Caroline Scholtes

Secretion of Hepatitis C Virus Envelope Glycoproteins Depends on Assembly of Apolipoprotein B Positive Lipoproteins

The density of circulating hepatitis C virus (HCV) particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB) positive and triglyceride rich lipoproteins (TRL) likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP) containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1–E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

Transactivation of the Hepatitis B Virus Core Promoter by the Nuclear Receptor FXRα

ABSTRACT Hepatitis B virus (HBV) core promoter activity is positively and negatively regulated by nuclear receptors, a superfamily of ligand-activated transcription factors, via cis-acting sequences located in the viral genome. In this study, we investigated the role of farnesoid X receptor alpha (FXRα) in modulating transcription from the HBV core promoter. FXRα is a liver-enriched nuclear receptor activated by bile acids recognizing hormone response elements by forming heterodimers with retinoid X receptor alpha (RXRα). Electrophoretic mobility shift assays demonstrated that FXRα-RXRα heterodimers can bind two motifs on the HBV enhancer II and core promoter regions, presenting high homology to the consensus (AGGTCA) inverted repeat FXRα response elements. In transient transfection of the human hepatoma cell line Huh-7, bile acids enhanced the activity of a luciferase reporter containing the HBV enhancer II and core promoter sequences through FXRα. Moreover, using a greater-than-genome-length HBV construct, we showed that FXRα also increased synthesis of the viral pregenomic RNA and DNA replication intermediates. The data strongly suggest that FXRα is another member of the nuclear receptor superfamily implicated in the regulation of HBV core promoter activity and that bile acids could play an important role in the natural history of HBV infection.

High plasma level of nucleocapsid‐free envelope glycoprotein‐positive lipoproteins in hepatitis C patients

Hepatitis C virus (HCV) particles associate viral and lipoprotein moieties to form hybrid lipoviral particles (LVPs). Cell culture–produced HCV (HCVcc) and ex vivo–characterized LVPs primarily differ by their apolipoprotein (apo) B content, which is low for HCVcc, but high for LVPs. Recombinant nucleocapsid‐free subviral LVPs are assembled and secreted by apoB‐producing cell lines. To determine whether such subviral particles circulate in HCV‐infected individuals, LVPs complexed with immunoglobulin were precipitated with protein A from low‐density plasma fractions of 36 hepatitis C patients, and their lipid content, apolipoprotein profile, and viral composition were determined. HCV RNA in LVPs was quantified and molar ratios of apoB and HCV genome copy number were calculated. LVPs lipidome from four patients was determined via electrospray ionization/tandem mass spectrometry. Protein A–purified LVPs contained at least the envelope glycoprotein E2 and E2‐specific antibodies. LVPs were present in every patient and were characterized by high lipid content, presence of apolipoproteins characteristic of triglyceride‐rich lipoproteins (TRLs), HCV RNA, and viral glycoprotein. Importantly, save for four patients, LVPs fractions contained large amounts of apoB, with on average more than 1 × 106 apoB molecules per HCV RNA genome. Because there is one apoB molecule per TRL, this ratio suggested that most LVPs are nucleocapsid‐free, envelope glycoprotein‐containing subviral particles. LVPs and TRLs had similar composition of triacylglycerol and phospholipid classes. Conclusion: LVPs are a mixed population of particles, comprising predominantly subviral particles that represent a distinct class of modified lipoproteins within the TRL family. (HEPATOLOGY 2012;56:39–48)

Hepatitis E virus mutations associated with ribavirin treatment failure result in altered viral fitness and ribavirin sensitivity

BACKGROUND & AIMS Ribavirin monotherapy is the preferred treatment for chronic hepatitis E, although occasional treatment failure occurs. We present a patient with chronic hepatitis E experiencing ribavirin treatment failure with a completely resistant phenotype. We aimed to identify viral mutations associated with treatment failure and explore the underlying mechanisms. METHODS Viral genomes were deep-sequenced at different time points and the role of identified mutations was assessed in vitro using mutant replicons, antiviral assays, cell culture of patient-derived virus and deep-sequencing. RESULTS Ribavirin resistance was associated with Y1320H, K1383N and G1634R mutations in the viral polymerase, but also an insertion in the hypervariable region comprising a duplication and a polymerase-derived fragment. Analysis of these genome alterations in vitro revealed replication-increasing roles for Y1320H and G1634R mutations and the hypervariable region insertion. In contrast, the K1383N mutation in the polymerase F1-motif suppressed viral replication and increased the in vitro sensitivity to ribavirin, contrary to the clinical phenotype. Analysis of the replication of mutant full-length virus and in vitro culturing of patient-derived virus confirmed that sensitivity to ribavirin was retained. Finally, deep-sequencing of hepatitis E virus genomes revealed that ribavirin is mutagenic to viral replication in vitro and in vivo. CONCLUSIONS Mutations Y1320H, G1634R and the hypervariable region insertion compensated for K1383N-associated replication defects. The specific role of the K1383N mutation remains enigmatic, but it appears to be of importance for the ribavirin resistant phenotype in this patient. LAY SUMMARY Ribavirin is the most common treatment for chronic hepatitis E and is mostly effective, although some cases of ribavirin treatment failure have been described. Here, we report on a particular case of ribavirin resistance and investigate the underlying causes of treatment failure. Mutations in the viral polymerase, an essential enzyme for viral replication, appear to be responsible.

The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription

Hepatitis B virus (HBV) genome transcription is highly dependent on liver‐enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor a (FXRa) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food‐withdrawal‐induced FXRα modulators, peroxisome proliferator‐activated receptor‐γ coactivator 1α (PGC‐1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC‐1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocar‐cinoma‐derived Huh‐7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter‐controlled luciferase expression by 25‐fold, compared with a 10‐fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα‐ and PGC‐1 α‐dependent manner. Maximal activation (> 150‐fold) was observed in FXRα‐and PGC‐1α‐overexpressing Huh‐7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full‐length HBV genomes, maximal induction (3.5‐fold) of core promoter‐controlled synthesis of 3.5‐kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.—Curtil, C., Enache, L. S., Radreau, P., Dron, A.‐G., Scholtès, C., Deloire, A., Roche, D., Lotteau, V., André, P., and Ramière, C. The metabolic sensors FXRα, PGC‐1 α, and SIRT1 cooperatively regulate hepatitis B virus transcription. FASEB J. 28, 1454–1463 (2014). www.fasebj.org

Naturally Occurring Resistance-Associated Variants of Hepatitis C Virus Protease Inhibitors in Poor Responders to Pegylated Interferon-Ribavirin

ABSTRACT The pretherapeutic presence of protease inhibitor (PI) resistance-associated variants (RAVs) has not been shown to be predictive of triple-therapy outcomes in treatment-naive patients. However, they may influence the outcome in patients with less effective pegylated interferon (pegIFN)-ribavirin (RBV) backbones. Using hepatitis C virus (HCV) population sequence analysis, we retrospectively investigated the prevalence of baseline nonstructural 3 (NS3) RAVs in a multicenter cohort of poor IFN-RBV responders (i.e., prior null responders or patients with a viral load decrease of <1 log IU/ml during the pegIFN-RBV lead-in phase). The impact of the presence of these RAVs on the outcome of triple therapy was studied. Among 282 patients, the prevalances (95% confidence intervals) of baseline RAVs ranged from 5.7% (3.3% to 9.0%) to 22.0% (17.3% to 27.3%), depending to the algorithm used. Among mutations conferring a >3-fold shift in 50% inhibitory concentration (IC50) for telaprevir or boceprevir, T54S was the most frequently detected mutation (3.9%), followed by A156T, R155K (0.7%), V36M, and V55A (0.35%). Mutations were more frequently found in patients infected with genotype 1a (7.5 to 23.6%) than 1b (3.3 to 19.8%) (P = 0.03). No other sociodemographic or viroclinical characteristic was significantly associated with a higher prevalence of RAVs. No obvious effect of baseline RAVs on viral load was observed. In this cohort of poor responders to IFN-RBV, no link was found with a sustained virological response to triple therapy, regardless of the algorithm used for the detection of mutations. Based on a cross-study comparison, baseline RAVs are not more frequent in poor IFN-RBV responders than in treatment-naive patients and, even in these difficult-to-treat patients, this study demonstrates no impact on treatment outcome, arguing against resistance analysis prior to treatment.

Low‐level viremia is associated with non‐B subtypes in patients infected with HIV with virological success following HAART introduction

This prospective study aimed to determine factors associated with detection of very low‐level viremia in patients infected with HIV‐1 with virological success following HAART introduction. Fifty‐seven patients, mostly (n = 51, 89%) treated with a protease inhibitor‐based regimen, were included and followed for 2 years. Viral loads were monitored by Abbott m2000 RealTime HIV‐1. Patients were classified as (i) HIV‐RNA‐negative if viral loads remained strictly undetectable (0 copies/ml), or (ii) HIV‐RNA‐positive if at least one HIV‐1 RNA could be detected in 1–49 copies/ml during follow‐up. At month 24, 44 patients (77%) were in the HIV‐RNA‐positive group, whereas 13 (23%) remained without very low‐level viremia. Univariate analysis, Kaplan–Meier curves and the Cox proportional hazard model revealed that B subtype was the only predictor of belonging to the HIV‐RNA‐negative group (HR 3.98; 95% CI 1.08–14.7). This association needs to be confirmed. Further study of the reservoir and the mechanisms of viral latency according to HIV‐subtype will also be necessary to develop new therapeutic strategies and eradicate HIV infection. J. Med. Virol. 85: 953–958, 2013.

Hepatitis C virus spread from HIV-positive to HIV-negative men who have sex with men

The aim of this study was to evaluate the potential transmission of HCV strains between HIV-positive men who have sex with men (MSM) and HIV-negative MSM. Since 2000, an ongoing epidemic of HCV infections is observed among HIV-positive MSM in high-income countries. However, HCV infections in HIV-negative MSM are investigated to a lesser extent due to the lack of follow-up in this population and only limited information is available on the risk of HCV transmission between HIV-positive MSM and HIV-negative MSM. We enrolled 49 MSM of which 43 were HIV-positive and 6 HIV-negative, including 4 being enrolled or waiting for enrolment in a preexposure prophylaxis (PrEP) program. All patients were diagnosed with acute HCV infection at the Infectious Disease Unit at the Hospices Civils de Lyon from 2014 to 2016. Risk factors for HCV infection were similar in both groups and included IV or nasal drug use, and rough sex practices. Typing and phylogenetic cluster analysis of HCV variants were performed by NS5B sequencing. Several clusters of infections were identified (genotype 1a: 3 clusters and 1 pair; genotype 4d: 1 cluster and 2 pairs), suggesting that several transmission events occurred within the study population. Every HCV strain identified in HIV-negative MSM was included in a cluster with HIV-positive MSM. Chronological analysis of contagiousness suggested the transmission of HCV from HIV-positive to HIV-negative patients. We conclude that recommendations for HCV surveillance should not be confined to HIV-positive MSM but should be extended to HIV-negative MSM with similar risk factors.

Hepatitis A outbreak in HIV-infected MSM and in PrEP-using MSM despite a high level of immunity, Lyon, France, January to June 2017

Since 2016, an increase in the number of hepatitis A cases affecting mainly men who have sex with men (MSM) has been reported in low endemic countries in Europe. We calculated the attack rate in Lyon, France, in populations considered at high-risk: HIV-infected MSM and HIV-negative MSM receiving HIV pre-exposure prophylaxis (PrEP). In these populations, high level of immunity did not prevent the outbreak, indicating that vaccination should be reinforced, particularly in younger individuals.

Multicenter Quality Control of Hepatitis C Virus Protease Inhibitor Resistance Genotyping

ABSTRACT Hepatitis C virus (HCV) protease inhibitor resistance-associated substitutions are selected during triple-therapy breakthrough. This multicenter quality control study evaluated the expertise of 23 French laboratories in HCV protease inhibitor resistance genotyping. A panel of 12 well-defined blinded samples comprising two wild-type HCV strains, nine transcripts from synthetic NS3 mutant samples or from clinical strains, and one HCV RNA-negative sample was provided to the participating laboratories. The results showed that any laboratory with expertise in sequencing techniques should be able to provide reliable HCV protease inhibitor resistance genotyping. Only a 0.7% error rate was reported for the amino acid sites studied. The accuracy of substitution identification ranged from 75% to 100%, depending on the laboratory. Incorrect results were mainly related to the methodology used. The results could be improved by changing the primers and modifying the process in order to avoid cross-contamination. This study underlines the value of quality control programs for viral resistance genotyping, which is required prior to launching observational collaborative multicenter studies on HCV resistance to direct-acting antiviral agents.