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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.

HIV-1 Load Comparison Using Four Commercial Real-Time Assays

ABSTRACT The HIV-1 RNA viral load is commonly used for the monitoring of disease progression and antiretroviral treatment of HIV-1-infected patients. Since the misestimating of values could lead to inappropriate therapeutical management, the comparative performances, especially the ability to span the genetic diversity of HIV-1, of available automated real-time assays need to be evaluated. We conducted a prospective study with 74 consenting patients enrolled between March 2007 and November 2008. A blood sample was obtained at the time of diagnosis of HIV seropositivity and blindly tested for HIV-1 RNA by at least 4 commercial tests: the Abbott m2000 RealTime HIV-1, bioMérieux NucliSens EasyQ HIV-1, version 1.2 (v1.2), and Cobas AmpliPrep/Cobas TaqMan (CAP/CTM) v1.0 and v2.0 assays. The means of difference were null between CAP/CTM v2.0 and Abbott for CRF02_AG subtypes but positive in favor of CAP/CTM v2.0 for genotype B and negative in favor of NucliSens for all genotypes. The standard deviation (SD) of difference ranged from 0.3 to 0.59, depending on the considered couples of assays. Reliabilities of these four tests, appreciated by the standard deviation of difference between the measurement and the estimated “true” viral load and by the coefficient of reliability, were significantly different (P < 10−4) among each other. Significant differences were also observed within each group of HIV-1 genotype. The global disparity was higher for CRF02_AG than for B subtypes. This study indicates a risk of viral load misestimating or discrepancies between techniques, depending on the HIV-1 subtype, and speaks in favor of using the same assay for the monitoring of HIV-1-infected patients.

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.

A case of Mayaro virus infection imported from French Guiana.

Emergence of arboviruses is a rising problem in several areas in the world. Here we report a case of Mayaro virus infection that was diagnosed in a French citizen presenting a dengue-like syndrome with prolonged arthralgia following a travel in French Guiana. Diagnosis was based on serological testing, a newly developed specific RT-PCR and sequencing. The real incidence of this viral infection among travelers is poorly known but this case is the first reported in a European area where Aedes albopictus mosquitoes are established, which underscores the necessity to determine the vector competence of the European strain of this mosquito species for Mayaro virus.

Morphological Characterization and Fusion Properties of Triglyceride-rich Lipoproteins Obtained from Cells Transduced with Hepatitis C Virus Glycoproteins

The density of hepatitis C virus (HCV) particles circulating in the blood of chronically infected patients and of cell-culture produced HCV is heterogeneous. Specific infectivity and fusion of low density particles are higher than those of high density particles. We recently characterized hybrid particles produced by Caco-2 colon or Huh-7.5 liver cells transduced with HCV E1 and E2 envelope glycoproteins. Caco-2-derived particles, called empty lipo-viral particles (eLVP), are composed of triglyceride-rich lipoproteins positive for apolipoproteins B (i.e. apoB100 and apoB48) and contain HCV E1 and E2. Here we aimed at characterizing the morphology and in vitro fusion properties of eLVP using electron microscopy and fluorescence spectroscopy. They displayed the aspect of β-lipoproteins, and immunogold labeling confirmed the presence of apoB and HCV E1 and E2 at their surface. These particles are able to fuse with lipid bilayers (liposomes) in a fusion process leading to the coalescence of internal contents of triglyceride-rich lipoproteins particles and liposomes. Fusion was pH-dependent and could be inhibited by either Z-fFG, a peptide known to inhibit viral fusion, or by monoclonal antibodies directed against HCV E2 or the apolipoprotein moiety of the hybrid particle. Interestingly, particles derived from Huh-7.5 cells failed to display equivalent efficient fusion. Optimal fusion activity is, thus, observed when HCV envelope proteins are associated to apoB-positive hybrid particles. Our results, therefore, point to a crucial role of the E1 and E2 proteins in HCV fusion with a subtle interplay with the apolipoprotein part of eLVP.

Discordance in HIV-1 Co-Receptor Use Prediction by Different Genotypic Algorithms and Phenotype Assay: Intermediate Profile in Relation to Concordant Predictions

Concordant and discordant genotypic predictions of HIV‐1 co‐receptor tropism were analyzed. V3 region was sequenced from plasma samples of patients screened for R5 tropism by the Trofile® assay, before CCR5 antagonist prescription. Ten tools including geno2pheno, PSSM, an “11/25” and “net charge” rule, and other published algorithms were used. Patients were grouped according to concordance or discordance between tools and Trofile® result. Trofile® tropism reports from 50 patient samples were R5 in 38 and Dual/Mixed (DM) in 12. Prediction with the genotypic tools were concordant for 23 R5 samples, and discordant for the 15 other ones. From Trofile® DM strains were concordant in 6 and discordant in 6. V3 sequences were not clearly distinct between R5 and DM strains, except a greater diversity in the later. Discordances were found with any tool or combination of them, so that no one can be proposed as better than the others. Predictive values of each algorithm were similar and rather good (efficacy ranged from 74% to 84%), but the rate of non‐confirmed prediction is greater when compelling the results of all tools with each individual sample. The mean of quantitative values obtained with one tool when another tool give the opposite prediction were different from those obtained when all tools agree with that prediction. The two discordant groups were often not distinguishable from each other. These results suggest that viruses giving discordant prediction with bioinformatic tools could be functionally distinct and/or in a different evolutionary state compared to those with concordant prediction. J. Med. Virol. 84:402–413, 2012.

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.

Variants With Different Mutation Patterns Persist in the Quasispecies of Enfuvirtide-Resistant HIV-1 Population During and After Treatment In Vivo

Background:Genotypic and phenotypic resistance in 11 HIV-1-infected patients receiving enfuvirtide (ENF), as part of a salvage regimen, has been evaluated. Methods:Resistance mutations were detected by sequencing the gp41 ectodomain from plasma samples. During treatment, longitudinal samples from 1 patient were sequenced after limiting dilution of complementary DNA to isolate single genomes. Phenotypic resistance was evaluated with a new recombinant virus assay (PHENOSCRIPT; VIRalliance, Paris, France), allowing the determination of coreceptor use. Results:All patients experienced ENF failure. One to 4 mutations in the 36-to-45 gp41 region appeared during ENF therapy in all patients and disappeared after ENF removal. Mixtures of wild type and mutants unexpectedly persisted under ENF treatment, however, despite continued replication, leading to discordant results between genotypic and phenotypic data. Sequencing of isolated genomes from 1 patient confirmed that a wild-type first heptad repeat region (HR1) region was still present at the end of therapy. Several mutated variants coexisted at different time points, despite a tendency toward quasispecies reduction with time. Conclusion:Individual variability of the mutation pattern and persistence of strains without mutation in the region mainly targeted by ENF resistance probably reflect the fact that resistance to ENF may rely on regions of gp41 or gp120 other than residues 36 to 45.

Comparison of HIV-1 drug-resistance genotyping by Ultra-Deep Sequencing and Sanger sequencing using clinical samples†

Sanger population sequencing (SPS) is the reference technique to monitor HIV‐1‐infected patients’ therapy. Ultra‐deep sequencing (UDS), which allows quantitative detection of drug resistance mutations, may be an alternative method. The study aimed to compare reproducibility and predictions of UDS versus SPS in a routine setting. A control containing low‐abundance variants was repeatedly tested and clinical plasma samples from 100 patients were prospectively assayed by SPS and UDS using the Roche 454 system. Complete analysis by UDS was available for 88% of samples with various viral loads and subtypes. Comparison of detection thresholds found that SPS sensitivity was variable. Variations found by UDS between 5% to >20% were detected by SPS in 25% to more than 80% of samples. At the 5% cut‐off, disagreements were rare and in most cases UDS detected an additional protease secondary mutation, suggesting a possible resistance to a protease inhibitor according to the 2015 ANRS algorithm. Mutations found on reverse transcriptase by only UDS were often explained by previous therapy. UDS with a variant detection threshold at 5% might allow therapy management with minimal differences compared to population sequencing while providing additional information for further determination of pertinent cutoff values for specific resistance mutations.

Relationship between discordant response to HAART, Tregs, immune activation and low-level viraemia.

The incomplete immune recovery upon effective long‐term highly active antiretroviral therapy (HAART) has been associated with increased morbidity and mortality in HIV infected patients [ 1 ]. Immune cellular activation, Tregs or very low‐level viraemia has been alternatively suspected, but never investigated simultaneously [ 2 ].