Arielle R. Rosenberg

Efficient hepatitis C virus particle formation requires diacylglycerol acyltransferase-1

Hepatitis C virus (HCV) infection is closely tied to the lipid metabolism of liver cells. Here we identify the triglyceride-synthesizing enzyme diacylglycerol acyltransferase-1 (DGAT1) as a key host factor for HCV infection. DGAT1 interacts with the viral nucleocapsid core and is required for the trafficking of core to lipid droplets. Inhibition of DGAT1 activity or RNAi-mediated knockdown of DGAT1 severely impairs infectious virion production, implicating DGAT1 as a new target for antiviral therapy.

Changing of hepatitis C virus genotype patterns in France at the beginning of the third millenium: The GEMHEP GenoCII Study.

Summary.  This cross‐sectional study aimed to investigate, during a short period between 2000 and 2001, in a large population of patients with chronic hepatitis C, the epidemiological characteristics of hepatitis C virus (HCV) genotypes in France. Data from 26 referral centres, corresponding to 1769 patients with chronic hepatitis C were collected consecutively during a 6‐month period. HCV genotyping in the 5′‐non‐coding region (NCR) was performed in each center using the line probe assay (LiPA, in 63% of cases), sequencing (25%) or primer‐specific polymerase chain reaction (PCR) (12%). HCV genotypes 1a, 1b, 2, 3, 4, 5, non‐subtyped 1 and mixed infection were found in 18, 27, 9, 21, 9, 3, 11 and 1% of our population, respectively. HCV genotype distribution was associated with gender, age, source and duration of infection, alanine aminotransferase (ALT) levels, cirrhosis, alcohol consumption, hepatitis B virus (HBV) and human immunodeficiency virus (HIV) coinfection. In multivariate analysis, only the source of infection was the independent factor significantly associated with genotype (P = 0.0001). In conclusion, this study shows a changing pattern of HCV genotypes in France, with i.v. drug abuse as the major risk factor, an increase of genotype 4, and to a lesser extent 1a and 5, and a decrease of genotypes 1b and 2. The modification of the HCV genotype pattern in France in the next 10 years may require new therapeutic strategies, and further survey studies.

Production of Infectious Hepatitis C Virus in Primary Cultures of Human Adult Hepatocytes

BACKGROUND & AIMS Although hepatitis C virus (HCV) can be grown in the hepatocarcinoma-derived cell line Huh-7, a cell-culture model is needed that supports its complete, productive infection cycle in normal, quiescent, highly differentiated human hepatocytes. We sought to develop such a system. METHODS Primary cultures of human adult hepatocytes were inoculated with HCV derived from Huh-7 cell culture (HCVcc) and monitored for expression of hepatocyte differentiation markers and replication of HCV. Culture supernatants were assayed for HCV RNA, core antigen, and infectivity titer. The buoyant densities of input and progeny virus were compared in iodixanol gradients. RESULTS While retaining expression of differentiation markers, primary hepatocytes supported the complete infectious cycle of HCV, including production of significant titers of new infectious progeny virus, which was called primary-culture-derived virus (HCVpc). Compared with HCVcc, HCVpc had lower average buoyant density and higher specific infectivity; this was similar to the characteristics of virus particles associated with the very-low-density lipoproteins that are produced during in vivo infection. These properties were lost after re-culture of HCVpc in poorly differentiated Huh-7 cells, suggesting that authentic virions can be produced only by normal hepatocytes that secrete authentic very-low-density lipoproteins. CONCLUSIONS We have established a cell-culture-based system that allows production of infectious HCV in physiologically relevant human hepatocytes. This provides a useful tool for the study of HCV interactions with its natural host cell and for the development of antiviral therapies.

Safety and Immunogenicity of 4 Intramuscular Double Doses and 4 Intradermal Low Doses vs Standard Hepatitis B Vaccine Regimen in Adults With HIV-1 A Randomized Controlled Trial

CONTEXT Alternative schedules more immunogenic than the standard hepatitis B vaccine regimen are needed in patients with human immunodeficiency virus 1 (HIV-1) infection. OBJECTIVE To compare the safety and immunogenicity of 4 intramuscular double-dose and 4 intradermal low-dose regimens vs the standard hepatitis B vaccine regimen. DESIGN, SETTING, AND PARTICIPANTS An open-label, multicenter, 1:1:1 parallel-group, randomized trial conducted between June 28, 2007, and October 23, 2008 (date of last patient visit, July 3, 2009) at 33 centers in France with patients enrolled in French National Agency for Research on AIDS and Viral Hepatitis trials in adults with HIV-1 infection who were hepatitis B virus (HBV) seronegative and having CD4 cell counts of more than 200 cells/μL. INTERVENTION Patients were randomly assigned to receive 3 intramuscular injections of the standard dose (20 μg) of recombinant HBV vaccine at weeks 0, 4, and 24 (IM20 × 3 group, n = 145); 4 intramuscular double doses (40 μg [2 injections of 20 μg]) of recombinant HBV vaccine at weeks 0, 4, 8, and 24 (IM40 × 4 group, n = 148); or 4 intradermal injections of low doses (4 μg [1/5 of 20 μg]) of recombinant HBV vaccine at weeks 0, 4, 8, and 24 (ID4 × 4 group, n = 144). MAIN OUTCOME MEASURES Percentage of responders at week 28, defined as patients with hepatitis B surface antibody (anti-HBs) of at least 10 mIU/mL in patients who received at least 1 dose of vaccine. Patients with missing anti-HBs titer measurement at the final follow-up visit at week 28 were considered as nonresponders in the primary (efficacy) analysis. RESULTS A total of 437 patients were randomized to the 3 study groups, of whom 11 did not receive any vaccine. Of these, 396 had available anti-HBs titers at week 28. The percentage of responders at week 28 was 65% (95% confidence interval [CI], 56%-72%) in the IM20 × 3 group (n = 91), 82% (95% CI, 77%-88%) in the IM40 × 4 group (n = 119) (P < .001 vs IM20 × 3 group), and 77% (95% CI, 69%-84%) in the ID4 × 4 group (n = 108) (P = .02 vs IM20 × 3 group). No safety signal and no effect on CD4 cell count or viral load were observed. CONCLUSION In adults with HIV-1, both the 4 intramuscular double-dose regimen and the 4 intradermal low-dose regimen improved serological response compared with the standard HBV vaccine regimen. TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00480792.

Hepatitis C virus (HCV) evades NKG2D-dependent NK cell responses through NS5A-mediated imbalance of inflammatory cytokines.

Understanding how hepatitis C virus (HCV) induces and circumvents the hosts natural killer (NK) cell-mediated immunity is of critical importance in efforts to design effective therapeutics. We report here the decreased expression of the NKG2D activating receptor as a novel strategy adopted by HCV to evade NK-cell mediated responses. We show that chronic HCV infection is associated with expression of ligands for NKG2D, the MHC class I-related Chain (MIC) molecules, on hepatocytes. However, NKG2D expression is downmodulated on circulating NK cells, and consequently NK cell-mediated cytotoxic capacity and interferon-γ production are impaired. Using an endotoxin-free recombinant NS5A protein, we show that NS5A stimulation of monocytes through Toll-like Receptor 4 (TLR4) promotes p38- and PI3 kinase-dependent IL-10 production, while inhibiting IL-12 production. In turn, IL-10 triggers secretion of TGFβ which downmodulates NKG2D expression on NK cells, leading to their impaired effector functions. Moreover, culture supernatants of HCV JFH1 replicating Huh-7.5.1 cells reproduce the effect of recombinant NS5A on NKG2D downmodulation. Exogenous IL-15 can antagonize the TGFβ effect and restore normal NKG2D expression on NK cells. We conclude that NKG2D-dependent NK cell functions are modulated during chronic HCV infection, and demonstrate that this alteration can be prevented by exogenous IL-15, which could represent a meaningful adjuvant for therapeutic intervention.

A Plant-Derived Flavonoid Inhibits Entry of All HCV Genotypes Into Human Hepatocytes

BACKGROUND & AIMS Interferon-based therapies for hepatitis C virus (HCV) infection are limited by side effects and incomplete response rates, particularly among transplant recipients. We screened a library of plant-derived small molecules to identify HCV inhibitors with novel mechanisms. METHODS We isolated phenolic compounds from Marrubium peregrinum L (Lamiaceae). Replication of HCV RNA, virus production, and cell entry were monitored using replicons and infectious HCV. Inhibition of HCV was measured in hepatoma cells and primary human hepatocytes using luciferase reporter gene assays, core enzyme-linked immunosorbent assays, or infectivity titration. We tested the bioavailability of the compound in mice. RESULTS We identified a flavonoid, ladanein (BJ486K), with unreported antiviral activity and established its oral bioavailability in mice. Natural and synthetic BJ486K inhibited a post-attachment entry step, but not RNA replication or assembly; its inhibitory concentration 50% was 2.5 μm. BJ486K was effective against all major HCV genotypes, including a variant that is resistant to an entry inhibitor; it prevented infection of primary human hepatocytes. Combined administration of BJ486K and cyclosporine A had a synergistic effect in inhibition of HCV infection. CONCLUSIONS BJ486K has oral bioavailability and interferes with entry of HCV into cultured human hepatocytes. It synergizes with cyclosporine A to inhibit HCV infection. Its inhibitory effects are independent of HCV genotype, including a variant that is resistant to an entry inhibitor against scavenger receptor class B type I. Flavonoid derivatives therefore might be developed as components of combination therapies because they are potent, broadly active inhibitors of HCV entry that could prevent graft reinfection after liver transplantation.

The PPPY motif of human T-cell leukemia virus type 1 Gag protein is required early in the budding process.

ABSTRACT Domains required late in the virus budding process (L domains) have been identified in the Gag proteins of a number of retroviruses. Here we show that the human T-cell leukemia virus type 1 candidate L domain motif PPPY is indeed required for virus production. Strikingly, however, mutation of this motif arrested virus particles at an earlier stage in the budding process than was seen for mutation of the L domain motifs thus far described for retroviruses. In view of the exchangeability of such domains, we propose that the retrovirus budding process may involve a continuum from bud formation to membrane fission.

Hepatitis C viral protein NS5A induces EMT and participates in oncogenic transformation of primary hepatocyte precursors.

BACKGROUND & AIMS Apicobasal polarity, which is essential for epithelial structure and function, is targeted by several tumour-related pathogens and is generally perturbed in the course of carcinogenesis. Hepatitis C virus (HCV) infection is associated with a strong risk of hepatocellular carcinoma, typically preceded by dysplastic alterations of cell morphology. We investigated the molecular mechanisms and the functional consequences of HCV-driven perturbations of epithelial polarity. METHODS We used biochemical, genetic, and cell biology approaches to assess the impact of hepatitis C viral protein NS5A on the polarity and function of hepatocytes and hepatic progenitors. Transgenic animals and xenograft models served for in vivo validation of the results obtained in cell culture. RESULTS We found that expression of HCV-NS5A in primary hepatic precursors and in immortalized hepatocyte cell lines gave rise to profound modifications of cell polarity, leading to epithelial to mesenchymal transition (EMT). NS5A, either alone or in the context of the full complement of viral proteins in the course of infection, acted through activating Twist2, a transcriptional regulator of EMT. The effects of NS5A were additive to those of TGF-β, a cytokine abundant in diseased liver and highly relevant to HCV-related pathology. Moreover, NS5A cooperates with oncogenic Ras, giving rise to transformed, invasive cells that are highly tumorigenic in vivo. CONCLUSIONS Our data suggest that in the context of HCV infection, NS5A favors formation of preneoplastic lesions by disrupting cell polarity and additional oncogenic events cooperate with the viral protein to give rise to motile and invasive tumour cells.

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding, but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.

Sequential processing of hepatitis C virus core protein by host cell signal peptidase and signal peptide peptidase: a reassessment

Summary.  Hepatitis C virus (HCV) core protein is believed to play critical roles in the virus morphogenesis and pathogenesis. In HCV polyprotein, core protein terminates with a signal peptide followed by E1 envelope protein. It has remained unclear whether cleavage by host cell signal peptidase (SP) at the core‐E1 junction to generate the complete form of core protein, which is anchored in the endoplasmic reticulum membrane, is absolutely required for cleavage within the signal peptide by host cell signal peptide peptidase (SPP) to liberate the mature form of core protein, which is then free for trafficking to lipid droplets. In this study, the possible sources of disagreement in published reports have been examined, and we conclude that a product generated upon inhibition of SP‐catalysed cleavage at the core‐E1 junction in heterologous expression systems was incorrectly identified as mature core protein. Moreover, inhibition of this cleavage in the most relevant model of human hepatoma cells replicating a full‐length HCV genome was shown to abolish interaction of core protein with lipid droplets and production of infectious progeny virus. These results firmly establish that SPP‐catalysed liberation of mature core protein is absolutely dependent on prior cleavage by SP at the correct core‐E1 site to generate the complete form of core protein, consistent with this obligatory order of processing playing a role in HCV infectious cycle.