associated with inflammatory activity and severity of fibrosis

The SR-BI Partner PDZK1 Facilitates Hepatitis C Virus Entry

Entry of hepatitis C virus (HCV) into hepatocytes is a multi-step process that involves a number of different host cell factors. Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes. Physiologically, SR-BI is the major receptor for high-density lipoproteins (HDL) in the liver, where its expression is primarily controlled at the post-transcriptional level by its interaction with the scaffold protein PDZK1. However, the importance of interaction with PDZK1 to the involvement of SR-BI in HCV entry is unclear. Here we demonstrate that stable shRNA-knockdown of PDZK1 expression in human hepatoma cells significantly reduces their susceptibility to HCV infection, and that this effect can be reversed by overexpression of full length PDZK1 but not the first PDZ domain of PDZK1 alone. Furthermore, we found that overexpression of a green fluorescent protein chimera of the cytoplasmic carboxy-terminus of SR-BI (amino acids 479–509) in Huh-7 cells resulted in its interaction with PDZK1 and a reduced susceptibility to HCV infection. In contrast a similar chimera lacking the final amino acid of SR-BI (amino acids 479–508) failed to interact with PDZK1 and did not inhibit HCV infection. Taken together these results indicate an indirect involvement of PDZK1 in HCV entry via its ability to interact with SR-BI and enhance its activity as an HCV entry factor

Within-Host Dynamics of the Hepatitis C Virus Quasispecies Population in HIV-1/HCV Coinfected Patients

HIV/HCV coinfected individuals under highly active antiretroviral therapy (HAART) represent an interesting model for the investigation of the role played by the immune system in driving the evolution of the HCV quasispecies. We prospectively studied the intra-host evolution of the HCV heterogeneity in 8 coinfected subjects, selected from a cohort of 32 patients initiating HAART: 5 immunological responders (group A) and 3 immunological non-responders (group B), and in two HCV singly infected controls not assuming drugs (group C). For all these subjects at least two serial samples obtained at the first observation (before HAART) and more than 1 year later, underwent clonal sequence analysis of partial E1/E2 sequences, encompassing the whole HVR1. Evolutionary rates, dated phylogenies and population dynamics were co-estimated by using a Bayesian Markov Chain Monte Carlo approach, and site specific selection pressures were estimated by maximum likelihood-based methods. The intra-host evolutionary rates of HCV quasispecies was 10 times higher in subjects treated with HAART than in controls without immunodeficiency (1.9 and 2.3×10−3 sub/site/month in group A and B and 0.29×10−3 sub/site/month in group C individuals). The within-host Bayesian Skyline plot analysis showed an exponential growth of the quasispecies populations in immunological responders, coinciding with a peak in CD4 cell counts. On the contrary, quasispecies population remained constant in group B and in group C controls. A significant positive selection pressure was detected in a half of the patients under HAART and in none of the group C controls. Several sites under significant positive selection were described, mainly included in the HVR1. Our data indicate that different forces, in addition to the selection pressure, drive an exceptionally fast evolution of HCV during HAART immune restoration. We hypothesize that an important role is played by the enlargement of the viral replicative space

siRNAs: Potential therapeutic agents against Hepatitis C Virus

Hepatitis C virus is a major cause of chronic liver diseases which can lead to permanent liver damage, hepatocellular carcinoma and death. The presently available treatment with interferon plus ribavirin, has limited benefits due to adverse side effects such as anemia, depression and "flu-like" symptoms. Needless to mention, the effectiveness of interferon therapy is predominantly, if not exclusively, limited to virus type 3a and 3b whereas in Europe and North America the majority of viral type is 1a and 2a. Due to the limited efficiency of current therapy, RNA interference (RNAi) a novel regulatory and powerful silencing approach for molecular therapeutics through a sequence-specific RNA degradation process represents an alternative option. Several reports have indicated the efficiency and specificity of synthetic and vector based siRNAs inhibiting HCV replication. In the present review, we focused that combination of siRNAs against virus and host genes will be a better option to treat HC

Novel Small-Molecule Inhibitors of Hepatitis C Virus Entry Block Viral Spread and Promote Viral Clearance in Cell Culture

Combinations of direct-acting anti-virals offer the potential to improve the efficacy, tolerability and duration of the current treatment regimen for hepatitis C virus (HCV) infection. Viral entry represents a distinct therapeutic target that has been validated clinically for a number of pathogenic viruses. To discover novel inhibitors of HCV entry, we conducted a high throughput screen of a proprietary small-molecule compound library using HCV pseudoviral particle (HCVpp) technology. We independently discovered and optimized a series of 1,3,5-triazine compounds that are potent, selective and non-cytotoxic inhibitors of HCV entry. Representative compounds fully suppress both cell-free virus and cell-to-cell spread of HCV in vitro. We demonstrate, for the first time, that long term treatment of an HCV cell culture with a potent entry inhibitor promotes sustained viral clearance in vitro. We have confirmed that a single amino acid variant, V719G, in the transmembrane domain of E2 is sufficient to confer resistance to multiple compounds from the triazine series. Resistance studies were extended by evaluating both the fusogenic properties and growth kinetics of drug-induced and natural amino acid variants in the HCVpp and HCV cell culture assays. Our results indicate that amino acid variations at position 719 incur a significant fitness penalty. Introduction of I719 into a genotype 1b envelope sequence did not affect HCV entry; however, the overall level of HCV replication was reduced compared to the parental genotype 1b/2a HCV strain. Consistent with these findings, I719 represents a significant fraction of the naturally occurring genotype 1b sequences. Importantly, I719, the most relevant natural polymorphism, did not significantly alter the susceptibility of HCV to the triazine compounds. The preclinical properties of these triazine compounds support further investigation of entry inhibitors as a potential novel therapy for HCV infection