Characterization of a novel type of HIV-1 particle assembly inhibitor using a quantitative Luciferase-Vpr packaging-based assay

The HIV-1 auxiliary protein Vpr and Vpr-fusion proteins can be copackaged with Gag precursor (Pr55Gag) into virions or membrane-enveloped virus-like particles (VLP). Taking advantage of this property, we developed a simple and sensitive method to evaluate potential inhibitors of HIV-1 assembly in a living cell system. Two proteins were coexpressed in recombinant baculovirus-infected Sf9 cells, Pr55Gag, which formed the VLP backbone, and luciferase fused to the N-terminus of Vpr (LucVpr). VLP-encapsidated LucVpr retained the enzymatic activity of free luciferase. The levels of luciferase activity present in the pelletable fraction recovered from the culture medium correlated with the amounts of extracellular VLP released by Sf9 cells assayed by conventional immunological methods. Our luciferase-based assay was then applied to the characterization of betulinic acid (BA) derivatives that differed from the leader compound PA-457 (or DSB) by their substituant on carbon-28. The beta-alanine-conjugated and lysine-conjugated DSB could not be evaluated for their antiviral potentials due to their high cytotoxicity, whereas two other compounds with a lesser cytotoxicity, glycine-conjugated and ε-NH-Boc-lysine-conjugated DSB, exerted a dose-dependent negative effect on VLP assembly and budding. A fifth compound with a low cytotoxicity, EP-39 (ethylene diamine-conjugated DSB), showed a novel type of antiviral effect. EP-39 provoked an aberrant assembly of VLP, resulting in nonenveloped, morula-like particles of 100-nm in diameter. Each morula was composed of nanoparticle subunits of 20-nm in diameter, which possibly mimicked transient intermediates of the HIV-1 Gag assembly process. Chemical cross-linking in situ suggested that EP-39 favored the formation or/and persistence of Pr55Gag trimers over other oligomeric species. EP-39 showed a novel type of negative effect on HIV-1 assembly, targeting the Pr55Gag oligomerisation. The biological effect of EP-39 underlined the critical role of the nature of the side chain at position 28 of BA derivatives in their anti-HIV-1 activity

Zinc Uptake by HIV-1 Viral Particles: An Isotopic Study

International audienceZinc, an essential trace element that serves as a cofactor for numerous cellular and viral proteins, plays a central role in the dynamics of HIV-1 infection. Among the viral proteins, the nucleocapsid NCp7, which contains two zinc finger motifs, is abundantly present viral particles and plays a crucial role in coating HIV-1 genomic RNA, thus concentrating zinc within virions. In this study, we investigated whether HIV-1 virus production impacts cellular zinc homeostasis and whether isotopic fractionation occurs between the growth medium, the producing cells, and the viral particles. We found that HIV-1 captures a significant proportion of cellular zinc in the neo-produced particles. Furthermore, as cells grow, they accumulate lighter zinc isotopes from the medium, resulting in a concentration of heavier isotopes in the media, and the viruses exhibit a similar isotopic fractionation to the producing cells. Moreover, we generated HIV-1 particles in HEK293T cells enriched with each of the five zinc isotopes to assess the potential effects on the structure and infectivity of the viruses. As no strong difference was observed between the HIV-1 particles produced in the various conditions, we have demonstrated that enriched isotopes can be accurately used in future studies to trace the fate of zinc in cells infected by HIV-1 particles. Comprehending the mechanisms underlying zinc absorption by HIV-1 viral particles offers the potential to provide insights for developing future treatments aimed at addressing this specific facet of the virus’s life cycle

Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes.

International audienceUNLABELLED: HepaRG human liver progenitor cells exhibit morphology and functionality of adult hepatocytes. We investigated the susceptibility of HepaRG hepatocytes to in vitro infection with serum-derived hepatitis C virus (HCV) particles (HCVsp) and the potential neutralizing activity of the E1E2-specific monoclonal antibody (mAb) D32.10. The infection was performed using HCVsp when the cells actively divided at day 3 postplating. HCV RNA, E1E2, and core antigens were quantified in HCV particles recovered from culture supernatants of differentiated cells for up to 66 days. The density distributions of particles were analyzed on iodixanol or sucrose gradients. Electron microscopy (EM) and immune-EM studies were performed for ultrastructural analysis of cells and localization of HCV E1E2 proteins in thin sections. HCV infection of HepaRG cells was documented by increasing production of E1E2-core-RNA(+) HCV particles from day 21 to day 63. Infectious particles sedimented between 1.06 and 1.12 g/mL in iodixanol gradients. E1E2 and core antigens were expressed in 50% of HCV-infected cells at day 31. The D32.10 mAb strongly inhibited HCV RNA production in HepaRG culture supernatants. Infected HepaRG cells frozen at day 56 were reseeded at low density. After only 1-3 subcultures and induction of a cell differentiation process the HepaRG cells produced high titer HCV RNA and thus showed to be sustainably infected. Apolipoprotein B-associated empty E1E2 and complete HCV particles were secreted. Characteristic virus-induced intracellular membrane changes and E1E2 protein-association to vesicles were observed. CONCLUSION: HepaRG progenitor cells permit HCVsp infection. Differentiated HepaRG cells support long-term production of infectious lipoprotein-associated enveloped HCV particles. The E1E2-specific D32.10 mAb neutralizes the infection and this cellular model could be used as a surrogate infection system for the screening of entry inhibitors

3D investigation of a PS/ABS polymer using different microscopy techniques

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Renal Involvement in Neuropathy, Ataxia, Retinitis Pigmentosa (NARP) Syndrome: A Case Report

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Rapeseed Lecithin Increases Lymphatic Lipid Output and α-Linolenic Acid Bioavailability in Rats

International audienceBackground: Soybean lecithin, a plant-based emulsifier widely used in food, is capable of modulating postprandial lipid metabolism. With arising concerns of sustainability, alternative sources of vegetal lecithin are urgently needed, and their metabolic effects must be characterized.Objectives: We evaluated the impact of increasing doses of rapeseed lecithin (RL), rich in essential alpha-linolenic acid (ALA), on postprandial lipid metabolism and ALA bioavailability in lymph-cannulated rats.Methods: Male Wistar rats (8 weeks old) undergoing a mesenteric lymph duct cannulation were intragastrically administered 1 g of an oil mixture containing 4% ALA and 0, 1, 3, 10, or 30% RL (5 groups). Lymph fractions were collected for 6 h. Lymph lipids and chylomicrons (CMs) were characterized. The expression of genes implicated in intestinal lipid metabolism was determined in the duodenum at 6 h. Data was analyzed using either sigmoidal or linear mixed-effects models, or one-way ANOVA, where appropriate.Results: RL dose-dependently increased the lymphatic recovery (AUC) of total lipids (1100 mu g/mL.h per additional RL%; P = 0.010) and ALA (50 mu g/mL.h per additional RL%; P = 0.0076). RL induced a faster appearance of ALA in lymph, as evidenced by the exponential decrease of the rate of appearance of ALA with RL (R-2 = 0.26; P= 0.0064). Although the number of CMs was unaffected by RL, CM diameter was increased in the 30%-RL group, compared to the control group (0% RL), by 86% at 3-4 h (P = 0.065) and by 81% at 4-6 h (P = 0.0002) following administration. This increase was positively correlated with the duodenal mRNA expression of microsomal triglyceride transfer protein (Mttp; rho= 0.63; P= 0.0052). The expression of Mttp and secretion-associated, ras-related GTPase 1 gene homolog B (Sarl b, CM secretion), carnitine palmitoyltransferase IA (Cpt1a) and acyl-coenzyme A oxidase 1 (Acoxl, beta-oxidation), and fatty acid desaturase 2 (Fads2, bioconversion of ALA into long-chain n-3 PUFAs) were, respectively, 49%, 29%, 74%, 48%, and 55% higher in the 30%-RL group vs. the control group (P < 0.05).Conclusions: In rats, RL enhanced lymphatic lipid output, as well as the rate of appearance of ALA, which may promote its subsequent bioavailability and metabolic fate

Quantification of VLP assembly and egress using luciferase assay.

<p>(<b>a</b>), <b><i>Ultracentrifugation analysis of VLP.</i></b> Cells coexpressing Pr55Gag and LucVpr were untreated (control 0) or treated with PA-457 in DMSO for 24 h at 24 h pi, at increasing concentrations as indicated. VLP were isolated from the culture medium at 48 h pi by isopycnic ultracentrifugation in sucrose-D₂0 density gradient, and assayed for luciferase activity, expressed as relative light units (RLU). (<b>b)</b>, <b><i>Dose-response curve of PA-457 inhibitory effect on VLP production</i></b>. The ratio of VLP-associated to intracellular luciferase activity was plotted versus PA-457 concentrations. The IC₅₀ value obtained was 2.2–2.4 µg/ml. <b><i>Inset</i></b> : VLP production (<b><i>top</i></b>) and intracellular expression of Pr55Gag (<b><i>bottom</i></b>) were evaluated in parallel by Western blot analysis using anti-Gag rabbit antibody and phosphatase-labeled conjugate.</p

Formyl Peptide Receptor 2 Plays a Deleterious Role During Influenza A Virus Infections

International audienceBackground: The pathogenesis of influenza A virus (IAV) infections is a multifactorial process that includes the replication capacity of the virus and a harmful inflammatory response to infection. Formyl peptide receptor 2 (FPR2) emerges as a central receptor in inflammatory processes controlling resolution of acute inflammation. Its role in virus pathogenesis has not been investigated yet.Methods, We used pharmacologic approaches to investigate the role of FPR2 during IAV infection in vitro and in vivo.Results: In vitro, FPR2 expressed on A549 cells was activated by IAV, which harbors its ligand, annexin A1, in its envelope. FPR2 activation by IAV promoted viral replication through an extracellular-regulated kinase (ERK)–dependent pathway. In vivo, activating FPR2 by administering the agonist WKYMVm-NH2 decreased survival and increased viral replication and inflammation after IAV infection. This effect was abolished by treating the mice with U0126, a specific ERK pathway inhibitor, showing that, in vivo, the deleterious role of FPR2 also occurs through an ERK-dependent pathway. In contrast, administration of the FPR2 antagonist WRW4 protected mice from lethal IAV infections.Conclusions: These data show that viral replication and IAV pathogenesis depend on FPR2 signaling and suggest that FPR2 may be a promising novel strategy to treat influenza

Electron microscopy of EP-39-treated, Pr55Gag-expressing cells.

<p>Samples of Sf9 cells coinfected with AcMNPV-Pr55Gag and AcMNPV-LucVpr at equal MOI, were treated at 24 h pi with 10 µg/ml of EP-39 inhibitor for 24 h, harvested at 48 h pi, and processed for observation under the EM. Note the morula-like shape of electron-dense particles of ca. 100 nm in diameter, some of which in the process of egressing into the extracellular milieu.</p

Structural analysis of extracellular EP-39-induced morula-like particles.

<p>Sf9 cells infected with AcMNPV-Pr55Gag were untreated (<b>a</b>) or treated (<b>b, c</b>) at 24 h pi with EP-39 (10 µg/ml) for 24 h. Cell culture medium was harvested at 48 h pi, VLP pelleted by ultracentrifugation, and processed for EM analysis. Electron-dense 100-nm particles are viewed at low (<b>b</b>) and high (<b>c</b>) magnification, respectively. Control, membrane-enveloped VLP released from untreated cells (<b>a</b>) are shown at the same magnification as in (<b>c</b>). (<b>d</b>), Hypothetical model of an EP-39-induced nanoparticle of ca. 20 nm in diameter, composed of 12 to 16 copies of Pr55Gag assembled with a dodecahedral or octahedral symmetry.</p