Recent years have seen the rapid advancement of new therapeutic agents against hepatitis C virus ( HCV ) in response to the need for treatment that is unmet by interferon-based therapies. Most antiviral drugs discovered to date are small molecules that modulate viral enzyme activities. In the search for highly selective protein-binding molecules capable of disrupting viral life cycle, we have identified a class of anionic tetraphenylporphyrins as potent and specific inhibitors of the HCV replicons. Based on the structure-activity relationship studies reported herein, meso-tetrakis-(3,5-dicarboxy-4,4'-biphenyl) porphyrin was found to be the most potent inhibitor of HCV genotype 1b ( Con1 ) replicon systems but was less effective against genotype 2a ( JFH-1 ) replicon. This compound induced a reduction of viral RNA and protein levels when acting in the low nanomolar range. Moreover the compound could suppress replicon rebound in drug-treated cells and exhibited additive to synergistic effects when combined with protease inhibitor BILN 2061 or with IFNalpha-2a. Our results demonstrate the potential use of tetracarboxyphenylporphyrins as potent anti-HCV agents.

The hepatitis C virus (HCV) NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly. Through forward genetic selection for variants capable of virus spread, we identified second-site mutations in E1, E2, NS2, NS3, and NS4A that suppressed NS2 defects in assembly. Two suppressor mutations, E1 A78T and NS3 Q221L, were further characterized by additional genetic and biochemical experiments. Both mutations were shown to suppress other NS2 defects, often with mutual exclusivity. Thus, several NS2 mutants were enhanced by NS3 Q221L and inhibited by E1 A78T, while others were enhanced by E1 A78T and inhibited by NS3 Q221L. Furthermore, we show that the NS3 Q221L mutation lowers the affinity of native, full-length NS3-4A for functional RNA binding. These data reveal a complex network of interactions involving NS2 and other viral structural and nonstructural proteins during virus assembly.

Non-structural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The non-structural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate co-factor for NS3 serine protease activity. Given its role in stimulating protease activity, we sought to determine if NS4A also influences the activity of NS3hel. Here we show that NS4A enhances the ability of NS3hel to bind RNA in the presence of ATP, thereby acting as a co-factor for helicase activity. This effect is mediated by amino acids in the C-terminal acidic domain of NS4A. When these residues are mutated, one observes drastic reductions in ATP-coupled RNA binding and duplex unwinding by NS3. These same mutations are lethal in HCV replicons, thereby establishing in-vitro and in-vivo that NS4A plays an important role in the helicase mechanism of NS3 and its function in replication.

Since the advent of efficient cell-culture methods for HCV replication and, more recently, infection, there has been a need to efficiently sequence the viral RNA in these systems. This need is especially urgent in light of the error-prone nature of HCV RNA replication, which leads to a variety of interesting mutations. The adaptation of hepatitis C replicons to cell culture, which greatly increased their replication capacity, and the subsequent identification of viral point mutations responsible for this adaptation are prime examples of the type of phenotype-genotype connection that viral RNA sequencing methods can provide. More recently, researchers have used similar sequencing methods to identify changes in replicons that represent viral adaptation to engineered mutations, adaptation to a variety of host cells, and viral evasion of antiviral compound susceptibility. Here, we describe the cloning and isolation of HCV replicon-bearing cells, the extraction of total RNA, the generation of cDNA, and the amplification of specific HCV replicon sequences for sequence analysis. The methods we describe permit rapid and robust determination of HCV RNA sequences from cell culture.

Recently identified hepatitis C virus (HCV) isolates that are infectious in cell culture provide a genetic system to evaluate the significance of virus-host interactions for HCV replication. We have completed a systematic RNAi screen wherein siRNAs were designed that target 62 host genes encoding proteins that physically interact with HCV RNA or proteins or belong to cellular pathways thought to modulate HCV infection. This includes 10 host proteins that we identify in this study to bind HCV NS5A. siRNAs that target 26 of these host genes alter infectious HCV production >3-fold. Included in this set of 26 were siRNAs that target Dicer, a principal component of the RNAi silencing pathway. Contrary to the hypothesis that RNAi is an antiviral pathway in mammals, as has been reported for subgenomic HCV replicons, siRNAs that target Dicer inhibited HCV replication. Furthermore, siRNAs that target several other components of the RNAi pathway also inhibit HCV replication. MicroRNA profiling of human liver, human hepatoma Huh-7.5 cells, and Huh-7.5 cells that harbor replicating HCV demonstrated that miR-122 is the predominant microRNA in each environment. miR-122 has been previously implicated in positively regulating the replication of HCV genotype 1 replicons. We find that 2'-O-methyl antisense oligonucleotide depletion of miR-122 also inhibits HCV genotype 2a replication and infectious virus production. Our data define 26 host genes that modulate HCV infection and indicate that the requirement for functional RNAi for HCV replication is dominant over any antiviral activity this pathway may exert against HCV.

Hepatitis C virus (HCV) nonstructural (NS) protein 4A is only 54 aa in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in eight of the fifteen C-terminal residues of NS4A, individual Ala substitutions or charge-reversal substitutions led to severe replication phenotypes as well as decreased NS5A hyperphosphorylation. By selecting for replication competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19-aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C-terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.

Superinfection exclusion is the ability of an established virus infection to interfere with infection by a second virus. In this study we found that Huh-7.5 cells acutely infected with genotype 2a hepatitis C virus (chimeric strain J6/JFH) and cells harboring genotype 1a, 1b, or 2a HCV full-length or subgenomic replicons were resistant to infection with cell culture produced HCV (HCVcc). Replicon-containing cells became permissive for HCVcc infection after treatment with an HCV-specific protease inhibitor. With the exception of cells harboring a J6/JFH-FLneo replicon, infected or replicon-containing cells were permissive for HCV pseudoparticle (HCVpp) entry, demonstrating a post-entry superinfection block, downstream of primary translation. The surprising resistance of J6/JFH-FLneo replicon cells to HCVpp infection suggested a defect in virus entry. This block was due to reduced expression of the HCV co-receptor, CD81. Further analyses indicated that J6/JFH may be toxic for cells expressing high levels of CD81, thus selecting for a CD81low population. CD81 down regulation was not observed in acutely infected cells suggesting that this may not be a general mechanism of HCV superinfection exclusion. Thus, HCV establishes superinfection exclusion at a post-entry step and this effect is reversible by treatment of infected cells with antiviral compounds.

Background & Aims: Modulation of the host innate immune response is an attractive means of inhibiting hepatitis C virus (HCV) replication. Having previously determined that expression of the interferon-sensitive gene (ISG)15 protease USP18 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-alfa therapy, we hypothesized that USP18 might hinder the ability of IFN to inhibit HCV replication. Methods: The role of USP18 in IFN antiviral activity was examined using an in vitro model of HCV replication that reproduces the full viral life cycle. USP18 was silenced specifically using small inhibitory RNAs (siRNAs), and the dose response of HCV replication and infectious virus production to IFN-alfa was measured. Results: The siRNA knockdown of USP18 in human cells consistently potentiated the ability of IFN to inhibit HCV-RNA replication and infectious virus particle production by a factor of 1-2 log(10). USP18 knockdown also resulted in a number of cellular changes consistent with increased sensitivity to IFN. Decreasing USP18 expression led to increased cellular protein ISGylation in response to exogenous IFN-alfa, prolonged tyrosine phosphorylation of signal transducer and activation of transcription (STAT1), and a general enhancement of IFN-stimulated gene expression. Conclusions: These data suggest that USP18 modulates the anti-HCV type I IFN response, and is a possible therapeutic target for the treatment of HCV infection.

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 IC(50) of 1.5 mug/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.(HEPATOLOGY 2006;43:932-942.).

Hepatitis B virus (HBV) and hepatitis C virus (HCV) are the major viruses known to cause viral hepatitis. Serological markers are commonly used as diagnostic and/or prognostic indicators of acute or chronic HBV or HCV infection. The ability to detect HBV DNA in serum has been reported to have prognostic value for the outcome of chronic HBV infection. A rapid and sustained drop in HBV DNA or HCV RNA levels in patients under therapy has been shown to be a predictive factor for a favourable treatment outcome. Various techniques for detecting HBV DNA or HCV RNA have already been described; however, there are various problems with the sensitivity or detection range of those methods. New virus measuring methods have recently been reported and used. The Cobas Taq Man HCV Test is a new method to detect HBV DNA and HCV RNA with higher sensitivity and a broader range of quantitation than conventional methods. Some reports have shown that these methods improve therapy monitoring and the management of HBV or HCV infection. Moreover, hepatitis E virus (HEV) infection has been reported in Japan. The clinical features and viral markers of HEV have also been described.

Systemic vasculitis is a rare but devastating problem in patients with human immunodeficiency virus (HIV). The coinfection with hepatitis C virus (HCV) further complicates the clinical management. We report a 46-year-old woman coinfected with HCV and HIV with a CD4 count of 950/mm who presented with a life-threatening vasculitis of the lungs, kidneys, and skin and who initially responded after use of corticosteroids and then 2 monthly pulses of i.v. cyclophosphamide. Her condition deteriorated when she was switched to azathioprine. Ultimately, the patient died of neutropenic sepsis. On the basis of our experience and an analysis of the literature, we suggest that monthly pulsed i.v. cyclophosphamide and steroids might be used as an induction therapy, followed by antiviral treatment for patients with HIV, HCV, and a life-threatening ischemic vasculitis if the CD4 count is >400/mm. For patients in this complex condition who are receiving immunosuppressants close surveillance for signs of secondary infection, and prophylactic trimethoprim/sulfamethoxazole, are advised. The use of interferon alpha, ribavirin, i.v. immunoglobulin, and plasmapheresis are alternatives for patients with milder vasculitis.