Catherine François

Expression of Hepatitis C Virus Proteins Interferes with the Antiviral Action of Interferon Independently of PKR-Mediated Control of Protein Synthesis

ABSTRACT Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2α (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208–5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262–1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2α after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2α phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.

Genetic recombination of the hepatitis C virus: clinical implications

Summary.  Genetic recombination is a well‐known feature of RNA viruses that plays a significant role in their evolution. Although recombination is well documented for Flaviviridae family viruses, the first natural recombinant strain of hepatitis C virus (HCV) was identified as recently as 2002. Since then, a few other natural inter‐genotypic, intra‐genotypic and intra‐subtype recombinant HCV strains have been described. However, the frequency of recombination may have been underestimated because not all known HCV recombinants are screened for in routine practice. Furthermore, the choice of treatment regimen and its predictive outcome remain problematic as the therapeutic strategy for HCV infection is genotype dependent. HCV recombination also raises many questions concerning its mechanisms and effects on the epidemiological and physiopathological features of the virus. This review provides an update on recombinant HCV strains, the process that gives rise to recombinants and clinical implications of recombination.

Ribavirin monitoring in chronic hepatitis C therapy: anaemia versus efficacy.

The standard treatment of HCV infection with pegylated interferon-alpha2a or -alpha2b and ribavirin is effective in <50% of HCV genotype-1-infected patients. To improve this figure, it might be desirable to obtain optimal plasma concentrations of the drug by increasing the dose. Unfortunately, there is great interpatient variability in ribavirin pharmacokinetics. In the present review, we describe the mechanism of ribavirin-induced anaemia in detail, evaluate host predictive factors for this harmful side effect and assess the literature data on attempts to improve the sustained virological response rate by increasing the dose of ribavirin. We suggest an optimal steady-state concentration range for ribavirin in monoinfected and coinfected patients. Lastly, we propose that it would be of particular value to monitor ribavirin concentrations in HCV genotype-1-infected patients and (regardless of the genotype) coinfected patients, haemodialyzed patients and obese patients.

The retinoblastoma (Rb) protein regulates ferroptosis induced by sorafenib in human hepatocellular carcinoma cells

Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC), the most frequent form of primary liver tumour. The loss of function of the retinoblastoma (Rb) protein is an important event during liver carcinogenesis, but it is unclear whether the Rb status modulates the response of HCC cells to sorafenib. Here, we examined this question in HCC cells with reduced levels of Rb achieved through stable RNA interference. We show that HCC cells with reduced levels of Rb exhibit a two- to threefold increase in cell death induction upon exposure to sorafenib compared with controls. Sorafenib treatment of Balb/c nude mice that received tumour xenografts derived from HCC cells with reduced Rb levels resulted in complete tumour regression in 50% of the animals treated, compared with tumour stabilization in mice that received control cells. We show that, upon exposure to sorafenib, the Rb-negative status of HCC cells promotes the occurrence of ferroptosis, a form of oxidative necrosis. The findings highlight the role of Rb in the response of HCC cells to sorafenib and the regulation of ferroptosis.

Emergence of a genomic variant of the recombinant 2k/1b strain during a mixed Hepatitis C infection: a case report.

To date, few natural intergenotypic recombinant hepatitis C virus (HCV) strains have been characterized. A recombinant strain 2k/1b was detected for one HCV RNA-positive individual who had just completed therapy for HCV 3a genotype infection. In the present report, five serum samples collected over the pre- and post-treatment periods were used to investigate all the present HCV strains and the change over time of the infection pattern. Interestingly, the 2k/1b strain was already present during the genotype 3a infection and persisted during treatment. In the specimen collected three months post-treatment, two distinct strains, 2k/1b and type 1, were found and then one 2k/1b strain in the subsequent ones. A genomic variant of the HCV RF1_2k/1b strain was identified. It was part of a mixed HCV infection and persisted and re-emerge after eradication of the dominant subtype 3a. This case indicates that HCV co-infection screening after relapse should be an alternative to explain the lack of response to treatment and the necessity to carefully study the epidemic spreading of this recombinant strain.

BAD, a proapoptotic member of the BCL2 family, is a potential therapeutic target in hepatocellular carcinoma.

Proteins of the BCL2 family are key regulators of apoptosis. Their expression levels are frequently altered in cancers, enabling tumor cells to survive. To gain insight into the pathogenesis of hepatocellular carcinoma (HCC), we performed a comprehensive survey of the expression of the members of the BCL2 family in samples obtained from surgically resected HCCs. Here, we report the occurrence of a new molecular anomaly, consisting of a strong reduction in the expression of the proapoptotic protein BAD in HCC compared with surrounding nontumoral tissue. We investigate the function of BAD in a panel of HCC cell lines. Using gene overexpression and RNA interference, we show that BAD is involved in the cytotoxic effects of sorafenib, a multikinase blocker, which is currently the sole therapeutic drug effective for the treatment of HCC. Finally, we report that ABT-737, a compound that interacts with proteins of the BCL2 family and exhibits a BAD-like reactivity, sensitizes HCC cells toward sorafenib-induced apoptosis. Collectively, our findings indicate that BAD is a key regulator of apoptosis in HCC and an important determinant of HCC cell response to sorafenib. Mol Cancer Res; 8(8); 1116–25. ©2010 AACR.

The antimalarial ferroquine is an inhibitor of hepatitis C virus

Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti‐HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)‐free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell‐cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell‐free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell‐to‐cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. Conclusion: FQ is a novel, interesting anti‐HCV molecule that could be used in combination with other direct‐acting antivirals. (HEPATOLOGY 2013)

Strong Correlation between Liver and Serum Levels of Hepatitis C Virus Core Antigen and RNA in Chronically Infected Patients

ABSTRACT HCV core antigen (Ag) and HCV RNA levels were evaluated in matched liver biopsy samples and sera from 22 patients with hepatitis C infection by using the quantitative Architect HCV Ag immunoassay and a real-time RT-qPCR assay, respectively. The data showed a strong correlation between liver and serum compartments of HCV Ag levels (r = 0.80) and HCV RNA levels (r = 0.87). In summary, the serum HCV Ag and RNA levels reflect the intrahepatic values.

Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication

ABSTRACT Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genus Orthohepevirus in the family Hepeviridae. HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I interferon (IFN) has been evaluated in a few infected transplant patients in vivo. In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication. Simultaneous determination of interferon-stimulated gene (ISG) expression levels for all IFN types demonstrated efficient downregulation by HEV. Furthermore, different IFN-α subtypes were also able to block viral replication in combination with ribavirin. The IFN-α subtypes 2a and 2b exerted the strongest antiviral activity against HEV. In conclusion, these data demonstrate for the first time moderate anti-HEV activities of types II and III IFNs and different IFN-α subtypes. As HEV employed a potent anti-interferon mechanism by restricting ISG expression, exogenous application of IFNs as immunotherapy should be carefully assessed.

Variability of the Nonstructural 5A Protein of Hepatitis C Virus Type 3a Isolates and Relation to Interferon Sensitivity

The nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) genotype 1 is thought to interact with several cellular proteins, including the double-stranded RNA-dependent protein kinase (PKR) induced by interferon (IFN). The PKR-binding domain (PKR-BD; aa 2209-2274), including the IFN sensitivity-determining region (aa 2209-2248) and other regions, could be linked to IFN resistance. Thus, the entire NS5A sequence of 27 isolates of HCV genotype 3a was investigated in relation to the clinical response to IFN. The NS5A 3a protein presented a low variability with some specific variable regions. Differential analysis between IFN-resistant and -sensitive isolates identified 5 regions in NS5A, 2 of them inside the PKR-BD and another around the variable 3 region. However, using the yeast growth suppression assay, no interaction was found between 5 resistant NS5A 3a proteins and PKR. Some amino acid changes of the NS5A protein of genotype 3a seemed to relate to IFN resistance independently of the PKR pathway.