The postbinding activity of scavenger receptor class B type I mediates initiation of hepatitis C virus infection and viral dissemination.

International audienceUNLABELLED: Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR-BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR-BI and HCV envelope glycoproteins has been reported to take place during this process. SR-BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR-BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR-BI remains largely unknown. To uncover the mechanistic role of SR-BI in viral initiation and dissemination, we generated a novel class of anti-SR-BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR-BI, we demonstrate that the postbinding function of SR-BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR-BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function. CONCLUSION: Taken together, our results uncover a crucial role of the SR-BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor-E2/HDL interactions. The dissection of the molecular mechanisms of SR-BI-mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR-BI

Methodological studies of the interaction between membrane-active peptides and membranes : structure/activity relationship and affinity photolabeling coupled to mass spectrometry

Les peptides membranotropes constituent une famille de peptides qui interagissent avec les membranes biologiques. Parmi ceux-ci, deux ont été étudiés : la Pénétratine (Pen), un peptide pénétrant (CPP), connu pour traverser les membranes sans les perturber et DMS-DA6-NH2, un peptide antimicrobien (AMP) de la famille des Dermaseptines, capable de cibler et détruire les membranes bactériennes. Une étude de relation structure/activité a été entreprise pour comprendre le rôle des différents résidus/régions de la séquence de DMS-DA6-NH2 et optimiser cette séquence en termes d’activité et d’ionisation (longueur et présence de résidu basique) pour son étude en spectrométrie de masse (MS). Différentes substitutions, insertions et/ou délétions ont été étudiées. En parallèle, l’identification des partenaires lipidiques d’interaction de Pen et DSM-DA6-NH2 a été entreprise par photomarquage d’affinité couplé à la MS. Des analogues photoactivables ont été synthétisés en faisant varier la position de la photosonde benzophénone (Bzp) et sa distance au squelette peptidique. Des études de calorimétrie différentielle à balayage ont montré que ces analogues conservaient des propriétés membranotropes. Pour une distance courte les photoadduits sont instables et conduisent à des réactions secondaires de la Bzp. La préférence lipidique des peptides pour des membranes modèles de composition variée est difficile à évaluer si on tient compte de la réactivité de la Bzp sur les positons α-allyliques. L’utilisation d’une photosonde diazirine (Diaz), dont la photoréactivité est différente de la Bzp montre qu’elle pourrait être utilisée en complément de la Bzp pour l’étude des interactions peptide/lipides.Membrane-active peptides are a family of peptides which interact with biological membranes. Amongst them, two have been studied: Penetratin (Pen), a cell-penetrating peptide (CPP), known to cross membranes in a non-disruptive way, and DMS-DA6-NH2, an antimicrobial peptide (AMP) from the Dermaseptin family, able to target and destroy bacterial membranes. A structure/activity relationship study was undertaken to understand the role of the different residues/regions of the DMS-DA6-NH2 sequence and to optimize this sequence in terms of activity and ionization for its study by mass spectrometry (MS) by modulating the length and amino-acid composition. Different substitutions, insertions and/or deletions have been studied. In parallel, the identification of the lipid interaction partners of Pen and DSM-DA6-NH2 was undertaken by affinity photolabeling coupled to MS. Photoactivatable analogues have been synthesized by varying the position of the benzophenone (Bzp) photoprobe and its distance from the peptide backbone. Differential scanning calorimetry confirmed these analogues retain membrane-active properties. For a short distance between the backbone and the Bzp, the photoadducts generated after UV irradiation are unstable. The lipid preference of peptides for model membranes of varying composition differs depending on the phase state of the membranes. However, Bzp reacts preferentially on α-allylic positions, which makes it impossible to distinguish this reactivity from the preference of peptides for unsaturated lipid partners. To make this distinction, the diazirine probe (Diaz) was used. The results show that Diaz is suitable for the study of peptide/lipid interactions

Benzophenone Photoreactivity in a Lipid Bilayer To Probe Peptide/Membrane Interactions: Simple System, Complex Information

International audienceAffinity photo-cross-linking coupled to mass spec-trometry, using benzophenone (Bzp)-functionalized peptides, was used to study the noncovalent interactions of cell-penetrating peptides and lipid membranes. Using biomimetic lipid vesicles composed of saturated and unsaturated negatively charged lipids, DMPG (14:0), DPPG (16:0), DOPG (18:1 cis Δ 9), 18:1 (trans Δ 9) PG, and DLoPG (18:2 cis Δ 9, 12), allowed observation of all the classical and less common reactivities of Bzp described in the literature by direct MS analysis: CC double bond formation on saturated fatty acids, covalent adducts formation via classical C−C bond, and Paterno-Buchi oxetane formation followed or not by fragmentation (retro-Paterno-Buchi) as well as photosensitization of unsaturated lipids leading to lipid dimers. All these reactions can occur concomitantly in a single complex biological system: a membrane-active peptide inserted within a phospholipid bilayer. We also detect oxidation species due to the presence of radical oxygen species. This work represents a noteworthy improvement for the characterization of interacting partners using Bzp photo-cross-linking, and it shows how to exploit in an original way the different reactivities of Bzp in the context of a lipid membrane. We propose an analytical workflow for the interpretation of MS spectra, giving access to information on the CPP/lipid interaction at a molecular level such as depth of insertion or membrane fluidity in the CPP vicinity. An application of this workflow illustrates the role of cholesterol in the CPP/lipids interaction

Structural bases for the involvement of phosphatidylinositol-4,5-bisphosphate in the internalization of the cell-penetrating peptide Penetratin

International audienceCell-penetrating peptides cross cell membranes through various parallel internalization pathways. Herein, we analyze the role of the negatively charged lipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) in the internalization of Penetratin. Contributions of both inner leaflet and outer leaflet pools of PI(4,5)P2 were revealed by quantifying the internalization of Penetratin in cells treated with PI(4,5)P2 binders. Studies on model systems showed that Penetratin has a strong affinity for PI(4,5)P2, and interacts selectively with this lipid, even in the presence of other negatively charged lipids, as demonstrated by affinity photocrosslinking experiments. Differential scanning calorimetry experiments showed that Penetratin induces lateral segregation in PI(4,5)P2-containing liposomes, which was confirmed by coarse-grained molecular dynamics simulations. NMR experiments indicated that Penetratin adopts a stabilized helical conformation in the presence of PI(4,5)P2-containing membranes, with an orientation parallel to the bilayer plane, which was also confirmed by all-atom simulations. NMR and photocrosslinking experiments also suggest a rather shallow insertion of the peptide in the membrane. Put together, our findings suggest that PI(4,5)P2 is a privileged interaction partner for Penetratin and that it plays an important role in Penetratin internalization

The postbinding activity of scavenger receptor class B type I mediates initiation of hepatitis C virus infection and viral dissemination

Scavenger receptor class B type I (SR-BI) is a high-density lipoprotein (HDL) receptor highly expressed in the liver and modulating HDL metabolism. Hepatitis C virus (HCV) is able to directly interact with SR-BI and requires this receptor to efficiently enter into hepatocytes to establish productive infection. A complex interplay between lipoproteins, SR-BI and HCV envelope glycoproteins has been reported to take place during this process. SR-BI has been demonstrated to act during binding and postbinding steps of HCV entry. Although the SR-BI determinants involved in HCV binding have been partially characterized, the postbinding function of SR-BI remains largely unknown. To uncover the mechanistic role of SR-BI in viral initiation and dissemination, we generated a novel class of anti-SR-BI monoclonal antibodies that interfere with postbinding steps during the HCV entry process without interfering with HCV particle binding to the target cell surface. Using the novel class of antibodies and cell lines expressing murine and human SR-BI, we demonstrate that the postbinding function of SR-BI is of key impact for both initiation of HCV infection and viral dissemination. Interestingly, this postbinding function of SR-BI appears to be unrelated to HDL interaction but to be directly linked to its lipid transfer function. Conclusion: Taken together, our results uncover a crucial role of the SR-BI postbinding function for initiation and maintenance of viral HCV infection that does not require receptor-E2/HDL interactions. The dissection of the molecular mechanisms of SR-BI-mediated HCV entry opens a novel perspective for the design of entry inhibitors interfering specifically with the proviral function of SR-BI. (HEPATOLOGY 2013).status: publishe

Mutations that alter use of hepatitis C virus cell entry factors mediate escape from neutralizing antibodies.

International audienceBACKGROUND & AIMS: The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS: We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture-derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS: By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus-antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS: We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines

Immunogenicity and efficacy of heterologous ChAdOx1-BNT162b2 vaccination

Following severe adverse reactions to the AstraZeneca ChAdOx1-S-nCoV-19 vaccine1,2, European health authorities recommended that patients under the age of 55?years who received one dose of ChAdOx1-S-nCoV-19 receive a second dose of the Pfizer BNT162b2 vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here we show that the heterologous ChAdOx1-S-nCoV-19 and BNT162b2 combination confers better protection against severe acute respiratory syndrome coronavirus?2 (SARS-CoV-2) infection than the homologous BNT162b2 and BNT162b2 combination in a real-world observational study of healthcare workers (n?=?13,121). To understand the underlying mechanism, we conducted a longitudinal survey of the anti-spike immunity conferred by each vaccine combination. Both combinations induced strong anti-spike antibody responses, but sera from heterologous vaccinated individuals displayed a stronger neutralizing activity regardless of the SARS-CoV-2 variant. This enhanced neutralizing potential correlated with increased frequencies of switched and activated memory B cells that recognize the SARS-CoV-2 receptor binding domain. The ChAdOx1-S-nCoV-19 vaccine induced a weaker IgG response but a stronger T cell response than the BNT162b2 vaccine after the priming dose, which could explain the complementarity of both vaccines when used in combination. The heterologous vaccination regimen could therefore be particularly suitable for immunocompromised individuals