Pestivirus-en p7 biroporinaren adierazpena eta purifikazioa bakterioetan

Hepacivirus eta Pestivirus generoen artean antzekotasun handia dago, bai genomaren egituran zein erreplikazio mekanismoetan. Antzekotasun hau ez da ematen aldiz, Flavivirus generoko birusekin. Hepacivirus zein Pestivirus generoko birusek euren RNA poliproteina aitzindari bakar batera itzultzen dute; jarraian, birus beraren zein ostalariaren proteasa espezifikoek aitzindari honen prozesamendu proteolitikoa burutuko dute, era honetan, birusaren infekzioa aurrera eraman ahal izateko proteina estruktural eta ez estrukturalak lortuz

Conformational Plasticity Underlies Membrane Fusion Induced by an HIV Sequence Juxtaposed to the Lipid Envelope

Envelope glycoproteins from genetically-divergent virus families comprise fusion peptides (FPs) that have been posited to insert and perturb the membranes of target cells upon activation of the virus-cell fusion reaction. Conserved sequences rich in aromatic residues juxtaposed to the external leaflet of the virion-wrapping membranes are also frequently found in viral fusion glycoproteins. These membrane-proximal external regions (MPERs) have been implicated in the promotion of the viral membrane restructuring event required for fusion to proceed, hence, proposed to comprise supplementary FPs. However, it remains unknown whether the structure-function relationships governing canonical FPs also operate in the mirroring MPER sequences. Here, we combine infrared spectroscopy-based approaches with cryo-electron microscopy to analyze the alternating conformations adopted, and perturbations generated in membranes by CpreTM, a peptide derived from the MPER of the HIV-1 Env glycoprotein. Altogether, our structural and morphological data support a cholesterol-dependent conformational plasticity for this HIV-1 sequence, which could assist cell-virus fusion by destabilizing the viral membrane at the initial stages of the processThis study was supported by the Spanish MCIU (Grants RTI2018-095624-B-C21; MCIU/AEI/FEDER, UE to JLN and BA; and PID2019-111096GA-I00; MCIU/AEI/FEDER, UE to AC) and Basque Government (Grant: IT1196-19). Technical assistance from MI Collado and M Carril with 31P-NMR measurements and data processing is greatly acknowledge

Functional Delineation of a Protein–Membrane Interaction Hotspot Site on the HIV-1 Neutralizing Antibody 10E8

Antibody engagement with the membrane-proximal external region (MPER) of the envelope glycoprotein (Env) of HIV-1 constitutes a distinctive molecular recognition phenomenon, the full appreciation of which is crucial for understanding the mechanisms that underlie the broad neutralization of the virus. Recognition of the HIV-1 Env antigen seems to depend on two specific features developed by antibodies with MPER specificity: (i) a large cavity at the antigen-binding site that holds the epitope amphipathic helix; and (ii) a membrane-accommodating Fab surface that engages with viral phospholipids. Thus, besides the main Fab–peptide interaction, molecular recognition of MPER depends on semi-specific (electrostatic and hydrophobic) interactions with membranes and, reportedly, on specific binding to the phospholipid head groups. Here, based on available cryo-EM structures of Fab–Env complexes of the anti-MPER antibody 10E8, we sought to delineate the functional antibody–membrane interface using as the defining criterion the neutralization potency and binding affinity improvements induced by Arg substitutions. This rational, Arg-based mutagenesis strategy revealed the position-dependent contribution of electrostatic interactions upon inclusion of Arg-s at the CDR1, CDR2 or FR3 of the Fab light chain. Moreover, the contribution of the most effective Arg-s increased the potency enhancement induced by inclusion of a hydrophobic-at-interface Phe at position 100c of the heavy chain CDR3. In combination, the potency and affinity improvements by Arg residues delineated a protein–membrane interaction site, whose surface and position support a possible mechanism of action for 10E8-induced neutralization. Functional delineation of membrane-interacting patches could open new lines of research to optimize antibodies of therapeutic interest that target integral membrane epitopes.This study was supported by the Spanish MCIN (Grants PID2021-126014OB-I00 MCIN/AEI/FEDER, UE to JLN and BA; and PID2021-122212OA-I00 MCIN/AEI/FEDER, UE to ER), Basque Government (Grant: IT1449-22) and JSPS KAKENHI 20H03228 (to J.M.M.C.)

Molecular recognition of a membrane-anchored HIV-1 pan-neutralizing epitope.

Antibodies against the carboxy-terminal section of the membrane-proximal external region (C-MPER) of the HIV-1 envelope glycoprotein (Env) are considered as nearly pan-neutralizing. Development of vaccines capable of producing analogous broadly neutralizing antibodies requires deep understanding of the mechanism that underlies C-MPER recognition in membranes. Here, we use the archetypic 10E8 antibody and a variety of biophysical techniques including single-molecule approaches to study the molecular recognition of C-MPER in membrane mimetics. In contrast to the assumption that an interfacial MPER helix embodies the entire C-MPER epitope recognized by 10E8, our data indicate that transmembrane domain (TMD) residues contribute to binding affinity and specificity. Moreover, anchoring to membrane the helical C-MPER epitope through the TMD augments antibody binding affinity and relieves the effects exerted by the interfacial MPER helix on the mechanical stability of the lipid bilayer. These observations support that addition of TMD residues may result in more efficient and stable anti-MPER vaccines.This study was supported by MCIN/AEI/10.13039/501100011033 - “ERDF A way of making Europe” (Grant PID2021-126014OB-I00 to J.L.N. and B.A.), MCIN/AEI/10.13039/501100011033 (Grant PID2020-112821GB-I00 to M.A.J.), Basque Government (Grant: IT1449-22 to J.L.N. and B.A.) and Kiban-B grant 20H03228 from JSPS to J.M.M.C. L.R.-M. acknowledges funding from the Agence National de la Recherche (ANR), as part of the ‘Investments d′Avenir’ Program (I-SITE ULNE/ANR-16-IDEX-0004 ULNE). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 895819 (to C.V.). Work at Pompeu Fabra University was supported by the María de Maeztu network of Units of Excellence of the Spanish Ministry of Science and Innovation. Technical assistance from Miguel García-Porras is greatly acknowledged. The NMR experiments were performed in the “Manuel Rico” NMR laboratory, LMR, CSIC, a node of the Spanish Large-Scale National Facility ICTS R-LRB

Pestivirus-en p7 biroporinaren adierazpena eta purifikazioa bakterioetan

Hepacivirus eta Pestivirus generoen artean antzekotasun handia dago, bai genomaren egituran zein erreplikazio mekanismoetan. Antzekotasun hau ez da ematen aldiz, Flavivirus generoko birusekin. Hepacivirus zein Pestivirus generoko birusek euren RNA poliproteina aitzindari bakar batera itzultzen dute; jarraian, birus beraren zein ostalariaren proteasa espezifikoek aitzindari honen prozesamendu proteolitikoa burutuko dute, era honetan, birusaren infekzioa aurrera eraman ahal izateko proteina estruktural eta ez estrukturalak lortuz

Molecular recognition of the HIV-1 neutralizing MPER epitope reconstituted in membranes. Contribution of lipid composition and transmembrane domain inclusion.

224 p.GIBaren gainazalean kokatzen den Env proteina da birusaren infekzioa hasteko gertatu behar den birusaren eta itu zelularen mintzen arteko fusioa eragiten duena eta immunitate sistemarentzako eskuragarri dagoen proteina bakarra. Beraz, GIBaren aurkako txerto profilaktiko bat garatzeko itu ezin hobea da. Halaber, GIBaren infekzioa blokeatzeko gaitasuna duten espektro zabaleko antigorputz neutralizatzaileak isolatu dira kutsatutako zenbait indibiduoen serumetik, birus honen aurkako erantzun immune babeslea sor daitekeela frogatuz. Antigorputz hauen artean, birusaren mintzean txertatuta dagoen MPER sekuentzia ezagutzen dutenak, espektro zabalena erakusten dute. Horrez gain, oso kontserbatua dagoen eskualdea da. Anti-MPER antigorputzek mintzarekin kontaktuan dagoen sekuentzia bat ezagutzen dutenez, MPER domeinuan oinarritutako antigenoa sortzeko mintz ingurune hori mantentzea garrantzitsua dela argudiatu da. Beraz, lan honetan 10E8 moduko antigorputz neutralizatzaileak sortzeko gai diren lipido-peptido formulazioen (LPF) diseinu arrazionalerako lagundu dezaketen zenbait baldintza definitu dira. Lehenbizi, N-MPER sekuentzia ezabatu beharko litzateke antigorputzen erantzuna 10E8aren epitopora bideratzeko. Gainera, transmintz domeinua gehitzean immunogeno egonkorragoak sor daitezkeela ikusi da eta domeinu honen sekuentziaren luzerak, epitopoaren irisgarritasuna eta ondorioz, antigorputzen afinitatea modula dezake. Azkenik, epitopoak mintzen gainazalean duen aurkezpena hobetzeko, LPFak kolesterolik gabe diseinatzea gomendagarriagoa dela ikusi da